Focus Issue Editorial: Synthetic BiologyHanson, Andrew D.; Hibberd, Julian M.; Koffas, Mattheos A.G.; Kopka, Joachim; Wurtzel, Eleanore T.
doi: 10.1104/pp.19.00074pmid: 30808713
Synthetic biology (SynBio) is a conceptual and operational revolution (Church et al., 2014) that’s coming soon to a branch of plant science near you, if it’s not there already (Liu and Stewart, 2015). The Synthetic Biology Focus Issue sets out to spread this disruptive news. SynBio is a transformative combination of DNA technology, engineering principles, and computational tools that makes it possible to design new life processes and to repurpose existing natural ones for useful purposes (Purnick and Weiss, 2009). SynBio will profoundly impact and empower how plant science is done and how plant science is used to sustainably solve global problems. SynBio is already creating new jobs and is likely to keep doing this for decades (Delebecque and Philp, 2015). SynBio is powerful for conceptual and operational reasons. The enormous conceptual power of SynBio is to open access to the vast “design space” that plants, and nature in general, have not explored (Bhatia et al., 2017). By discovering and deploying useful design space that evolution has “missed,” SynBio enables plants: to make familiar compounds by new pathways, and make new-to-nature compounds; to supply genes needed to make high-value compounds in microorganisms; to manipulate familiar morphological structures, and build totally new-to-nature ones; to respond in new ways to old stimuli, and sense and respond to totally new stimuli; and to be managed based on weather forecasts and other predictions that plants cannot make themselves. The transformative operational power of SynBio lies in its drive to industrialize biology: that is, to replace high-skill, slow, and costly artisanal work such as cloning and custom assays with computationally guided, automated, and standardized engineering procedures (Cameron et al., 2014; Chao et al., 2017). Although the industrial-scale engineering potential of SynBio is far from fully realized at this point (Davies, 2019), it is very much here to stay and has begun to dictate major changes in biology education and training (National Research Council, 2015). Whether you are a trainee or a trainer, these trends are seriously worth considering for your future career’s sake. A hallmark of SynBio is to rethink biological molecules, genes and proteins, as engineering “parts” that can be standardized, quantitatively characterized, and used to build a range of devices, much as standard resistors and capacitors are used as components in countless different electrical circuits (de Lorenzo and Schmidt, 2018). In the case of metabolic pathways, for instance, enzyme parts from plants or other organisms can be used as is and combined in novel ways to build new pathways, or used as the starting point for directed evolution to create an enzyme part with a new-to-nature activity (Erb et al., 2017). Directed evolution is one of SynBio’s most iconic achievements, as recognized by the 2018 Nobel Prize in Chemistry to Frances H. Arnold “for the directed evolution of enzymes” (Ranganathan, 2018). However, reconceiving genes and proteins as potential SynBio parts spotlights how massively incomplete our “parts lists” for organisms still are. Even in the minimal Mycoplasma mycoides JCVI-syn3.0 genome, 149 genes out of a total of 473 (32%) have no known biological function (Hutchison et al., 2016), and the situation is far worse in plants, with Arabidopsis (Arabidopsis thaliana) having ∼7,000 enzymes and transporters of unknown function (Niehaus et al., 2015). The curiosity-driven research that has been accustomed to sifting through these proteins to assign function with few if any concrete objectives can now be reframed and refocused as purposeful bioprospecting or “parts discovery” for SynBio, a highly defensible justification for continued public support of what can seem to some like an aimless academic exercise. Principal investigators might want to consider this argument. The collection of Update Reviews, Research Articles, and Breakthrough Technologies Articles in this Focus Issue cover the above conceptual and operational possibilities and more, as we now detail. Starting with SynBio-enabled engineering of plant metabolism, three Update Reviews and one Research Article cover photosynthesis and plastid metabolism. The Update by Leister (2019) explores progress and prospects in applying SynBio to the light reactions of photosynthesis, both to enhance the efficiency of light use in crops and to couple the light reactions in novel ways to enzymes or nonbiological components that use the reducing power from the light reactions (see also the News and Views article in this issue by Moses, 2019). The Update by Boehm and Bock (2019) explains the promise of the plastid genome as a platform for engineering plant metabolism, describes tools and technologies for plastid SynBio, and highlights challenges for future research in this area. The Research Article by Occhialini et al. (2019) expands on tools and technologies by describing the development and validation of a modular chloroplast cloning system, MoChlo, for constructing heterologous metabolic pathways in plastids. The Update by Weber and Bar-Even (2019) discusses the huge potential for SynBio to raise crop yields by increasing the CO2 concentration around Rubisco to reduce photorespiration and by rerouting photorespiratory metabolism via synthetic bypasses or new pathways that convert photorespiration from a carbon-loss process into a carbon-gain process. Two Update Reviews and a Research Article then look toward the SynBio-enabled future of plant metabolic engineering. Mitchell and Weng (2019) review the structure-function relationships of cytochrome P450 monooxygenases and other plant oxygenases and show how these enzymes are a versatile toolset for SynBio to develop biocatalysts and manipulate plant traits. The review by Wurtzel (2019) explores how applying SynBio to carotenoid pathways could alter plant form and function for agricultural and industrial purposes and emphasizes the ongoing need for discovery research to drive the SynBio era forward. Illustrating the utility of discovery research, Sun et al. (2019) report success in a targeted parts-prospecting project to discover genes from which to start the directed evolution of a highly efficient thiamin synthesis enzyme. Two articles come from the very active and commercially important field of rebuilding plant biosynthetic pathways in tractable laboratory microbes. The Update by Pyne et al. (2019) provides a broad overview of the field and its challenges, with particular emphasis on pathways for total de novo synthesis of high-value plant isoprenoids, alkaloids, phenylpropanoids, and polyketides from sugar feedstocks. The Research Article by Kallscheuer et al. (2019) first establishes that salidroside (a phenylpropanoid glucoside from raspberry [Rubus idaeus]) is bioactive, then details the reconstruction of salidroside biosynthesis in Saccharomyces cerevisiae and Corynebacterium glutamicum. Two Updates and three Articles cover orthogonal (i.e. nonnative) synthetic sensing and regulatory circuits and their application to plant development. The review by Andres et al. (2019) introduces and illustrates the key concepts of synthetic switches, logic gates, and regulatory circuits, which leads neatly to the Research Articles by Iacopino et al. (2019), on the design and testing of a synthetic oxygen sensor for plants based on bioengineered versions of a mammalian oxygen sensor, and by Schreiber et al. (2019), on design and optimization of a two-component AND-gate system for synthetic circuits in plants based on bacterial transcription activator-like effectors. Wright and Nemhauser (2019) review how SynBio can help uncover the parts and logic mediating plant development, leading to predictive models that specify the molecular circuitry needed to change cell state. This review connects to the Breakthrough Technologies Article by Faden et al. (2019) on the use of a temperature-switchable version of a toxic bacterial protein to control trichome development as a test case. Lastly, three Updates and two Articles address core technologies that enable SynBio. One such technology, and another hallmark of full-blown SynBio, is computational design, which Küken and Nikoloski (2019) review in relation to synthetic metabolic pathways using a case study approach. Two further core SynBio technologies are protein engineering and directed evolution, both of which are reviewed by Engqvist and Rabe (2019). Another core technology is the construction of synthetic enzyme complexes, comprehensively reviewed by Smirnoff (2019) and illustrated by Camagna et al. (2019) in a Research Article detailing a fused-enzyme metabolon to boost flux to phytoene synthesis. In closing their Update, Weber and Bar-Even (2019) quote Nobel Peace Prize winner Norman Borlaug: “Then I wake up and become disillusioned to find that mutation genetics programs are still engaged mostly in such minutiae as putting beards on wheat plants and taking off the hairs.” Weber and Bar-Even then add on their own account, “we should leave the minutiae behind and reap the full potential of synthetic biology to overcome one of the major challenges of the 21st century, sustainably feeding a growing population without destroying the environment.” Amen. LITERATURE CITED Andres J , Blomeier T, Zurbriggen MD ( 2019 ) Synthetic switches and regulatory circuits in plants . Plant Physiol 179 : 862–884 Google Scholar OpenURL Placeholder Text WorldCat Bhatia SP , Smanski MJ, Voigt CA, Densmore DM ( 2017 ) Genetic design via combinatorial constraint specification . ACS Synth Biol 6 : 2130 – 2135 Google Scholar Crossref Search ADS PubMed WorldCat Boehm CR , Bock R ( 2019 ) Recent advances and current challenges in synthetic biology of the plastid genetic system and metabolism . Plant Physiol 179 : 794–802 Google Scholar OpenURL Placeholder Text WorldCat Camagna M , Grundmann A, Bär C, Koschmieder J, Beyer P, Welsch R ( 2019 ) Enzyme fusion removes competition for geranylgeranyl diphosphate in carotenogenesis . Plant Physiol 179 : 1013–1027 Google Scholar OpenURL Placeholder Text WorldCat Cameron DE , Bashor CJ, Collins JJ ( 2014 ) A brief history of synthetic biology . Nat Rev Microbiol 12 : 381 – 390 Google Scholar Crossref Search ADS PubMed WorldCat Chao R , Mishra S, Si T, Zhao H ( 2017 ) Engineering biological systems using automated biofoundries . Metab Eng 42 : 98 – 108 Google Scholar Crossref Search ADS PubMed WorldCat Church GM , Elowitz MB, Smolke CD, Voigt CA, Weiss R ( 2014 ) Realizing the potential of synthetic biology . Nat Rev Mol Cell Biol 15 : 289 – 294 Google Scholar Crossref Search ADS PubMed WorldCat Davies JA ( 2019 ) Real-world synthetic biology: Is it founded on an engineering approach, and should it be? Life (Basel) 9 : E6 Google Scholar PubMed OpenURL Placeholder Text WorldCat Delebecque C , Philp J ( 2015 ) Training for synthetic biology jobs in the new bioeconomy. http://www.sciencemag.org/careers/2015/06/training-synthetic-biology-jobs-new-bioeconomy de Lorenzo V , Schmidt M ( 2018 ) Biological standards for the knowledge-based bioeconomy: What is at stake . N Biotechnol 40 : 170 – 180 Google Scholar Crossref Search ADS PubMed WorldCat Engqvist MKM , Rabe KS ( 2019 ) Applications of protein engineering and directed evolution in plant research . Plant Physiol 179 : 907–917 Google Scholar OpenURL Placeholder Text WorldCat Erb TJ , Jones PR, Bar-Even A ( 2017 ) Synthetic metabolism: Metabolic engineering meets enzyme design . Curr Opin Chem Biol 37 : 56 – 62 Google Scholar Crossref Search ADS PubMed WorldCat Faden F , Mielke S, Dissmeyer N ( 2019 ) Switching toxic protein function in life cells . Plant Physiol 179 : Google Scholar OpenURL Placeholder Text WorldCat Hutchison CA III , Chuang RY, Noskov VN, Assad-Garcia N, Deerinck TJ, Ellisman MH, Gill J, Kannan K, Karas BJ, Ma L, et al. ( 2016 ) Design and synthesis of a minimal bacterial genome . Science 351 : aad6253 Google Scholar Crossref Search ADS PubMed WorldCat Iacopino S , Jurinovich S, Cupellini L, Piccinini L, Cardarelli F, Perata P, Mennucci B, Giuntoli B, Licausi F ( 2019 ) A synthetic oxygen sensor for plants based on animal hypoxia signalling . Plant Physiol 179 : 986–1000 Google Scholar Crossref Search ADS PubMed WorldCat Kallscheuer N , Menezes R, Foito A, Silva M, Braga A, Dekker W, Sevillano DM, Rosado-Ramos R, Jardim C, Oliveira J, et al. ( 2019 ) Identification and microbial production of the raspberry phenol salidroside that is active against Huntington’s disease . Plant Physiol 179 : 969–985 Google Scholar OpenURL Placeholder Text WorldCat Küken A , Nikoloski Z ( 2019 ) Design of synthetic metabolic pathways: Challenges and opportunities for plant synthetic biology . Plant Physiol 179 : 894–906 Google Scholar OpenURL Placeholder Text WorldCat Leister D ( 2019 ) Genetic engineering, synthetic biology and the light reactions of photosynthesis . Plant Physiol 179 : 778–793 Google Scholar OpenURL Placeholder Text WorldCat Liu W , Stewart CN Jr ( 2015 ) Plant synthetic biology . Trends Plant Sci 20 : 309 – 317 Google Scholar Crossref Search ADS PubMed WorldCat Mitchell AJ , Weng JK ( 2019 ) Unleashing the synthetic power of plant oxygenases: From mechanism to application . Plant Physiol 179 : 813–829 Google Scholar OpenURL Placeholder Text WorldCat Moses T ( 2019 ) Shedding light on the power of light . Plant Physiol 179 : 775–777 Google Scholar OpenURL Placeholder Text WorldCat National Research Council ( 2015 ) Industrialization of Biology: A Roadmap to Accelerate the Advanced Manufacturing of Chemicals. National Academies Press , Washington, DC Google Scholar PubMed OpenURL Placeholder Text Google Preview WorldCat COPAC Niehaus TD , Thamm AM, de Crécy-Lagard V, Hanson AD ( 2015 ) Proteins of unknown biochemical function: A persistent problem and a roadmap to help overcome it . Plant Physiol 169 : 1436 – 1442 Google Scholar PubMed OpenURL Placeholder Text WorldCat Occhialini A , Piatek A, Pfotenhauer AC, Frazier TP, Stewart CN Jr. , Lenaghan SC ( 2019 ) MoChlo: A versatile modular cloning toolbox for chloroplast biotechnology . Plant Physiol 179 : 943–957 Google Scholar OpenURL Placeholder Text WorldCat Purnick PE , Weiss R ( 2009 ) The second wave of synthetic biology: From modules to systems . Nat Rev Mol Cell Biol 10 : 410 – 422 Google Scholar Crossref Search ADS PubMed WorldCat Pyne ME , Narcross L, Martin VJJ ( 2019 ) Engineering plant secondary metabolism in microbial systems . Plant Physiol 179 : 844–861 Google Scholar OpenURL Placeholder Text WorldCat Ranganathan R ( 2018 ) Putting evolution to work . Cell 175 : 1449 – 1451 Google Scholar Crossref Search ADS PubMed WorldCat Schreiber T , Prange A, Tissier AF ( 2019 ) Split-TALE: A TALE-based two-component system for synthetic biology applications in planta . Plant Physiol 179 : 1001–1012 Google Scholar OpenURL Placeholder Text WorldCat Smirnoff N ( 2019 ) Engineering of metabolic pathways using synthetic enzyme complexes . Plant Physiol 179 : 918–928 Google Scholar OpenURL Placeholder Text WorldCat Sun J , Sigler CL, Beaudoin GA, Joshi J, Patterson JA, Cho KH, Ralat MA, Gregory JF, Clark DG, Deng Z, et al. ( 2019 ) Parts-prospecting for a high-efficiency thiamin thiazole biosynthesis pathway . Plant Physiol 179 : 958–968 Google Scholar OpenURL Placeholder Text WorldCat Weber APM , Bar-Even A ( 2019 ) Improving the efficiency of photosynthetic carbon reactions . Plant Physiol 179 : 803–812 Google Scholar OpenURL Placeholder Text WorldCat Wright RC , Nemhauser J ( 2019 ) How can synthetic biology help quantify and discover the unknowns in plant development? Plant Physiol 179 : 885–893 Google Scholar OpenURL Placeholder Text WorldCat Wurtzel ET ( 2019 ) Changing form and function through carotenoids and synthetic biology . Plant Physiol 179 : 830–843 Google Scholar OpenURL Placeholder Text WorldCat Author notes 1 Author for contact: [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.19.00074 © 2019 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Shedding Light on the Power of LightMoses, Tessa
doi: 10.1104/pp.19.00045pmid: 30808714
Photosynthesis is the process in which solar energy is used to fix atmospheric carbon dioxide (CO2) to chemical energy in phototrophs. Oxygenic photosynthesis is the principle producer of oxygen (O2) and organic matter that sustains life on earth. In reality, only a fraction of the solar energy that reaches the earth’s surface is actually fixed. This is because, of the average solar spectrum that reaches the earth’s surface, only 48.7% falls within the photosynthetically active band of 400 to 740 nm, and oxygenic photosynthesis is highly inefficient. The maximum conversion efficiency of solar energy to biomass in plants is estimated to be 4.6% for C3 photosynthesis and 6% for C4 photosynthesis at 30°C and 380 ppm atmospheric CO2 (Zhu et al., 2008). In comparison, solar photovoltaic panels convert light to electrical energy at an efficiency of 6% to 20% (Gul et al., 2016). Three kinds of photosynthesis, C3, C4, and Crassulacean acid metabolism, are identified in plants, based on their natural habitat, first molecule produced by the fixation of CO2, and photorespiration rate. Approximately 85% of all known plants, including crops like rice (Oryza sativa), wheat (Triticum aestivum), and soybean (Glycine max), have C3 photosynthesis, which is less efficient than C4 photosynthesis that occurs in vascular plants (∼3%), like sugarcane (Saccharum officinarum), crabgrass (Digitaria sanguinalis), and corn (Zea mays). With the advances in genetic engineering, it is now possible to engineer plants for improved photosynthetic efficiency, which could in turn boost crop yield and agricultural productivity and significantly contribute to recapturing atmospheric CO2 (Foyer et al., 2017). The ongoing multipartner Photosynthesis 2.0 Program was initiated to exploit the natural variation in photosynthetic efficiencies of plants and to engineer robust crop plants of the future that can withstand negative effects of global climate change, extreme weather, and soil deterioration, while efficiently utilizing resources, like water and minerals (https://www.wur.nl/upload_mm/2/4/5/0a26c527-34e4-4c95-8e58-4f343abdbce9_WUR_Brochure_Photosynthesis2018_vdef.pdf). The well-known ribulose-1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) enzyme catalyses the carboxylation reaction of RuBP (CO2 fixation) in photosynthesis. The Rubisco-catalyzed reaction is accelerated with increasing concentrations of atmospheric CO2. However, the regeneration of RuBP (the acceptor of CO2) by the Calvin cycle enzyme sedoheptulose-1,7-biphosphatase (SBPase) is kinetically limited, which hinders photosynthetic efficiency at higher concentrations of atmospheric CO2. With increasing levels of global atmospheric CO2, attempts to improve photosynthetic efficiency in model and crop plants have been reported. The first successful photosynthetic carbon metabolism engineering was demonstrated in tobacco (Nicotiana tabacum) by overexpressing the Arabidopsis (Arabidopsis thaliana) SBPase gene, which resulted in higher SBPase activity, increased photosynthetic rate, higher levels of sucrose and starch accumulation, and total biomass increment of ∼30% in controlled (Lefebvre et al., 2005) and field (Rosenthal et al., 2011) conditions. Elevating SBPase activity in the food crops tomato (Solanum lycopersicum; Ding et al., 2016) and rice (Feng et al., 2007a, 2007b) also resulted in similar increases in photosynthesis rate and growth under abiotic stress. In addition, transgenic wheat lines overexpressing Brachypodium distachyon SBPase have also been reported to show higher rates of CO2 assimilation and ∼40% increase in total biomass and seed weight per plant (Driever et al., 2017). Recently, an alternative synthetic biology strategy to enhance photosynthetic efficiency by tackling the bottleneck of photorespiration that occurs in most C3 crops was reported. Photorespiration is the process in which Rubisco oxygenates the substrate RuBP, which leads to costly processing of toxic byproducts, such as glycolic acid, and 20% to 50% reduction in photosynthetic efficiency. To overcome this bottleneck, South et al. installed a synthetic glycolic acid metabolic pathway in tobacco chloroplasts by expressing pumpkin (Cucurbita maxima) malate synthase and Chlamydomonas reinhardtii glycolate dehydrogenase and inhibited glycolic acid export from the chloroplast by down-regulating the expression of a native glycolate transporter. The resulting homozygous transgenic tobacco lines showed > 40% increase in biomass in field trials (South et al., 2019). These limited examples of engineering photosynthesis for improved efficiency highlight the complex nature of the light reactions, which are carried out by highly conserved structural components that are organized into evolutionarily conserved multiprotein complexes. Photosynthesis in plants require the interaction of two photosystems that are excited by light of wavelengths ≤ 700 nm and < 680 nm, referred to as PSI and PSII, respectively. The reaction centers of each photosystem contain chlorophyll molecules that differ in their light absorption maxima, because of which they are denoted as P700 (PSI) and P680 (PSII). To maintain the flow of electrons, the photosystems are connected by a multiprotein complex called cytochrome b6f (Cyt b6f), and the electron carriers plastoquinone and plastocyanin transport electrons from PSII to Cyt b6f and from Cyt b6f to PSI, respectively. The reaction center in each photosystem is associated with a light-harvesting complex that consists of different proteins, as a result of which PSII and PSI have defined functions in plants, with the former involved in the splitting of water to release O2 and the latter involved in the reduction of NADP+ to NADPH. Cyanobacteria and algae also use water as an electron donor and have two photosystems, which contrasts with purple and green photosynthetic bacteria that have only one photosystem (Blankenship, 2010). However, among the two photosystems, PSI has an inherent higher photosynthetic efficiency than PSII (Caffarri et al., 2014). In this special issue on synthetic biology, Leister (2018) sheds light on the synthetic biology strategies that have applied the light reactions of photosynthesis for not only increasing biomass and crop yield, but also for coupling evolutionarily unconnected pathways to produce high-value compounds in vivo and for the generation of hydrogen or electricity in vitro. The evolutionarily conserved photosynthetic modules in plants, algae, and cyanobacteria have encouraged many groups to study the effect of swapping homologous proteins between species. However, these simple genetic engineering feats have resulted in reduced photosynthetic efficiency or a total loss of photoautotrophy, highlighting the incompatibility of such approaches, despite the high similarity between core subunit proteins of photosystems. As discussed in Leister (2018), this inability to exchange conserved modules is a prime example of how evolution has optimized individual components of multicomponent systems to adapt to their intrinsic environments for optimal interactions and thus optimum function. As a result, exchanging single proteins disturbs the evolved optimal state and causes suboptimal photoautotrophy, a phenomenon that was initially coined as the “frozen metabolic accident” (Shi et al., 2005) and then generalized to the “frozen metabolic state” (Gimpel et al., 2016). To prevent disturbing established networks, it is proposed that future engineering of highly integrated systems, like photosynthesis, should consider exchanging the entire complex together with its coevolved networks. In fact, this was demonstrated with the successful assembly of Arabidopsis Rubisco in Escherichia coli when chaperones were coexpressed, highlighting that the associated network and embedded interactions are crucial for complex integrity (Aigner et al., 2017). The significance of auxiliary factors in transferring synthetic photosynthetic modules was evidenced in the study of Aigner et al., because of the distant evolutionary nature of E. coli. However, this revelation adds another facet to the concept of the “frozen metabolic state” and encourages consideration of cofactors, chaperones, and assembly factors required for the assembly of multiprotein complexes in future engineering efforts. It is also interesting to note that the reintroduction of photosynthetic proteins lost in the evolutionary transition from cyanobacteria to chloroplasts in algae and plants, has demonstrated enhanced stress tolerance and an advantage to growth and photosynthesis in the transgenic plant, which contrasts with the unsuccessful expression of plant proteins in cyanobacteria. Leister (2018) also extensively discusses the various synthetic biology applications of exploiting the photosynthetic machinery for driving unrelated biological and nonbiological processes. The light reactions have an inherent capacity to generate reducing power (NADPH), which can be coupled to reactions that have a high demand for electrons. In plants, the cytochrome P450 family of enzymes are well known for their subcellular localization to the endoplasmic reticulum, involvement in biosynthetic pathways of high-value compounds and their demand for NADPH as a cofactor for catalyzing various reactions. Several studies have relocated cytochrome P450s to the chloroplasts to harness the reducing power of the light reactions, which was successful in both in vivo and in vitro reactions. On the other hand, hybrids of PSI and cytochrome P450 or hydrogenase enzymes have been generated and demonstrated to be functional in vitro. The PSI-hydrogenase hybrids were engineered for the generation of H2, which is a sustainable and clean source of energy. However, their extensive use is inhibited by the O2 sensitivity of the hydrogenase enzymes, and extensive efforts are directed toward markedly enhancing the lifetime and efficiency of these hybrids for H2 production. An alternative approach is to use hybrids of PSI with an abiotic catalyst, like platinum or earth-abundant molecular catalysts, which unfortunately are less efficient compared to the PSI-hydrogenase hybrids. Although in its infancy, PSI-based photocurrent-generating artificial systems are also being developed to generate electricity, and the progress on these have been elaborated in Leister (2018). The light reactions of photosynthesis provide ample targets of opportunity for synthetic biology applications. Leister (2018) sheds light on the power of sunlight and photosynthesis in addressing food and energy sustainability challenges of the future. LITERATURE CITED Aigner H , Wilson RH, Bracher A, Calisse L, Bhat JY, Hartl FU, Hayer-Hartl M ( 2017 ) Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2 . Science 358 : 1272 – 1278 Google Scholar Crossref Search ADS PubMed WorldCat Blankenship RE ( 2010 ) Early evolution of photosynthesis . Futur Perspect Plant Biol 154 : 434 – 438 Google Scholar OpenURL Placeholder Text WorldCat Caffarri S , Tibiletti T, Jennings RC, Santabarbara S ( 2014 ) A comparison between plant photosystem I and photosystem II architecture and functioning . Curr Protein Pept Sci 15 : 296 – 331 Google Scholar Crossref Search ADS PubMed WorldCat Ding F , Wang M, Zhang S, Ai X ( 2016 ) Changes in SBPase activity influence photosynthetic capacity, growth, and tolerance to chilling stress in transgenic tomato plants . Sci Rep 6 : 32741 Google Scholar Crossref Search ADS PubMed WorldCat Driever SM , Simkin AJ, Alotaibi S, Fisk SJ, Madgwick PJ, Sparks CA, Jones HD, Lawson T, Parry MAJ, Raines CA ( 2017 ) Increased SBPase activity improves photosynthesis and grain yield in wheat grown in greenhouse conditions . Philos Trans R Soc Lond B Biol Sci 372 : 20160384 Google Scholar Crossref Search ADS PubMed WorldCat Feng L , Han Y, Liu G, An B, Yang J, Yang G, Li Y, Zhu Y ( 2007 a ) Overexpression of sedoheptulose-1,7-bisphosphatase enhances photosynthesis and growth under salt stress in transgenic rice plants . Funct Plant Biol 34 : 822 – 834 Google Scholar Crossref Search ADS PubMed WorldCat Feng L , Wang K, Li Y, Tan Y, Kong J, Li H, Li Y, Zhu Y ( 2007 b ) Overexpression of SBPase enhances photosynthesis against high temperature stress in transgenic rice plants . Plant Cell Rep 26 : 1635 – 1646 Google Scholar Crossref Search ADS PubMed WorldCat Foyer CH , Ruban AV, Nixon PJ ( 2017 ) Photosynthesis solutions to enhance productivity . Philos Trans R Soc Lond B Biol Sci 372 : 20160374 Google Scholar Crossref Search ADS PubMed WorldCat Gimpel JA , Nour-Eldin HH, Scranton MA, Li D, Mayfield SP ( 2016 ) Refactoring the six-gene photosystem II core in the chloroplast of the green algae Chlamydomonas reinhardtii . ACS Synth Biol 5 : 589 – 596 Google Scholar Crossref Search ADS PubMed WorldCat Gul M , Kotak Y, Muneer T ( 2016 ) Review on recent trend of solar photovoltaic technology . Energy Explor Exploit 34 : 485 – 526 Google Scholar Crossref Search ADS WorldCat Lefebvre S , Lawson T, Zakhleniuk OV, Lloyd JC, Raines CA, Fryer M ( 2005 ) Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development . Plant Physiol 138 : 451 – 460 Google Scholar Crossref Search ADS PubMed WorldCat Leister D ( 2018 ) Genetic engineering, synthetic biology and the light reactions of photosynthesis . Plant Physiol 179 : 778–793 Google Scholar OpenURL Placeholder Text WorldCat Rosenthal DM , Locke AM, Khozaei M, Raines CA, Long SP, Ort DR ( 2011 ) Over-expressing the C(3) photosynthesis cycle enzyme Sedoheptulose-1-7 Bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO(2) fumigation (FACE) . BMC Plant Biol 11 : 123 Google Scholar Crossref Search ADS PubMed WorldCat Shi T , Bibby TS, Jiang L, Irwin AJ, Falkowski PG ( 2005 ) Protein interactions limit the rate of evolution of photosynthetic genes in cyanobacteria . Mol Biol Evol 22 : 2179 – 2189 Google Scholar Crossref Search ADS PubMed WorldCat South PF , Cavanagh AP, Liu HW, Ort DR ( 2019 ) Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field . Science 363 : eaat9077 Google Scholar Crossref Search ADS PubMed WorldCat Zhu XG , Long SP, Ort DR ( 2008 ) What is the maximum efficiency with which photosynthesis can convert solar energy into biomass? Curr Opin Biotechnol 19 : 153 – 159 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 Author for contact: [email protected]. 2 Senior author. www.plantphysiol.org/cgi/doi/10.1104/pp.19.00045 © 2019 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Genetic Engineering, Synthetic Biology and the Light Reactions of PhotosynthesisLeister, Dario
doi: 10.1104/pp.18.00360pmid: 29991483
Oxygenic photosynthesis is imperfect, and the evolutionarily conditioned patchwork nature of the light reactions in plants provides ample scope for their improvement (Leister, 2012; Blankenship and Chen, 2013). In fact, only around 40% of the incident solar energy is used for photosynthesis. Two obvious ways of reducing energy loss are to expand the spectral band used for photosynthesis and to shift saturation of the process to higher light intensities. Indeed, even minor enhancements to the efficiency or stress resistance of the light reactions of photosynthesis should have a positive impact on biomass production and yield (Leister, 2012; Blankenship and Chen, 2013; Long et al., 2015). However, modifications to the essential structure of the light reactions of plant photosynthesis are currently limited by two main factors. One is the high degree of conservation of their structural components, which limits the efficiency gains attainable by conventional breeding approaches (Dann and Leister, 2017). The second is that the organization of these structural components into multiprotein complexes requires the simultaneous tailoring of several proteins, some of them encoded by different genetic systems in different subcellular compartments (nucleus and chloroplasts) in land plants. Therefore, the successful modification of the light reactions of plant photosynthesis has been limited to a few cases. Similarly, modification of the activity of auxiliary proteins involved in the regulation of the light reactions to enhance plant growth and yield only recently resulted in successful outcomes and is limited to a small subset of regulatory proteins (Pribil et al., 2010; Kromdijk et al., 2016; Głowacka et al., 2018). In addition to enhancing the light reactions for improved biomass production and yield, concepts have been developed for the direct coupling of photosynthesis to other important pathways that are connected indirectly to photosynthesis in natural systems, for instance, because they reside in different subcellular compartments (Lassen et al., 2014b). Direct coupling would be expected to boost the production of rare compounds in cells and contribute to the biotechnological production of high-value compounds in vivo. In vitro, it is possible to functionally link components of photosynthesis with entirely unrelated biotic or abiotic catalysts or with abiotic electrode materials. In fact, photosystem I (PSI) is naturally adapted for highly efficient light harvesting and charge separation (Kargul et al., 2012; Nguyen and Bruce, 2014; Martin and Frymier, 2017) and has been described as the most efficient natural nano-photochemical machine (Nelson, 2009). Upon light excitation, PSI produces the most powerful naturally occurring reducing agent, P700*, which, together with an exceptionally long-lived charge-separated state, provides sufficient driving force to reduce protons to H2 at neutral pH. PSI operates with a quantum yield close to 1, and currently, no synthetic system has approached its remarkable efficiency. Moreover, PSI preparations generally are robust, especially those obtained from extremophilic microalgae (Kubota et al., 2010; Haniewicz et al., 2018). The superior qualities of PSI have stimulated strategies designed to generate in vitro hybrids of PSI and various types of redox-active catalysts or other materials. In sum, the light reactions of photosynthesis are a prime target for genetic engineering and synthetic biology approaches for three major reasons. (1) Enhancement of the process in vivo to increase the efficiency of light use promises to increase biomass and crop yields. (2) Coupling of the light reactions of photosynthesis to previously unconnected pathways will enable us to utilize the reducing power of the light reactions directly to produce large amounts of high-value compounds in vivo. (3) The high efficiency and robustness of PSI should allow it to be used in hybrids with biotic or abiotic components to generate hydrogen, simple carbon-based solar fuels, or electricity in vitro. In this review, the background to and recent developments in these three strategies are discussed. Open in new tabDownload slide Open in new tabDownload slide NATURAL BUILDING BLOCKS OF PHOTOSYNTHESIS During photosynthesis, carbon dioxide (CO2) is converted into organic compounds, principally sugars, using sunlight as energy. In plants, algae, and cyanobacteria, photosynthesis uses water as the electron donor for the chemical reduction of CO2 and releases oxygen. Algae and plants derive from a lineage that arose from an endosymbiotic relationship between a protist and a cyanobacterium. Chloroplasts, the photosynthetic organelles in modern plants, are in fact the descendants of this ancient symbiotic cyanobacterium and possess an internal membrane system that resembles the thylakoid membranes of modern-day cyanobacteria. Indeed, during the evolutionary transition from cyanobacteria to chloroplasts, the overall organization and mode of action of the photosynthetic machinery was retained (Box 1). However, significant changes have occurred in the subunit composition of photosystems (Fig. 1), their posttranslational modification (Pesaresi et al., 2011), the harvesting of light energy, pigment composition, and the regulation of photosynthesis (Box 2; Holt et al., 2004; Mullineaux and Emlyn-Jones, 2005; Rochaix, 2007; de Bianchi et al., 2010). Open in new tabDownload slide Open in new tabDownload slide Figure 1. Open in new tabDownload slide Subunit composition of cyanobacterial (Synechocystis) and plant (Arabidopsis) thylakoid multiprotein complexes. Subunits specific to either the cyanobacterium Synechocystis or the flowering plant Arabidopsis are indicated by black shading; subunits with domains specific to one group of organisms are shown in dark gray, and conserved subunits are shown in light gray. c6, Cytochrome c 6; Cyt b6f, cytochrome b 6 f complex; Fd, ferredoxin; FLV, flavodiiron protein; FNR, ferredoxin-NADP reductase; Fv, flavodoxin; LHCI (II), light-harvesting complex I (II). For reasons of clarity, the ATP synthase and NAD(P)H dehydrogenase complex are not shown. Figure 1. Open in new tabDownload slide Subunit composition of cyanobacterial (Synechocystis) and plant (Arabidopsis) thylakoid multiprotein complexes. Subunits specific to either the cyanobacterium Synechocystis or the flowering plant Arabidopsis are indicated by black shading; subunits with domains specific to one group of organisms are shown in dark gray, and conserved subunits are shown in light gray. c6, Cytochrome c 6; Cyt b6f, cytochrome b 6 f complex; Fd, ferredoxin; FLV, flavodiiron protein; FNR, ferredoxin-NADP reductase; Fv, flavodoxin; LHCI (II), light-harvesting complex I (II). For reasons of clarity, the ATP synthase and NAD(P)H dehydrogenase complex are not shown. Open in new tabDownload slide Open in new tabDownload slide Its evolutionary history makes photosynthesis well suited for synthetic biology strategies. The evolutionary diversification of the photosynthetic machinery has provided a set of building blocks, ranging from single proteins such as the soluble electron transporters (plastocyanin, cytochrome c 6, flavodoxin, and ferredoxin) to multiprotein complexes like photosystems and antenna complexes (phycobiliosomes and light-harvesting complexes [LHCs]). In principle, the building blocks should be interchangeable between cyanobacteria, algae, and plants. Instances of the swapping of homologous photosynthetic proteins between species by means of genetic engineering are discussed in the next section to highlight the complications associated with apparently straightforward approaches. The focus then shifts to genuinely synthetic approaches, in which specific photosynthetic building blocks have been introduced into photosynthetic species that lack them. In the subsequent two sections, the combination of nonphotosynthetic (bio-bio hybrids, using photosynthesis as an electron source for unrelated biological processes) and nonbiological (bio-nano hybrids, using photosynthesis as an electron source for nonbiological processes) building blocks with components of the photosynthetic machinery is described. An overview of these approaches is provided in Figure 2. Figure 2. Open in new tabDownload slide Overview of genetic engineering and synthetic biology approaches related to the light reactions of photosynthesis. Different shading is used to indicate cyanobacterial (blue) and plant (green) proteins (complexes) and proteins (complexes) that are typically not associated directly with photosynthesis (red). Yellow shading indicates nonbiological materials. For designations of proteins, see Figure Box 1. Figure 2. Open in new tabDownload slide Overview of genetic engineering and synthetic biology approaches related to the light reactions of photosynthesis. Different shading is used to indicate cyanobacterial (blue) and plant (green) proteins (complexes) and proteins (complexes) that are typically not associated directly with photosynthesis (red). Yellow shading indicates nonbiological materials. For designations of proteins, see Figure Box 1. EXCHANGE OF CONSERVED PHOTOSYNTHETIC MODULES Theoretically, it should be relatively easy to exchange individual conserved photosynthetic proteins between different species, even in such distantly related organisms as cyanobacteria and plants. However, in practice, these simple genetic engineering exercises can be problematic (Table 1). Several individual proteins from cyanobacterial PSII (D1, CP43, CP47, and PsbH) and PSI (PsaA) have indeed been genetically replaced by their plant counterparts with varying success. Synechocystis PCC6803 (Synechocystis) strains expressing the D1 protein from Poa annua or PsbH from maize (Zea mays) could still perform photosynthesis, albeit with less efficiency than the wild-type strain (Nixon et al., 1991; Chiaramonte et al., 1999), but the replacement of Synechocystis CP43 or CP47 by their homologs from spinach (Spinacia oleracea) was incompatible with photoautotrophy (Carpenter et al., 1993; Vermaas et al., 1996). Similarly, in Synechocystis strains equipped with Arabidopsis (Arabidopsis thaliana) PsaA, PSI function was severely disrupted (Viola et al., 2014). Replacement of conserved individual or multiple photosynthetic proteins Table 1. Replacement of conserved individual or multiple photosynthetic proteins Gene/Protein . Description . Protein Identity . Reference . psbA/D1 Synechocystis D1 was replaced by its homolog from Poa annua, which resulted in slower growth. This is likely to be due to increased charge recombination between the donor and acceptor sides of the reaction center. 86% Nixon et al. (1991) psbB/CP47 Synechocystis CP47 was replaced by its homolog from spinach and photoautotrophy was lost. 80% Vermaas et al. (1996) psbC/CP43 Synechocystis CP43 was replaced by its homolog from spinach and photoautotrophy was lost. 85% Carpenter et al. (1993) psbH/PsbH Synechocystis PsbH was replaced by its counterpart from maize. The resulting strain displayed increased light sensitivity and lower chlorophyll content. 78% Chiaramonte et al. (1999) psaA/PsaA Synechocystis PsaA was replaced by its equivalent from Arabidopsis. This resulted in reduced photoautotrophic growth and a drastically reduced chlorophyll-phycocyanin ratio. 80% Viola et al. (2014) psbA, -B, -C, -D, -E, -F/D1, CP47, CP43, D2, cytochrome b559-α, cytochrome b559-β All six core subunits of C. reinhardtii were replaced by their counterparts from two different green algae (Volvox carteri and Scenedesmus obliquus). The resulting strains were photoautotrophic but showed reduced photosynthetic efficiency, and the heterologous proteins reached only between 10% and 20% of the levels of those they replaced. 82%–99% Gimpel et al. (2016) Gene/Protein . Description . Protein Identity . Reference . psbA/D1 Synechocystis D1 was replaced by its homolog from Poa annua, which resulted in slower growth. This is likely to be due to increased charge recombination between the donor and acceptor sides of the reaction center. 86% Nixon et al. (1991) psbB/CP47 Synechocystis CP47 was replaced by its homolog from spinach and photoautotrophy was lost. 80% Vermaas et al. (1996) psbC/CP43 Synechocystis CP43 was replaced by its homolog from spinach and photoautotrophy was lost. 85% Carpenter et al. (1993) psbH/PsbH Synechocystis PsbH was replaced by its counterpart from maize. The resulting strain displayed increased light sensitivity and lower chlorophyll content. 78% Chiaramonte et al. (1999) psaA/PsaA Synechocystis PsaA was replaced by its equivalent from Arabidopsis. This resulted in reduced photoautotrophic growth and a drastically reduced chlorophyll-phycocyanin ratio. 80% Viola et al. (2014) psbA, -B, -C, -D, -E, -F/D1, CP47, CP43, D2, cytochrome b559-α, cytochrome b559-β All six core subunits of C. reinhardtii were replaced by their counterparts from two different green algae (Volvox carteri and Scenedesmus obliquus). The resulting strains were photoautotrophic but showed reduced photosynthetic efficiency, and the heterologous proteins reached only between 10% and 20% of the levels of those they replaced. 82%–99% Gimpel et al. (2016) Open in new tab Table 1. Replacement of conserved individual or multiple photosynthetic proteins Gene/Protein . Description . Protein Identity . Reference . psbA/D1 Synechocystis D1 was replaced by its homolog from Poa annua, which resulted in slower growth. This is likely to be due to increased charge recombination between the donor and acceptor sides of the reaction center. 86% Nixon et al. (1991) psbB/CP47 Synechocystis CP47 was replaced by its homolog from spinach and photoautotrophy was lost. 80% Vermaas et al. (1996) psbC/CP43 Synechocystis CP43 was replaced by its homolog from spinach and photoautotrophy was lost. 85% Carpenter et al. (1993) psbH/PsbH Synechocystis PsbH was replaced by its counterpart from maize. The resulting strain displayed increased light sensitivity and lower chlorophyll content. 78% Chiaramonte et al. (1999) psaA/PsaA Synechocystis PsaA was replaced by its equivalent from Arabidopsis. This resulted in reduced photoautotrophic growth and a drastically reduced chlorophyll-phycocyanin ratio. 80% Viola et al. (2014) psbA, -B, -C, -D, -E, -F/D1, CP47, CP43, D2, cytochrome b559-α, cytochrome b559-β All six core subunits of C. reinhardtii were replaced by their counterparts from two different green algae (Volvox carteri and Scenedesmus obliquus). The resulting strains were photoautotrophic but showed reduced photosynthetic efficiency, and the heterologous proteins reached only between 10% and 20% of the levels of those they replaced. 82%–99% Gimpel et al. (2016) Gene/Protein . Description . Protein Identity . Reference . psbA/D1 Synechocystis D1 was replaced by its homolog from Poa annua, which resulted in slower growth. This is likely to be due to increased charge recombination between the donor and acceptor sides of the reaction center. 86% Nixon et al. (1991) psbB/CP47 Synechocystis CP47 was replaced by its homolog from spinach and photoautotrophy was lost. 80% Vermaas et al. (1996) psbC/CP43 Synechocystis CP43 was replaced by its homolog from spinach and photoautotrophy was lost. 85% Carpenter et al. (1993) psbH/PsbH Synechocystis PsbH was replaced by its counterpart from maize. The resulting strain displayed increased light sensitivity and lower chlorophyll content. 78% Chiaramonte et al. (1999) psaA/PsaA Synechocystis PsaA was replaced by its equivalent from Arabidopsis. This resulted in reduced photoautotrophic growth and a drastically reduced chlorophyll-phycocyanin ratio. 80% Viola et al. (2014) psbA, -B, -C, -D, -E, -F/D1, CP47, CP43, D2, cytochrome b559-α, cytochrome b559-β All six core subunits of C. reinhardtii were replaced by their counterparts from two different green algae (Volvox carteri and Scenedesmus obliquus). The resulting strains were photoautotrophic but showed reduced photosynthetic efficiency, and the heterologous proteins reached only between 10% and 20% of the levels of those they replaced. 82%–99% Gimpel et al. (2016) Open in new tab Such complications resulting from the replacement of core subunits of photosystems, despite their high similarity (78%–86% identity between the cyanobacterial and plant proteins; Table 1), reflect the so-called "frozen metabolic state" of the photosynthetic multiprotein complexes. This term was coined by Gimpel et al. (2016) but was introduced originally as "frozen metabolic accident" by Shi et al. (2005). The frozen metabolic accident concept refers to the observation that selection has not significantly altered biophysically and physiologically inefficient photosynthetic proteins (including the D1 protein of PSII or Rubisco of the Calvin-Benson cycle) over billions of years of evolution. In fact, bioinformatic analysis of photosynthetic cyanobacterial genes suggests that the evolution rate of proteins at the core of the photosynthetic apparatus is highly constrained by protein-protein, protein-lipid, and protein-cofactor interactions (Shi et al., 2005). This provides an internal selection pressure, conserving the sequence of proteins in photosynthetic multiprotein complexes and, in the case of prokaryotes, also the genomic organization of their genes (Shi et al., 2005). The term frozen metabolic accident was generalized to frozen metabolic state by Gimpel et al. (2016) and implies that, in each photosynthetic species, slightly distinct modules of the cores of photosynthetic multiprotein complexes have evolved that are optimized with respect to their intrinsic interactions and that rarely tolerate the alteration of single proteins by exchange or mutation. In consequence, the simultaneous exchange of entire sets of core proteins (or modules) with all their intrinsic interactions should be more feasible than substituting individual proteins that may disrupt the frozen metabolic state. Such an experiment has been performed in the green alga Chlamydomonas reinhardtii, where the six PSII core proteins D1, CP47, CP43, D2, cytochrome b 559-α, and cytochrome b 559-β were swapped for their homologs from two other green algal species (Gimpel et al., 2016). Photoautotrophy was not affected by the exchange of this synthetic biology module, although the fully altered strains performed suboptimally compared with strains in which only one to five genes were exchanged. However, in control experiments where the synthetic C. reinhardtii six-gene module was reintroduced to the C. reinhardtii deletion strain that lacked all six genes, only about 86% of wild-type PSII functionality was rescued. Tentative explanations for this decreased efficiency include the following: (1) off-target effects of the PSII gene deletions on the operon components and tRNA genes associated with these PSII loci; (2) misregulation of the transferred PSII genes because their expression cassettes lacked unidentified cis-acting DNA elements; and (3) perturbation of polycistron-dependent posttranscriptional regulation of the transformed genes, since they were no longer part of an operon (Gimpel et al., 2016). Taken together, the outcomes of these replacement experiments indicate that exchanging conserved photosynthetic proteins from multiprotein complexes can be problematic, given the highly integrated nature of the photosynthetic machinery. Therefore, approaches designed to enhance photosynthesis, such as introducing the high light-resistant D1 protein from a green alga that lives under extreme conditions (Treves et al., 2016) into crop plants, do not appear promising. Instead, entire (sub)complexes with their internal network of evolutionarily optimized interactions should be transferable between species. EXCHANGE OF NONCONSERVED PHOTOSYNTHETIC MODULES Inspection of the repertoire of subunits of the photosynthetic machinery in the three model species Synechocystis PCC6803, C. reinhardtii, and Arabidopsis, which represent different stages in the evolution of oxygen-generating photosynthesis, shows that the most dramatic changes in the photosynthetic proteome occurred during the transition from the cyanobacterial endosymbiont (for which Synechocystis serves as proxy) to the chloroplast of unicellular algae (with C. reinhardtii as proxy; Fig. 3; Supplemental Table S1). In particular, phycobilisomes, flavodoxin, and several photosystem subunits were lost, while LHCs, some novel photosystem subunits, and several proteins involved in alternative electron pathways or photoprotection evolved. During the transition from algal chloroplasts to those of flowering plants, relatively few proteins were lost (e.g. flavodiiron proteins and the canonical cytochrome c 6) or acquired (Lhcb6/CP24; Fig. 3; Supplemental Table S1). The NAD(P)H dehydrogenase (NDH) complex involved in antimycin A-insensitive cyclic electron flow (Box 1) is a special case, since the chloroplast NDH from flowering plants traces back to the cyanobacterial complex, but the complex was lost during evolution in C. reinhardtii (Fig. 3; Supplemental Table S1). Figure 3. Open in new tabDownload slide Evolutionary plasticity of the photosynthetic proteome. The changes in the composition of the inventory of photosynthetic proteins (top) during evolution from the cyanobacterial endosymbiont to the chloroplast in flowering plants (bottom) are shown. As proxies for the original endosymbiont and the unicellular chloroplast-containing protist that gave rise to flowering plants, the model cyanobacterium Synechocystis PCC6803 (left), the model green alga C. reinhardtii (middle), and the model flowering plant Arabidopsis (right) are used. At top left, the entire inventory of photosynthetic proteins in Synechocystis PCC6803 is listed and assigned to the five different classes PSII, PSI, other electron transport components (other ET), antenna, and photoprotection, whereby the transition from other ET to photoprotection is fluid. The proteins that have been acquired or lost during evolution in C. reinhardtii and Arabidopsis are listed in the middle and right sections, respectively, of the top part. Note that, for reasons of simplicity, we have not considered the ATP synthase complex here. The NDH complex (or Nda2 in the case of C. reinhardtii) appears only as a whole (without its individual subunits) here. NDH listed in parentheses indicates that the NDH complex is specifically lost only in C. reinhardtii and, therefore, that the plant NDH complex is not a reacquisition. Accordingly, Nda2 replaces the NDH complex in C. reinhardtii. A detailed catalog of the indicated proteins with their full names, functions, and further literature links is available in Supplemental Table S1. The bottom part provides a sketch of the evolution of flowering plants that traces back to the endosymbiosis between a unicellular eukaryote and a cyanobacterium, resulting (besides the red algal and glaucophyte lineages that are not shown) in chloroplast-containing protists that evolved further to plants. Figure 3. Open in new tabDownload slide Evolutionary plasticity of the photosynthetic proteome. The changes in the composition of the inventory of photosynthetic proteins (top) during evolution from the cyanobacterial endosymbiont to the chloroplast in flowering plants (bottom) are shown. As proxies for the original endosymbiont and the unicellular chloroplast-containing protist that gave rise to flowering plants, the model cyanobacterium Synechocystis PCC6803 (left), the model green alga C. reinhardtii (middle), and the model flowering plant Arabidopsis (right) are used. At top left, the entire inventory of photosynthetic proteins in Synechocystis PCC6803 is listed and assigned to the five different classes PSII, PSI, other electron transport components (other ET), antenna, and photoprotection, whereby the transition from other ET to photoprotection is fluid. The proteins that have been acquired or lost during evolution in C. reinhardtii and Arabidopsis are listed in the middle and right sections, respectively, of the top part. Note that, for reasons of simplicity, we have not considered the ATP synthase complex here. The NDH complex (or Nda2 in the case of C. reinhardtii) appears only as a whole (without its individual subunits) here. NDH listed in parentheses indicates that the NDH complex is specifically lost only in C. reinhardtii and, therefore, that the plant NDH complex is not a reacquisition. Accordingly, Nda2 replaces the NDH complex in C. reinhardtii. A detailed catalog of the indicated proteins with their full names, functions, and further literature links is available in Supplemental Table S1. The bottom part provides a sketch of the evolution of flowering plants that traces back to the endosymbiosis between a unicellular eukaryote and a cyanobacterium, resulting (besides the red algal and glaucophyte lineages that are not shown) in chloroplast-containing protists that evolved further to plants. Several attempts have been made to introduce photosynthetic proteins into species that lack the corresponding homolog (Table 2). These heterologous expression approaches have been successful for the soluble electron transporters flavodoxin, cytochrome c 6, and flavodiiron proteins. Indeed, cyanobacterial flavodoxin can at least partially replace plant ferredoxin and confer enhanced stress tolerance when expressed in addition to ferredoxin (Tognetti et al., 2006, 2007; Blanco et al., 2011). Similarly, red algal cytochrome c 6 enhances the growth and photosynthesis of Arabidopsis plants (Chida et al., 2007). Since Arabidopsis lacks a functional cytochrome c 6 that can transfer electrons from the cytochrome b 6 f complex to PSI (Molina-Heredia et al., 2003; Weigel et al., 2003), it is plausible that the more oxidized plastoquinone pool in the transgenic plants is a direct consequence of additional PSI reduction mediated by the algal protein (Chida et al., 2007). Interestingly, it seems to make no difference if an endogenous soluble electron transport protein (Box 3) or its heterologous equivalent from a distant species is overexpressed. For instance, overexpression of the endogenous soluble proteins plastocyanin and ferredoxin also can enhance growth in plants (Pesaresi et al., 2009; Lin et al., 2013; Chang et al., 2017; Zhou et al., 2018). Interestingly and rather unexpectedly, this concept also can work for certain proteins that are part of multiprotein complexes (Simkin et al., 2017; see Box 3). This indicates that the quantity of such proteins is relevant for growth enhancement and not their evolutionary origin. Introduction of heterologous photosynthetic proteins (protein complexes) Table 2. Introduction of heterologous photosynthetic proteins (protein complexes) Gene/Protein . Description . Reference . PetJ/cytochrome c 6 Growth and photosynthesis of Arabidopsis plants was enhanced by the expression of a red algal (Porphyra yezoensis) cytochrome c 6 gene. Chida et al. (2007) Fld/flavodoxin Tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin in addition to endogenous ferredoxin display increased tolerance to environmental stress. Flavodoxin can at least partially replace ferredoxin in tobacco. Tognetti et al. (2006, 2007; Blanco et al. (2011 FlvA+B/flavodiiron protein (FLV) FlvA and FlvB from the moss Physcomitrella patens mediate pseudocyclic electron flow in Arabidopsis. Yamamoto et al. (2016) Lhcb/LHCII Pea Lhcb protein is synthesized in Synechocystis and integrated into the membrane, but it is then degraded, such that it cannot be detected by immunoblot analysis. He et al. (1999) Gene/Protein . Description . Reference . PetJ/cytochrome c 6 Growth and photosynthesis of Arabidopsis plants was enhanced by the expression of a red algal (Porphyra yezoensis) cytochrome c 6 gene. Chida et al. (2007) Fld/flavodoxin Tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin in addition to endogenous ferredoxin display increased tolerance to environmental stress. Flavodoxin can at least partially replace ferredoxin in tobacco. Tognetti et al. (2006, 2007; Blanco et al. (2011 FlvA+B/flavodiiron protein (FLV) FlvA and FlvB from the moss Physcomitrella patens mediate pseudocyclic electron flow in Arabidopsis. Yamamoto et al. (2016) Lhcb/LHCII Pea Lhcb protein is synthesized in Synechocystis and integrated into the membrane, but it is then degraded, such that it cannot be detected by immunoblot analysis. He et al. (1999) Open in new tab Table 2. Introduction of heterologous photosynthetic proteins (protein complexes) Gene/Protein . Description . Reference . PetJ/cytochrome c 6 Growth and photosynthesis of Arabidopsis plants was enhanced by the expression of a red algal (Porphyra yezoensis) cytochrome c 6 gene. Chida et al. (2007) Fld/flavodoxin Tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin in addition to endogenous ferredoxin display increased tolerance to environmental stress. Flavodoxin can at least partially replace ferredoxin in tobacco. Tognetti et al. (2006, 2007; Blanco et al. (2011 FlvA+B/flavodiiron protein (FLV) FlvA and FlvB from the moss Physcomitrella patens mediate pseudocyclic electron flow in Arabidopsis. Yamamoto et al. (2016) Lhcb/LHCII Pea Lhcb protein is synthesized in Synechocystis and integrated into the membrane, but it is then degraded, such that it cannot be detected by immunoblot analysis. He et al. (1999) Gene/Protein . Description . Reference . PetJ/cytochrome c 6 Growth and photosynthesis of Arabidopsis plants was enhanced by the expression of a red algal (Porphyra yezoensis) cytochrome c 6 gene. Chida et al. (2007) Fld/flavodoxin Tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin in addition to endogenous ferredoxin display increased tolerance to environmental stress. Flavodoxin can at least partially replace ferredoxin in tobacco. Tognetti et al. (2006, 2007; Blanco et al. (2011 FlvA+B/flavodiiron protein (FLV) FlvA and FlvB from the moss Physcomitrella patens mediate pseudocyclic electron flow in Arabidopsis. Yamamoto et al. (2016) Lhcb/LHCII Pea Lhcb protein is synthesized in Synechocystis and integrated into the membrane, but it is then degraded, such that it cannot be detected by immunoblot analysis. He et al. (1999) Open in new tab Open in new tabDownload slide Open in new tabDownload slide More recently, the two Physcomitrella patens flavodiiron protein (FLV) genes FlvA and FlvB were introduced into Arabidopsis, which, like other angiosperms, has lost FLVs during evolution (Yamamoto et al., 2016). FLVs are the main mediators of pseudocyclic electron flow in photosynthetic organisms, but heterologous expression of FLVs in Arabidopsis had no effect on steady-state photosynthesis and growth of the transgenic plants (Yamamoto et al., 2016). However, the Arabidopsis FLV lines displayed higher photosynthetic yields just after the onset of actinic light following a long dark adaptation, suggesting that the FLVs mediated a large electron sink during the induction of photosynthesis. In fluctuating light experiments, the Arabidopsis FLV lines had much less PSI acceptor side limitation, implying that the large FLV-mediated electron sink makes photosynthesis more resistant to the fluctuating light. Consequently, this protective effect of FLVs is more pronounced in Arabidopsis lines that are more sensitive to fluctuating light, like the pgr5 mutant defective in antimycin A-sensitive cyclic electron flow (Box 1; Leister and Shikanai, 2013; Yamamoto et al., 2016). Similarly, the expression of FLVs in a rice (Oryza sativa) line with markedly reduced cyclic electron flow restored CO2 assimilation and growth rate to wild-type levels (Wada et al., 2018). Like FLVs, LHcx/LHCSR proteins play a role in photoprotection in green algae and mosses, but they have been lost in angiosperms during evolution. The heterologous expression of P. patens LHCSR1 in Nicotiana benthamiana and Nicotiana tabacum yielded an active protein that has properties similar if not identical to those of moss LHCSR1 (Pinnola et al., 2015). Efforts to create LHCII complexes like those in plants by heterologously expressing the membrane-spanning light-harvesting chlorophyll a/b-binding protein Lhcb from Pisum spp. (pea) in Synechocystis were unsuccessful. Although the pea Lhcb protein was synthesized in Synechocystis and integrated into the membrane, it did not accumulate to steady-state levels detectable by immunoblot analysis (He et al., 1999). Possible explanations are that Lhcb is degraded rapidly, either because its unfamiliar structure makes it a good substrate for the cyanobacterial proteolytic system or it cannot fold/assemble properly due to the lack of plant-specific pigments or assembly factors. Interestingly, chlorophyll b production (Box 2) after the introduction of plant chlorophyll a oxygenase is boosted when Lhcb is expressed, even though LHCII does not accumulate in detectable amounts (Xu et al., 2001). Even soluble multiprotein complexes can be expressed heterologously, as has been demonstrated impressively from the carbon-fixation end of photosynthesis. For example, by coexpression of five auxiliary factors, a functional plant Rubisco complex was assembled successfully in Escherichia coli (Aigner et al., 2017). This result is in line with the frozen metabolic state concept, showing that photosynthetic proteins embedded in a network of interactions with other proteins and auxiliary factors can be introduced into distantly related species, but only with their own interaction networks. While Gimpel et al. (2016) showed that efficient gene expression needs to be accounted for when designing synthetic modules for the transfer of photosystem subunits from one species to another, Aigner et al. (2017) demonstrated that auxiliary factors required for the biogenesis of photosynthetic (sub)complexes need to be considered in such experiments, adding additional facets to the frozen metabolic state concept. Auxiliary factors required for the accumulation of photosynthetic multiprotein complexes include the following: (1) cofactors like iron-sulfur clusters and pigments (Box 2); (2) chaperones that are required for the insertion of cofactors (e.g. pigments into LHCs; Schmid, 2008); and (3) assembly factors. Assembly factors are required to support the stepwise assembly and functionality of the multiprotein/pigment complexes, and many of these are conserved between photosynthetic species (Nixon et al., 2010; Nickelsen and Rengstl, 2013; Jensen and Leister, 2014). Therefore, the exchange of entire multiprotein complexes between distantly related species will not be simple due to the repertoire of auxiliary factors that has changed markedly during evolution. Since the species that Gimpel et al. (2016) studied were closely related, this aspect could be neglected. In consequence, the impact of these auxiliary factors on the formation of multiprotein/pigment complexes will have to be fully elucidated to understand which factors are sufficient to assemble a photosynthetic multiprotein complex; previous (genetic) approaches only identified factors required for this process. Hence, the transfer of entire photosynthetic (sub)complexes between distantly related species by a synthetic photosynthetic module must include entire sets of strongly interacting proteins (to address the frozen metabolic state), as well as all genetic elements and auxiliary factors sufficient for the efficient expression, biogenesis, and function of the proteins in the module. BIO-BIO HYBRIDS: USING PHOTOSYNTHESIS AS AN ELECTRON SOURCE FOR UNRELATED BIOLOGICAL PROCESSES The idea of using the reducing power of photosynthesis to drive unrelated metabolic reactions has inspired several biotechnological concepts. The photosynthesis-driven formation of secondary metabolites has been demonstrated in vivo, whereas the light-driven generation of H2 by bio-bio hybrids so far works only in vitro and is closely related to bio-nano approaches. Therefore, the coupling of photosynthesis to previously unrelated pathways is discussed here first, followed by bio-bio systems for H2 generation. The redirection of PSI-reducing equivalents to drive reactions catalyzed by cytochrome P450 enzymes has been achieved in vivo by genetic modifications of plants and cyanobacteria (Lassen et al., 2014b; Nielsen et al., 2016; Mellor et al., 2017). Cytochrome P450s constitute the largest family of plant enzymes that act on various endogenous and xenobiotic molecules (Rasool and Mohamed, 2016). Their extreme versatility and irreversibility of catalyzed reactions make these enzymes very attractive for use in biotechnology, medicine, and phytoremediation. P450s are monooxygenases that insert an oxygen atom into hydrophobic molecules, which enhances their reactivity and hydrophilicity. Most eukaryotic P450s require NADPH:cytochrome P450 reductase as the electron donor. The endoplasmic reticulum (ER) membrane generally is accepted to be the primary subcellular repository of eukaryotic P450s and their NADPH:cytochrome P450 reductase. By relocating cytochrome P450s to the chloroplasts, the reducing power of photosynthesis can be targeted directly to the reactions catalyzed by these enzymes, thus providing the basis for the large-scale production of valuable products. Initial attempts to couple P450s with photosynthesis were conducted in vitro (Table 3; Fig. 4A). Spinach chloroplasts were combined with microsomes from yeast cells that had been stably transformed with a fusion gene expressing the rat CYP1A1-NADPH:cytochrome P450 reductase fusion enzyme (Kim et al., 1996). These experiments confirmed that it is feasible to drive P450-mediated reactions using electrons derived from photosynthetically generated NADPH. Intriguingly, the NADPH:cytochrome P450 reductase is not always essential, as demonstrated by coincubating CYP79A1 from sorghum (Sorghum bicolor) with barley (Hordeum vulgare) PSI and employing ferredoxin to deliver electrons directly from PSI to the P450 (Jensen et al., 2011). This approach also was shown to be practicable in vivo. CYP79A1, either alone or in combination with CYP71E1 and the UDP-glucosyltransferase UGT85B1, was first targeted in vivo to cyanobacterial or plant thylakoid membranes, where they catalyzed the same reactions as in their original cellular compartment (the ER), utilizing photosynthetically reduced ferredoxin as the electron donor (Nielsen et al., 2013; Lassen et al., 2014a; Gnanasekaran et al., 2016; Wlodarczyk et al., 2016). Genetic fusions also have been employed for the coupling of P450s to PSI. CYP79A1 has been fused to either cyanobacterial PsaM or Arabidopsis ferredoxin, and the engineered enzyme showed light-driven activity both in vivo and in vitro (Lassen et al., 2014a; Mellor et al., 2016). In the latter case, the efficiency of the system was enhanced because the fusion could compete better with endogenous electron sinks coupled to metabolic pathways (Mellor et al., 2016). Combination of PSI with nonphotosynthetic proteins or complexes: bio-bio hybrids Table 3. Combination of PSI with nonphotosynthetic proteins or complexes: bio-bio hybrids Gene/Protein . Description . Reference . -----------------------------------------------------------------------------PSI-P450 hybrids---------------------------------------------------------------------- Fusion enzyme of rat CYP1A1 and yeast NADPH-P450 reductase (in vitro) Spinach chloroplasts were combined with microsomes from yeast expressing the rat CYP1A1/CPR fusion enzyme. In this system, NADP+ was photosynthetically reduced to NADPH to supply the electrons for P450, thus enabling the light-driven conversion of 7-ethoxycoumarin to 7-hydroxycoumarin. Kim et al. (1996) CP79A1 from sorghum (in vitro and in vivo) The ER-derived P450 CYP79A1 was capable of converting Tyr to hydroxyphenyl-acetaldoxime, employing ferredoxin reduced by barley PSI (without the need for NADPH and CPR). In the next step, CYP79A1 was fused to cyanobacterial PsaM or Arabidopsis ferredoxin. The engineered fusions exhibited light-driven activity both in vivo and in vitro. Jensen et al. (2011); Lassen et al. (2014a); Mellor et al. (2016) CYP79A1, CYP71E1, and UGT85B1 from sorghum (in vivo) The two P450s CYP79A1 and CYP71E1 and the UDP-glucosyltransferase UGT85B1 were targeted to N. benthamiana or Synechocystis thylakoid membranes, where they converted Tyr to dhurrin employing photosynthetically reduced ferredoxin. Nielsen et al. (2013); Wlodarczyk et al. (2016) ---------------------------------------------------------------------PSI-hydrogenase hybrids--------------------------------------------------------------------- Proteobacterial [NiFe] hydrogenase genetically fused to cyanobacterial PSI (in vitro) A fusion of cyanobacterial PsaE to an [NiFe] hydrogenase from the β-proteobacterium Ralstonia eutropha was reconstituted into a PsaE-deficient PSI from Synechocystis by self-assembly. In a modified version of this system, cytochrome c 3 from Desulfovibrio vulgaris was cross-linked to the docking site of ferredoxin in PsaE, targeting electrons directly from PSI via cytochrome c 3 to the hydrogenase. This gave the hydrogenase a competitive advantage over the natural acceptors of electrons from PSI. In a third variant of this system, the R. eutropha hydrogenase was genetically fused to Synechocystis PsaE and reconstituted into a PsaE-deficient PSI from Synechocystis. His tagging of PsaF enabled the assembly of the PSI-hydrogenase hybrid onto a gold electrode. Ihara et al. (2006a, 2006b; Krassen et al. (2009 Green algal [FeFe] hydrogenase genetically fused to ferredoxin (in vitro) The C. reinhardtii hydrogenase was fused to ferredoxin. This switched the bias of electron transfer from FNR to hydrogenase and resulted in an increased rate of hydrogen photoproduction in vitro. Yacoby et al. (2011) Bacterial [FeFe] hydrogenase wired to cyanobacterial PSI (in vitro) To enable the transfer of electrons from the terminal Fe4S4 cluster in PSI of Synechococcus sp. PCC 7002 to the distal Fe4S4 cluster in the hydrogenase from Clostridium acetobutylicum, the two components were covalently coupled to each other via a molecular wire, the thiolated organic molecule octanedithiol. This allowed electrons to tunnel through the wire from PSI to the hydrogenase. Self-assembly of the PSI-wire-hydrogenase complex was obtained in vitro. Lubner et al. (2010, 2011 Gene/Protein . Description . Reference . -----------------------------------------------------------------------------PSI-P450 hybrids---------------------------------------------------------------------- Fusion enzyme of rat CYP1A1 and yeast NADPH-P450 reductase (in vitro) Spinach chloroplasts were combined with microsomes from yeast expressing the rat CYP1A1/CPR fusion enzyme. In this system, NADP+ was photosynthetically reduced to NADPH to supply the electrons for P450, thus enabling the light-driven conversion of 7-ethoxycoumarin to 7-hydroxycoumarin. Kim et al. (1996) CP79A1 from sorghum (in vitro and in vivo) The ER-derived P450 CYP79A1 was capable of converting Tyr to hydroxyphenyl-acetaldoxime, employing ferredoxin reduced by barley PSI (without the need for NADPH and CPR). In the next step, CYP79A1 was fused to cyanobacterial PsaM or Arabidopsis ferredoxin. The engineered fusions exhibited light-driven activity both in vivo and in vitro. Jensen et al. (2011); Lassen et al. (2014a); Mellor et al. (2016) CYP79A1, CYP71E1, and UGT85B1 from sorghum (in vivo) The two P450s CYP79A1 and CYP71E1 and the UDP-glucosyltransferase UGT85B1 were targeted to N. benthamiana or Synechocystis thylakoid membranes, where they converted Tyr to dhurrin employing photosynthetically reduced ferredoxin. Nielsen et al. (2013); Wlodarczyk et al. (2016) ---------------------------------------------------------------------PSI-hydrogenase hybrids--------------------------------------------------------------------- Proteobacterial [NiFe] hydrogenase genetically fused to cyanobacterial PSI (in vitro) A fusion of cyanobacterial PsaE to an [NiFe] hydrogenase from the β-proteobacterium Ralstonia eutropha was reconstituted into a PsaE-deficient PSI from Synechocystis by self-assembly. In a modified version of this system, cytochrome c 3 from Desulfovibrio vulgaris was cross-linked to the docking site of ferredoxin in PsaE, targeting electrons directly from PSI via cytochrome c 3 to the hydrogenase. This gave the hydrogenase a competitive advantage over the natural acceptors of electrons from PSI. In a third variant of this system, the R. eutropha hydrogenase was genetically fused to Synechocystis PsaE and reconstituted into a PsaE-deficient PSI from Synechocystis. His tagging of PsaF enabled the assembly of the PSI-hydrogenase hybrid onto a gold electrode. Ihara et al. (2006a, 2006b; Krassen et al. (2009 Green algal [FeFe] hydrogenase genetically fused to ferredoxin (in vitro) The C. reinhardtii hydrogenase was fused to ferredoxin. This switched the bias of electron transfer from FNR to hydrogenase and resulted in an increased rate of hydrogen photoproduction in vitro. Yacoby et al. (2011) Bacterial [FeFe] hydrogenase wired to cyanobacterial PSI (in vitro) To enable the transfer of electrons from the terminal Fe4S4 cluster in PSI of Synechococcus sp. PCC 7002 to the distal Fe4S4 cluster in the hydrogenase from Clostridium acetobutylicum, the two components were covalently coupled to each other via a molecular wire, the thiolated organic molecule octanedithiol. This allowed electrons to tunnel through the wire from PSI to the hydrogenase. Self-assembly of the PSI-wire-hydrogenase complex was obtained in vitro. Lubner et al. (2010, 2011 Open in new tab Table 3. Combination of PSI with nonphotosynthetic proteins or complexes: bio-bio hybrids Gene/Protein . Description . Reference . -----------------------------------------------------------------------------PSI-P450 hybrids---------------------------------------------------------------------- Fusion enzyme of rat CYP1A1 and yeast NADPH-P450 reductase (in vitro) Spinach chloroplasts were combined with microsomes from yeast expressing the rat CYP1A1/CPR fusion enzyme. In this system, NADP+ was photosynthetically reduced to NADPH to supply the electrons for P450, thus enabling the light-driven conversion of 7-ethoxycoumarin to 7-hydroxycoumarin. Kim et al. (1996) CP79A1 from sorghum (in vitro and in vivo) The ER-derived P450 CYP79A1 was capable of converting Tyr to hydroxyphenyl-acetaldoxime, employing ferredoxin reduced by barley PSI (without the need for NADPH and CPR). In the next step, CYP79A1 was fused to cyanobacterial PsaM or Arabidopsis ferredoxin. The engineered fusions exhibited light-driven activity both in vivo and in vitro. Jensen et al. (2011); Lassen et al. (2014a); Mellor et al. (2016) CYP79A1, CYP71E1, and UGT85B1 from sorghum (in vivo) The two P450s CYP79A1 and CYP71E1 and the UDP-glucosyltransferase UGT85B1 were targeted to N. benthamiana or Synechocystis thylakoid membranes, where they converted Tyr to dhurrin employing photosynthetically reduced ferredoxin. Nielsen et al. (2013); Wlodarczyk et al. (2016) ---------------------------------------------------------------------PSI-hydrogenase hybrids--------------------------------------------------------------------- Proteobacterial [NiFe] hydrogenase genetically fused to cyanobacterial PSI (in vitro) A fusion of cyanobacterial PsaE to an [NiFe] hydrogenase from the β-proteobacterium Ralstonia eutropha was reconstituted into a PsaE-deficient PSI from Synechocystis by self-assembly. In a modified version of this system, cytochrome c 3 from Desulfovibrio vulgaris was cross-linked to the docking site of ferredoxin in PsaE, targeting electrons directly from PSI via cytochrome c 3 to the hydrogenase. This gave the hydrogenase a competitive advantage over the natural acceptors of electrons from PSI. In a third variant of this system, the R. eutropha hydrogenase was genetically fused to Synechocystis PsaE and reconstituted into a PsaE-deficient PSI from Synechocystis. His tagging of PsaF enabled the assembly of the PSI-hydrogenase hybrid onto a gold electrode. Ihara et al. (2006a, 2006b; Krassen et al. (2009 Green algal [FeFe] hydrogenase genetically fused to ferredoxin (in vitro) The C. reinhardtii hydrogenase was fused to ferredoxin. This switched the bias of electron transfer from FNR to hydrogenase and resulted in an increased rate of hydrogen photoproduction in vitro. Yacoby et al. (2011) Bacterial [FeFe] hydrogenase wired to cyanobacterial PSI (in vitro) To enable the transfer of electrons from the terminal Fe4S4 cluster in PSI of Synechococcus sp. PCC 7002 to the distal Fe4S4 cluster in the hydrogenase from Clostridium acetobutylicum, the two components were covalently coupled to each other via a molecular wire, the thiolated organic molecule octanedithiol. This allowed electrons to tunnel through the wire from PSI to the hydrogenase. Self-assembly of the PSI-wire-hydrogenase complex was obtained in vitro. Lubner et al. (2010, 2011 Gene/Protein . Description . Reference . -----------------------------------------------------------------------------PSI-P450 hybrids---------------------------------------------------------------------- Fusion enzyme of rat CYP1A1 and yeast NADPH-P450 reductase (in vitro) Spinach chloroplasts were combined with microsomes from yeast expressing the rat CYP1A1/CPR fusion enzyme. In this system, NADP+ was photosynthetically reduced to NADPH to supply the electrons for P450, thus enabling the light-driven conversion of 7-ethoxycoumarin to 7-hydroxycoumarin. Kim et al. (1996) CP79A1 from sorghum (in vitro and in vivo) The ER-derived P450 CYP79A1 was capable of converting Tyr to hydroxyphenyl-acetaldoxime, employing ferredoxin reduced by barley PSI (without the need for NADPH and CPR). In the next step, CYP79A1 was fused to cyanobacterial PsaM or Arabidopsis ferredoxin. The engineered fusions exhibited light-driven activity both in vivo and in vitro. Jensen et al. (2011); Lassen et al. (2014a); Mellor et al. (2016) CYP79A1, CYP71E1, and UGT85B1 from sorghum (in vivo) The two P450s CYP79A1 and CYP71E1 and the UDP-glucosyltransferase UGT85B1 were targeted to N. benthamiana or Synechocystis thylakoid membranes, where they converted Tyr to dhurrin employing photosynthetically reduced ferredoxin. Nielsen et al. (2013); Wlodarczyk et al. (2016) ---------------------------------------------------------------------PSI-hydrogenase hybrids--------------------------------------------------------------------- Proteobacterial [NiFe] hydrogenase genetically fused to cyanobacterial PSI (in vitro) A fusion of cyanobacterial PsaE to an [NiFe] hydrogenase from the β-proteobacterium Ralstonia eutropha was reconstituted into a PsaE-deficient PSI from Synechocystis by self-assembly. In a modified version of this system, cytochrome c 3 from Desulfovibrio vulgaris was cross-linked to the docking site of ferredoxin in PsaE, targeting electrons directly from PSI via cytochrome c 3 to the hydrogenase. This gave the hydrogenase a competitive advantage over the natural acceptors of electrons from PSI. In a third variant of this system, the R. eutropha hydrogenase was genetically fused to Synechocystis PsaE and reconstituted into a PsaE-deficient PSI from Synechocystis. His tagging of PsaF enabled the assembly of the PSI-hydrogenase hybrid onto a gold electrode. Ihara et al. (2006a, 2006b; Krassen et al. (2009 Green algal [FeFe] hydrogenase genetically fused to ferredoxin (in vitro) The C. reinhardtii hydrogenase was fused to ferredoxin. This switched the bias of electron transfer from FNR to hydrogenase and resulted in an increased rate of hydrogen photoproduction in vitro. Yacoby et al. (2011) Bacterial [FeFe] hydrogenase wired to cyanobacterial PSI (in vitro) To enable the transfer of electrons from the terminal Fe4S4 cluster in PSI of Synechococcus sp. PCC 7002 to the distal Fe4S4 cluster in the hydrogenase from Clostridium acetobutylicum, the two components were covalently coupled to each other via a molecular wire, the thiolated organic molecule octanedithiol. This allowed electrons to tunnel through the wire from PSI to the hydrogenase. Self-assembly of the PSI-wire-hydrogenase complex was obtained in vitro. Lubner et al. (2010, 2011 Open in new tab Figure 4. Open in new tabDownload slide Design of bio-bio hybrids. A, Harnessing the reducing power of photosynthesis for cytochrome P450 enzymes (red) has been demonstrated in vitro and in vivo. In vitro approaches were based either on a CPR-CYP1A1 fusion protein that could use NADPH produced by photosynthesis or on CYP79A1 that can utilize photoreduced ferredoxin directly. The latter approach also was realized in vivo by fusing CYP79A1 to ferredoxin (not shown) or the PSI subunit PsaM. The most elaborate approach reported utilizes three enzymes (CYP79A1, CYP71E1, and UGT85B1) to couple dhurrin synthesis with photosynthesis. B, PSI-hydrogenase complexes are based either on genetic fusions of the hydrogenase (Hyd) to PsaE or ferredoxin or on covalent linking of the hydrogenase and PSI via a molecular wire. Currently, these bio-bio hybrids function only in vitro. Figure 4. Open in new tabDownload slide Design of bio-bio hybrids. A, Harnessing the reducing power of photosynthesis for cytochrome P450 enzymes (red) has been demonstrated in vitro and in vivo. In vitro approaches were based either on a CPR-CYP1A1 fusion protein that could use NADPH produced by photosynthesis or on CYP79A1 that can utilize photoreduced ferredoxin directly. The latter approach also was realized in vivo by fusing CYP79A1 to ferredoxin (not shown) or the PSI subunit PsaM. The most elaborate approach reported utilizes three enzymes (CYP79A1, CYP71E1, and UGT85B1) to couple dhurrin synthesis with photosynthesis. B, PSI-hydrogenase complexes are based either on genetic fusions of the hydrogenase (Hyd) to PsaE or ferredoxin or on covalent linking of the hydrogenase and PSI via a molecular wire. Currently, these bio-bio hybrids function only in vitro. In contrast to PSI-P450 hybrids, PSI-hydrogenase hybrids currently function only in vitro. Unlike fossil fuels, H2 is environmentally benign, as it produces only water when combusted. Therefore, in principle, harnessing of the reducing power of photosynthesis for the direct production of H2 (i.e. ultimately using sunlight and water) would yield a fully sustainable system of energy generation. PSI, but not PSII, provides a standard midpoint potential that is sufficiently negative to power the reduction of protons to H2 (Utschig et al., 2015). Hence, approaches have been developed to engineer PSI to produce H2 either as a replacement or in addition to its natural product NADPH (see Figure Box 1) by redirecting PSI electrons to a catalytic component. This catalytic component can be abiotic or biotic (hydrogenases). The feasibility of coupling H2 generation to photosynthesis was demonstrated 45 years ago by mixing chloroplast preparations with ferredoxin and hydrogenase (Benemann et al., 1973). Twenty-five years later, hydrogen evolution by direct electron transfer (without ferredoxin) from PSI to hydrogenases was accomplished for the first time (McTavish, 1998). Hydrogenases catalyze the reversible interconversion of H2 into protons and electrons and are widespread in nature; they occur in bacteria and archaea but also in some eukarya. In vivo, hydrogenases can mediate photosynthetic H2 production, albeit mostly indirectly or under anaerobic conditions due to their oxygen sensitivity (Ghirardi, 2015; Oey et al., 2016). Hydrogenases can be classified according to their metal-ion composition (e.g. [NiFe] and [FeFe] hydrogenases; Lubitz et al., 2014; Martin and Frymier, 2017). [FeFe] hydrogenases preferentially catalyze proton reduction and can evolve H2 at high rates but are extremely sensitive to oxygen and are the only type of hydrogenases found in eukaryotic microorganisms. [NiFe] hydrogenases are less sensitive to oxygen but preferentially oxidize H2 under physiological conditions (Lubitz et al., 2014). In vitro, both types of hydrogenases have been linked directly to PSI (Table 3; Fig. 4B). PSI-[NiFe] hydrogenase complexes have been generated by fusing the hydrogenase genetically to the PsaE protein (Ihara et al., 2006b). PSI-[FeFe] hydrogenase complexes were obtained by genetically fusing the hydrogenase to ferredoxin (Yacoby et al., 2011) or covalently linking the FeS clusters present in the hydrogenase to PSI via a molecular wire (Lubner et al., 2010, 2011). Two parameters characterize the efficiency of these in vitro systems: their H2 production rate and longevity. While low rates of H2 production (in the range from 0.1 to 10 μmol H2 mg−1 chlorophyll h−1) were described for the early chloroplast extract experiments and genetic fusions of PSI to [NiFe] or [FeFe] hydrogenases (McTavish, 1998; Ihara et al., 2006b; Yacoby et al., 2011), higher rates of between 2,000 and 3,000 μmol H2 mg−1 chlorophyll h−1 have been achieved with wired PSI-[FeFe] hydrogenase complexes and genetically fused PSI-[NiFe] hydrogenase complexes assembled on a gold electrode (Krassen et al., 2009; Lubner et al., 2011). Few data are available with respect to the longevity of the PSI-hydrogenase systems; however, a minimum lifetime of 64 d was reported for a wired [FeFe] hydrogenase-PSI complex at room temperature and under ambient illumination (Lubner et al., 2010). The future use of hydrogenases in photosynthesis-driven H2 production will depend strongly on whether it is possible to overcome the oxygen sensitivity of many hydrogenases, for instance by employing oxygen-tolerant [NiFe] hydrogenases (Burgdorf et al., 2005; Schiffels et al., 2013). If this is not possible, their efficient use in vivo in thylakoids, which inevitably generate oxygen during linear electron flow, will be impossible. However, as demonstrated with the gold surface system (Krassen et al., 2009), the design of novel matrices into which the hybrid system can be incorporated may enhance the efficiency markedly. BIO-NANO HYBRIDS: USE OF PHOTOSYNTHESIS AS A SOURCE OF ELECTRONS FOR NONBIOLOGICAL PROCESSES From bio-bio hybrids, it is only a small step to developing bio-nano hybrids, as demonstrated by PSI hybrids that employ abiotic catalysts for photosynthetic hydrogen production instead of hydrogenase. In fact, abiotic catalysts have the advantage of bypassing the lability of hydrogenases in the presence of oxygen. PSI-platinum hybrids have been produced by combining PSI with platinum nanoparticles (either directly or via tethering by nanowires) or platinum nanoclusters (Kargul et al., 2012; Fukuzumi, 2015; Utschig et al., 2015; Fig. 5A; Table 4). Current PSI-platinum hybrids are less efficient than the most advanced PSI-hydrogenase systems but are extremely robust (Utschig et al., 2015). However, future widespread usage of the PSI-platinum system will be limited by the high cost of platinum. A more economical alternative to precious metals are earth-abundant molecular catalysts. However, hybrids consisting of PSI and earth-abundant molecular catalysts have a much shorter working life than platinum-based configurations (Utschig et al., 2015), likely due to the instability of the molecular catalyst. Figure 5. Open in new tabDownload slide Design of selected bio-nano hybrids. A, PSI-platinum hybrids are generated by combining platinum nanoparticles (dots) or nanoclusters (star) with PSI. Platinum nanoparticles also can be linked to PSI via nanowires. B, PSI-based photocurrent-generating system. A variety of such systems have been developed, which consist of PSI molecules immobilized on electrodes and implement electron transfer by means of diffusible redox mediators or nanowires. Moreover, all-solid-state PSI-based solar cells and systems in which cytochrome c was employed to interface PSI with electrode materials have been generated (Gordiichuk et al., 2014; Gizzie et al., 2015; Ciornii et al., 2017; Janna Olmos et al., 2017). The cross-containing circle indicates a current-using device, and the yellow rectangles symbolize the electrodes. Figure 5. Open in new tabDownload slide Design of selected bio-nano hybrids. A, PSI-platinum hybrids are generated by combining platinum nanoparticles (dots) or nanoclusters (star) with PSI. Platinum nanoparticles also can be linked to PSI via nanowires. B, PSI-based photocurrent-generating system. A variety of such systems have been developed, which consist of PSI molecules immobilized on electrodes and implement electron transfer by means of diffusible redox mediators or nanowires. Moreover, all-solid-state PSI-based solar cells and systems in which cytochrome c was employed to interface PSI with electrode materials have been generated (Gordiichuk et al., 2014; Gizzie et al., 2015; Ciornii et al., 2017; Janna Olmos et al., 2017). The cross-containing circle indicates a current-using device, and the yellow rectangles symbolize the electrodes. Combination of PSI with nonbiological materials: bio-nano hybrids Table 4. Combination of PSI with nonbiological materials: bio-nano hybrids System/Nonbiological Components . Description . Reference . H2-producing PSI bio-nano hybrids PSI-biohybrid photocatalytic systems for H2 production contain cyanobacterial PSI and precious metal catalysts, including platinum nanoparticles, platinum nanowires, and platinum nanoclusters. Examples for earth-abundant molecular catalysts in PSI-biohybrids include cobaloxime, nickel diphosphine, and nickel apoflavodoxin. Reviewed in Kargul et al. (2012); Fukuzumi (2015); Utschig et al. (2015) PSI-based photocurrent-generating bio-nano hybrids PSI-based photocurrent-generating devices contain PSI from either plants or cyanobacteria immobilized on electrode materials such as gold, graphene, indium tin oxide, fluorine-doped tin oxide, TiO2, glass, ZnO, or alumina. The most commonly utilized immobilization strategies involve the usage of organothiol-based self-assembled monolayers. Electron donors to PSI include sodium ascorbate, 2,6-dichlorophenolindophenol, reduced ferricyanide, osmium complexes, ruthenium hexamine trichloride, or the immobilization wire (NTA-Ni-His6-PSI). Acceptors of PSI electrons include naphthoquinone-derivative molecular wire, methyl viologen, oxidized ferricyanide, composite Bis-aniline nanoparticle-ferredoxin, and Methylene Blue. Also, all-solid-state PSI-based solar cells that do not employ any exogenous redox mediators or buffer solutions have been generated. Reviewed in Gordiichuk et al. (2014); Nguyen and Bruce (2014); Gizzie et al. (2015); Janna Olmos et al. (2017) System/Nonbiological Components . Description . Reference . H2-producing PSI bio-nano hybrids PSI-biohybrid photocatalytic systems for H2 production contain cyanobacterial PSI and precious metal catalysts, including platinum nanoparticles, platinum nanowires, and platinum nanoclusters. Examples for earth-abundant molecular catalysts in PSI-biohybrids include cobaloxime, nickel diphosphine, and nickel apoflavodoxin. Reviewed in Kargul et al. (2012); Fukuzumi (2015); Utschig et al. (2015) PSI-based photocurrent-generating bio-nano hybrids PSI-based photocurrent-generating devices contain PSI from either plants or cyanobacteria immobilized on electrode materials such as gold, graphene, indium tin oxide, fluorine-doped tin oxide, TiO2, glass, ZnO, or alumina. The most commonly utilized immobilization strategies involve the usage of organothiol-based self-assembled monolayers. Electron donors to PSI include sodium ascorbate, 2,6-dichlorophenolindophenol, reduced ferricyanide, osmium complexes, ruthenium hexamine trichloride, or the immobilization wire (NTA-Ni-His6-PSI). Acceptors of PSI electrons include naphthoquinone-derivative molecular wire, methyl viologen, oxidized ferricyanide, composite Bis-aniline nanoparticle-ferredoxin, and Methylene Blue. Also, all-solid-state PSI-based solar cells that do not employ any exogenous redox mediators or buffer solutions have been generated. Reviewed in Gordiichuk et al. (2014); Nguyen and Bruce (2014); Gizzie et al. (2015); Janna Olmos et al. (2017) Open in new tab Table 4. Combination of PSI with nonbiological materials: bio-nano hybrids System/Nonbiological Components . Description . Reference . H2-producing PSI bio-nano hybrids PSI-biohybrid photocatalytic systems for H2 production contain cyanobacterial PSI and precious metal catalysts, including platinum nanoparticles, platinum nanowires, and platinum nanoclusters. Examples for earth-abundant molecular catalysts in PSI-biohybrids include cobaloxime, nickel diphosphine, and nickel apoflavodoxin. Reviewed in Kargul et al. (2012); Fukuzumi (2015); Utschig et al. (2015) PSI-based photocurrent-generating bio-nano hybrids PSI-based photocurrent-generating devices contain PSI from either plants or cyanobacteria immobilized on electrode materials such as gold, graphene, indium tin oxide, fluorine-doped tin oxide, TiO2, glass, ZnO, or alumina. The most commonly utilized immobilization strategies involve the usage of organothiol-based self-assembled monolayers. Electron donors to PSI include sodium ascorbate, 2,6-dichlorophenolindophenol, reduced ferricyanide, osmium complexes, ruthenium hexamine trichloride, or the immobilization wire (NTA-Ni-His6-PSI). Acceptors of PSI electrons include naphthoquinone-derivative molecular wire, methyl viologen, oxidized ferricyanide, composite Bis-aniline nanoparticle-ferredoxin, and Methylene Blue. Also, all-solid-state PSI-based solar cells that do not employ any exogenous redox mediators or buffer solutions have been generated. Reviewed in Gordiichuk et al. (2014); Nguyen and Bruce (2014); Gizzie et al. (2015); Janna Olmos et al. (2017) System/Nonbiological Components . Description . Reference . H2-producing PSI bio-nano hybrids PSI-biohybrid photocatalytic systems for H2 production contain cyanobacterial PSI and precious metal catalysts, including platinum nanoparticles, platinum nanowires, and platinum nanoclusters. Examples for earth-abundant molecular catalysts in PSI-biohybrids include cobaloxime, nickel diphosphine, and nickel apoflavodoxin. Reviewed in Kargul et al. (2012); Fukuzumi (2015); Utschig et al. (2015) PSI-based photocurrent-generating bio-nano hybrids PSI-based photocurrent-generating devices contain PSI from either plants or cyanobacteria immobilized on electrode materials such as gold, graphene, indium tin oxide, fluorine-doped tin oxide, TiO2, glass, ZnO, or alumina. The most commonly utilized immobilization strategies involve the usage of organothiol-based self-assembled monolayers. Electron donors to PSI include sodium ascorbate, 2,6-dichlorophenolindophenol, reduced ferricyanide, osmium complexes, ruthenium hexamine trichloride, or the immobilization wire (NTA-Ni-His6-PSI). Acceptors of PSI electrons include naphthoquinone-derivative molecular wire, methyl viologen, oxidized ferricyanide, composite Bis-aniline nanoparticle-ferredoxin, and Methylene Blue. Also, all-solid-state PSI-based solar cells that do not employ any exogenous redox mediators or buffer solutions have been generated. Reviewed in Gordiichuk et al. (2014); Nguyen and Bruce (2014); Gizzie et al. (2015); Janna Olmos et al. (2017) Open in new tab PSI-based photocurrent-producing devices constitute another class of photosynthesis-derived nano-bio systems (Table 4; Fig. 5). In such devices, PSI is immobilized onto electrodes. Many variants of this concept have been tested, such as varying the electrode materials, immobilization/orientation strategies, and/or artificial redox mediators (Nguyen and Bruce, 2014; Janna Olmos and Kargul, 2015; Plumeré and Nowaczyk, 2016; Kargul et al., 2018). PSI must be immobilized on the electrode surface in such a way that electron transfers between the electrodes and the oxidizing (P700) and reducing (FB, the terminal [4Fe-4S] cluster, or an intermediate electron transporter) sides of PSI can proceed with the required efficiency. Electrons are transferred between the electrodes and the oxidizing or reducing sides of PSI either by a diffusible redox mediator or molecular wires. Examples of electrode materials and redox mediators are provided in Table 4. After P700 photoexcitation, electrons are transferred from P700 via several factors (P700 → A0 → A1 → FX → FA → FB) to the iron-sulfur cluster FB (Box 1). As in the case of PSI-hydrogenase and PSI-platinum nanoparticles, PSI can be wired to its electrodes. This has been achieved by wiring the A1 cofactor (phylloquinone) to the substrate surface, such that electrons are transferred directly from A1 to the electrode, thus bypassing the downstream FeS clusters (FX, FA, and FB) in the stromal domain of PSI (Terasaki et al., 2007, 2009; Miyachi et al., 2009, 2010). Modifications of PSI have been utilized to enhance the efficiency of the photocurrent-generating system (Das et al., 2004; Frolov et al., 2005; Carmeli et al., 2010). As mentioned previously, fusions to the stroma-faced PSI subunit PsaE can be used to link new components to PSI; however, in this case, PsaD fusions were used. To this end, recombinant His-tagged PsaD was immobilized on the functionalized electrode surface, which then was exposed to native PSI complexes, resulting in immobilized PSI with P700 facing away from the electrode (Das et al., 2004). Another way to control the orientation of PSI during immobilization involves introducing Cys mutations. To allow for direct thiol coupling to an gold surface, various residues on the lumen-exposed face of PSI were replaced by Cys and tested (Frolov et al., 2005). PSI attachment was achieved with all single mutants, even those placed farther from the P700 site, suggesting that a specific location is not required as long as the Cys is exposed at the luminal surface of PSI. The concept of targeted attachment via introduced Cys residues was exploited in subsequent studies to link PSI to maleimide-functionalized gallium arsenide (Frolov et al., 2008), to immobilize PSI between the substrate and a metallized scanning near-field optical microscopy tip (Gerster et al., 2012), and to bind PSI to carbon nanotubes (Kaniber et al., 2010). Another modification of PSI is represented by the attachment of plasmonic metal nanoparticles, which resulted in enhanced light absorption (Carmeli et al., 2010) even in the green part of the solar spectrum that is not absorbed normally (Szalkowski et al., 2017). This suggests that it is possible to enhance light absorption by PSI in vitro through the attachment of abiotic components that act as optical antennae to extend the spectrum of photons available for P700 activation. Taken together, these efforts demonstrate that PSI-based photocurrent-generating systems are still in an exploratory phase, with many variants under development. In the next phase, viable building blocks and reference systems should be established that can serve as starting points for the systematic engineering of superior systems. This will require the modification of PSI for optimal effect in artificial systems and undoubtedly will differ substantially from the original environment in which PSI was molded by biological evolution. Open in new tabDownload slide Open in new tabDownload slide CONCLUSION AND OUTLOOK Enhancing photosynthesis and growth can be achieved by a simple overexpression of certain photosynthetic proteins, and PSI can be coupled to previously unconnected biotic or abiotic components to generate valuable compounds, hydrogen, or electricity. Moreover, entire photosynthetic complexes can be expressed functionally in distantly related species (as demonstrated for Rubisco; Aigner et al., 2017). Thus, what are the next goals and which challenges need to be overcome? Two challenges for the in vivo systems are obvious: (1) enhancing the efficiency of in vivo bio-bio hybrids; and (2) upscaling the size of synthetic photosynthetic modules to cover entire photosystems or complex antenna systems. With respect to the enhancement of in vivo cyanobacterial and algal systems harboring hybrid configurations in which photosynthesis directly drives previously unconnected pathways, the use of laboratory evolution offers a unique opportunity to tailor these systems for their intended purpose. Laboratory evolution utilizes the high rate of evolution typical in microbial systems (in particular, if suitable selection conditions can be designed) to fine-tune and optimize processes through the selection of advantageous genetic variation. While this strategy has been employed in E. coli and yeast with impressive success, now photosynthetic microbial systems are emerging as attractive targets for this approach (Leister, 2018). The exchange of entire multiprotein complexes between distantly related species will not be a simple exercise, because the frozen metabolic state of the core photosynthetic complexes will necessitate the exchange of major parts of photosystems rather than individual subunits. The Rubisco case study by Aigner et al. (2017) represents a promising proof of principle, but one needs to consider that the synthetic Rubisco module comprised only the two different subunits present in the mature complex and five auxiliary factors for its assembly. Entire photosystems will require much larger synthetic photosynthetic modules, and many of the auxiliary factors have not been identified yet. Therefore, when designing these complex synthetic photosynthetic modules, we will identify the set of components that are sufficient (and not only necessary) to drive photosynthesis, providing an unprecedentedly deep understanding of this fundamental and complex process. Given that the problems described above can be solved, what will be the next steps in the synthetic biology of the light reactions of photosynthesis (see Outstanding Questions)? The combination of synthetic photosynthetic modules from diverse species could allow the design of novel variants of photosynthesis. Numerous instances of such recombined photosynthetic variants can be imagined, including plants that employ cyanobacteria-derived phycobilisomes for highly sufficient photosynthesis under low-light conditions, cyanobacteria that employ plant-derived LHCs as antennae to shift the light saturation of photosynthesis to higher intensities, or the integration of cyanobacterial chlorophyll d and chlorophyll f (which can absorb far-red and near infrared light) into algal or plant photosynthesis to expand the spectral region available to drive photosynthesis (Loughlin et al., 2013; Ho et al., 2016). Moreover, such variants of recombined photosynthesis could be optimized further by laboratory evolution within a suitable microbial chassis. However, such recombined photosynthetic variants cannot be considered a truly novel type of photosynthesis because they would only bring together preexisting pieces that evolution has separated. Nevertheless, they could be an important step toward the ambitious goal to design truly novel synthetic photosynthetic modules that contain more efficient substitutes of the frozen metabolic accidents discussed above. Ample proof of functionality for in vivo hybrids (between photosynthesis and previously unconnected metabolic pathways) and in vitro hybrids (between PSI and biotic or abiotic catalysts) has been obtained, but such systems will only be commercially successful if they can compete with established nonbiological systems. Tailoring of PSI in cyanobacteria, where techniques like gene replacement and gene modification are routine, may contribute to further improving the efficiency and robustness of in vitro and in vivo hybrids. One promising route could be to identify those parts of the original PSI (which evolved under constraints imposed by the cyanobacterial cell) that are necessary for hybrid devices (a minimal PSI). Such bio-nano systems can be optimized to include novel nonbiological components that replace or complement natural pigments and/or the protein-based backbone of photosystems, going far beyond the limited toolbox of nature’s chemistry. Such novel systems, inspired by natural photosynthesis, could be used to engage biologists in the design of fundamentally different types of photosynthesis in living organisms. Supplemental Data The following supplemental materials are available. Supplemental Table S1. 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Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Recent Advances and Current Challenges in Synthetic Biology of the Plastid Genetic System and MetabolismBoehm, Christian R.; Bock, Ralph
doi: 10.1104/pp.18.00767pmid: 30181342
Abstract Building on recombinant DNA technology, leaps in synthesis, assembly, and analysis of DNA have revolutionized genetics and molecular biology over the past two decades (Kosuri and Church, 2014). These technological advances have accelerated the emergence of synthetic biology as a new discipline (Cameron et al., 2014). Synthetic biology is characterized by efforts targeted at the modification of existing and the design of novel biological systems based on principles adopted from information technology and engineering (Andrianantoandro et al., 2006; Khalil and Collins, 2010). As in more traditional engineering disciplines such as mechanical, electrical and civil engineering, synthetic biologists utilize abstraction, decoupling and standardization to make the design of biological systems more efficient and scalable. To facilitate the management of complexity, synthetic biology relies on an abstraction hierarchy composed of multiple levels (Endy, 2005): DNA as genetic material, “parts” as elements of DNA encoding basic biological functions (e.g. promoter, ribosome-binding site, terminator sequence), “devices” as any combination of parts implementing a human-defined function, and “systems” as any combination of devices fulfilling a predefined purpose. Parts are designated to perform predictable and modular functions in the context of higher-level devices or systems, which are successively refined through a cycle of designing, building, and testing. Within the past two decades, the synthetic biology approach has produced several notable successes, especially in microbial systems. These include, for example, the design of a minimal bacterial genome (Hutchison et al., 2016) and a highly modified yeast genome (Richardson et al., 2017), as well as the metabolic engineering of yeast for the biosynthesis of the antimalarial drug precursor artemisinic acid (Ro et al., 2006) and the opioid compounds thebaine and hydrocodone (Galanie et al., 2015). Compared to synthetic biology in bacteria and yeast, synthetic biology in algae and plants is still lagging behind. While the potential of photoautotrophic organisms for environmentally sustainable bioproduction has long been recognized (Georgianna and Mayfield, 2012; Fesenko and Edwards, 2014; Liu and Stewart, 2015; Boehm et al., 2017), their relatively slow growth, scarcely available tools for genetic manipulation, and the physiological as well as genomic complexity of plant systems have delayed their widespread adoption as synthetic biology chassis. However, especially the small genome of the plastid (chloroplast) represents a highly promising platform for engineering the sophisticated metabolism and physiology of the eukaryotic cell it is embedded in (Fig. 1). Figure 1. Open in new tabDownload slide Biological properties and existing technical capacities for synthetic biology of plastids compared to bacteria, yeast and the plant nucleus. The number of asterisks roughly illustrates the relative degree of (top) presence of a biological feature, (middle) availability of a tool or technique, and (bottom) current implementation of a type of application across the different chassis. Figure 1. Open in new tabDownload slide Biological properties and existing technical capacities for synthetic biology of plastids compared to bacteria, yeast and the plant nucleus. The number of asterisks roughly illustrates the relative degree of (top) presence of a biological feature, (middle) availability of a tool or technique, and (bottom) current implementation of a type of application across the different chassis. The chloroplast originated through the endosymbiotic uptake of a cyanobacterium by a heterotrophic eukaryote more than a billion years ago (Palmer, 2003). Following this event, the endosymbiont evolved mechanisms for facilitated exchange of metabolites with the host cell, underwent radical streamlining of its genome (by gene loss and large-scale transfer of genes to the host nuclear genome) and established an import machinery for the uptake of nucleus-encoded proteins. The resulting organelle serves as the major biosynthetic compartment in photoautotrophic organisms, and has been exploited as a platform for metabolic engineering and molecular farming since the successful development of transformation technologies in the late 1980s (Boynton et al., 1988; Svab et al., 1990). Compared to nuclear genetic engineering, plastid transformation offers several notable advantages relevant to plant biotechnology. These include (1) the high precision of genetic engineering enabled by efficient homologous recombination, (2) the possibility of transgene stacking in synthetic operons, (3) the potential for high-level expression of gene products, (4) the absence of epigenetic transgene silencing, and (5) the reduced risk of unwanted transgene transmission due to maternal inheritance of plastid DNA (Bock, 2015). In this article, we provide an update on tools and technologies available for extending the synthetic biology approach to plastids and highlight key challenges to be addressed through future research. Guided by an abstraction hierarchy of biological design, we identify a scarcity of well-characterized genetic parts, tightly controlled expression devices, and quantitative knowledge of plastid gene expression as current key limitations to plastid synthetic biology. We highlight recent technological developments narrowing the existing complexity gap between bacterial and plastid synthetic biology and provide an outlook to the implementation of complex systems such as synthetic metabolic feedback loops, designer subcompartments and tailor-made genomes in chloroplasts. Open in new tabDownload slide Open in new tabDownload slide Parts The Registry of Standard Biological Parts (http://parts.igem.org) currently contains over 20,000 genetic elements which can be requested by researchers for use in synthetic biology applications. From this collection, approximately 100 parts each have been designed for use in the unicellular green alga Chlamydomonas reinhardtii and in multicellular plants (e.g. the seed plants Nicotiana tabacum and Arabidopsis thaliana, the moss Physcomitrella patens and the liverwort Marchantia polymorpha). The majority of these parts are designated for nuclear engineering, with only about two dozen suitable for gene expression from the chloroplast genome. One explanation for the relative paucity of plastid genetic elements in the Registry of Standard Biological Parts lies in the half-year timeframe of projects pursued as part of the international Genetically Engineered Machine (iGEM) competition (Smolke, 2009), which is barely compatible with the generation and characterization of stable plastid-engineered (transplastomic) organisms. Beyond iGEM, the repertoire of regulatory sequences routinely used for transgene expression in plastids has remained similarly small: it is comprised of a few preferred promoters (e.g. from the plastid rRNA operon, Prrn; the gene for the large subunit of Rubisco, PrbcL; and the gene for the D1 protein of photosystem II, PpsbA) and a handful of 5′-and 3′-UTRs (Jin and Daniell, 2015). In addition, the bacterial hybrid promoter Ptrc (Newell et al., 2003) and several bacteriophage-derived expression elements (McBride et al., 1994; Kuroda and Maliga, 2001; Yang et al., 2013) have been successfully used for plastid transgene expression. A greater variety of parts available for controlled expression of plastid transgenes is desirable for several reasons. First, multiple use of the same genetic element within the chloroplast genome is problematic due to the risk of unwanted homologous recombination between sequence stretches as short as 50 bp (Dauvillee et al., 2004; Rogalski et al., 2006). Second, synthetic genetic circuits commonly require precise tuning of the activity of their constitutive parts for optimal function (Brophy and Voigt, 2014). For synthetic biology applications in plastids to catch up in versatility and complexity with those already demonstrated in bacteria, gene expression elements covering a wider activity range will be required. Natural plastid genomes represent an obvious source of such elements. The small size and low coding capacity of chloroplast genomes (in most seed plants, approximately 130 genes in an ∼ 150 kb genome) should allow refactoring of all coding and regulatory regions into standardized genetic parts. The sequences of over 800 chloroplast genomes have been determined (Daniell et al., 2016), and the functions of most of their (widely conserved) genes are known (Scharff and Bock, 2014). Plastid genetic elements contained within this wealth of sequence data can be domesticated according to a recently proposed common syntax for plant synthetic biology (Patron et al., 2015). This scheme promises to facilitate sharing of genetic resources among the community and, although developed for a eukaryotic system, is also compatible with GoldenBraid-based modular cloning of chloroplast transformation vectors (Vafaee et al., 2014). Plastid parts containing internal recognition sites for type IIS restriction enzymes (e.g. BsaI, BsmBI, BbsI) that cannot be synonymously changed (e.g. because they constitute essential sequence motifs in a promoter or UTR sequence) may alternatively be assembled using long-overlap-based methods such as Gibson Assembly (Gibson et al., 2009). Gene Expression Devices Gene expression devices send or receive signals in the form of levels of gene expression. A basic device of this kind may be composed of four parts: a promoter, a ribosome-binding site, a coding sequence and a terminator. This device architecture is commonly used for the quantification of part performance to inform the rational design of genetic circuits. Hundreds of prokaryotic gene-expression elements (including promoters, ribosome-binding sites and terminators) have been characterized in bacterial hosts using reporter gene-based assays (Salis et al., 2009; Cambray et al., 2013; Chen et al., 2013; Kosuri et al., 2013; Mutalik et al., 2013), and standards have been formulated for quantifying their activities (Canton et al., 2008; Kelly et al., 2009; Rudge et al., 2016). To reduce the context dependence of part activity, standardized flanking sequences (Mutalik et al., 2013), strong terminators (Chen et al., 2013) and enzymatic cleavage of UTRs (Lou et al., 2012; Qi et al., 2012) have been successfully employed as insulators in bacteria. In plastids, not more than two dozen combinations of regulatory elements (i.e. promoters, 5′-UTRs and 3′-UTRs) have been systematically characterized for their impact on transgene expression using GFP (Barnes et al., 2005; Caroca et al., 2013), GUS (Eibl et al., 1999; Herz et al., 2005; Gerasymenko et al., 2017) or other reporter proteins (Ruhlman et al., 2010; Zhang et al., 2012). Compared to part characterization in microbes, that in plastids involves several notable challenges. First, relatively long timescales are required to generate transplastomic organisms ready for characterization. While only a few days are needed for transformation of the microbial models Escherichia coli or Saccharomyces cerevisiae by a genetic part, several months of selection are needed to recover homoplasmic plastid transformants (i.e. transplastomic cells or plants that are devoid of residual copies of the wild-type plastid genome). In theory, the establishment of homoplasmy could be accelerated through inducible expression of endonucleases that selectively target the wild-type chloroplast genome, but it remains to be tested how much time this approach can save. Alternatively, measurement fidelity can be traded for high-throughput, transient assays to quantify part performance within days of particle bombardment of algal cells or plant tissues. Such assays will require (1) high transient transformation frequencies, (2) high sensitivity, and (3) a robust way of normalizing the primary reporter signal to the copy number of transformed plastomes. The latter could be achieved by using a ratiometric approach (Rudge et al., 2016; Boehm et al., 2018). If a suitable reporter system can be developed, the activities of hundreds of plastid parts could rapidly be measured in algal cells or plant protoplasts using microtiter plate-based assays (Schaumberg et al., 2016) or microfluidic devices (Yu et al., 2018). Second, the plastome exhibits abundant read-through transcription due to inefficient termination (Stern and Gruissem, 1987; Rott et al., 1996; Legen et al., 2002; Shi et al., 2016). Consequently, part behavior is, by default, poorly insulated from its specific genetic context: both upstream promoters and downstream antisense promoters may significantly affect the expression level of a target gene (Quesada-Vargas et al., 2005; Sharwood et al., 2011). However, some sequences such as the endogenous tRNA genes trnS and trnH (Stern and Gruissem, 1987) or the heterologous E. coli Thr attenuator (thra; Chen and Orozco, 1988) have been shown to terminate plastid transcription with at least 85% efficiency. Use of insulators based on these parts or new synthetic terminators can potentially enhance the robustness of gene expression levels generated by plastid synthetic biology devices. Third, plastid transgene expression has been shown to be primarily determined by posttranscriptional control and protein stability rather than by the accumulation of mRNA (Eberhard et al., 2002; Birch-Machin et al., 2004; Bellucci et al., 2005; Kahlau and Bock, 2008; Valkov et al., 2009; Zoschke and Bock, 2018). Chloroplast transcripts are subject to a series of complex processing steps which include intercistronic cleavage, 5′-and 3′-end maturation, intron splicing and mRNA editing (Stern et al., 2010). These steps are largely mediated by nucleus-encoded and organelle-targeted factors, including a large family of modular proteins known as pentatricopeptide repeat (PPR) proteins that site-specifically bind to one or several premRNAs (Barkan and Small, 2014). Plastid gene expression levels can, therefore, vary considerably between different transgenes even if the same promoter and 3′-UTR are used, limiting the informative value of part characterization based on standard reporter protein assays. While the amino acid sequence of the N-terminus is thought to substantially influence protein stability in the chloroplast (Apel et al., 2010; De Marchis et al., 2012), our general knowledge of plastid proteostasis remains limited. A better understanding of the molecular determinants of plastid protein (in)stability may in the future allow the design of protective amino acid sequences (Elghabi et al., 2011) that level the stabilities of different plastid-expressed proteins and make transgene expression from the plastid genome more predictable. Metabolic Devices Metabolic devices send or receive signals in the form of levels of metabolites. Accordingly, a synthetic metabolic pathway represents a metabolic device carrying out a specific series of enzyme-catalyzed reactions. A variety of metabolic devices have been successfully implemented in plastids for the production of molecules such as polyhydroxybutyrate (Bohmert-Tatarev et al., 2011), carotenoids (Wurbs et al., 2007; Hasunuma et al., 2008; Apel and Bock, 2009), fatty acids (Madoka et al., 2002; Craig et al., 2008), artemisinic acid (Fuentes et al., 2016), vitamin E (Lu et al., 2013) and dhurrin (Gnanasekaran et al., 2016). These applications have been reviewed in more detail elsewhere (Bock, 2015; Fuentes et al., 2018). While heterologous pathways composed of 20 genes or more have been expressed in bacteria and yeast (Temme et al., 2012; Galanie et al., 2015; Li et al., 2018), no more than seven transgenes have to date been simultaneously expressed from the plastome (Krichevsky et al., 2010). The complexity of plastid-based metabolic devices has primarily been limited by a scarcity of available expression signals (see Gene Expression Devices) rather than by the physical size of the introduced DNA (Adachi et al., 2007). Recently, the complexity and number of pathway variants accessible to experimental interrogation has been expanded through combinatorial supertransformation of transplastomic recipient lines (COSTREL). Using this approach, an up to 77-fold increase in artemisinic acid production has been demonstrated in transplastomic tobacco plants combinatorially supertransformed by five additional nuclear transgenes (Fuentes et al., 2016). There is no in-principle limitation to the number of transgenes that can be simultaneously introduced into the plant nucleus using combinatorial transformation (Naqvi et al., 2009). However, handling hundreds to thousands of plants resulting from combinatorial transformation with several dozen transgenes will require an effective screening pipeline. In plastid-based metabolic devices containing multicistronic operons, intercistronic expression elements (IEEs) can be used to facilitate correct processing of polycistronic transcripts into monocistronic mRNAs and their efficient translation (Fig. 2A; Zhou et al., 2007). To avoid defects in mRNA stabilization upon repeated use of the same IEE, more complex future metabolic devices may feature a variety of different such elements and/or additionally overexpress their cognate RNA-binding proteins (Legen et al., 2018). Figure 2. Open in new tabDownload slide Design of plastid-based metabolic devices. A, Intercistronic expression elements (IEEs; Zhou et al., 2007) can be used to design synthetic operons composed of n genes of interest (GOIs) under the control of a single promoter. Alternatively, each transgene can be controlled by its own promoter. SD, Shine-Dalgarno sequence; SMG, selectable marker gene. B, Expression of a GOI can be controlled by a synthetic 5′-UTR that is specifically stabilized by a designer PPR protein (that recognizes a different binding sequence than all other RNA-binding proteins present in the plastid). Figure 2. Open in new tabDownload slide Design of plastid-based metabolic devices. A, Intercistronic expression elements (IEEs; Zhou et al., 2007) can be used to design synthetic operons composed of n genes of interest (GOIs) under the control of a single promoter. Alternatively, each transgene can be controlled by its own promoter. SD, Shine-Dalgarno sequence; SMG, selectable marker gene. B, Expression of a GOI can be controlled by a synthetic 5′-UTR that is specifically stabilized by a designer PPR protein (that recognizes a different binding sequence than all other RNA-binding proteins present in the plastid). Genetic Circuits Genetic circuits mimic logical functions commonly found in their electronic counterparts. A genetic circuit can be used to control the activity of other devices (such as the gene expression devices or metabolic devices discussed above) in response to external stimuli. A wide range of genetic circuits implementing Boolean logic functions such as yes, not, and, or, nand, nor, xor and n-imply has been reported for bacteria, yeast and mammalian cells (Miyamoto et al., 2013). In plastids, only the simplest logic function yes has been implemented in the form of chemically inducible transgene expression. Chloroplast transcription is natively controlled by two different types of RNA polymerases in seed plants. The nucleus-encoded RNA polymerase (NEP) is a chloroplast-targeted bacteriophage-type single subunit enzyme, while the plastid-encoded RNA polymerase (PEP) is a eubacteria-type multisubunit enzyme (Barkan, 2011; Börner et al., 2015). The promoter specificity of PEP is modulated by nucleus-encoded and plastid-targeted sigma factors in response to light, hormones and biotic as well as abiotic stresses. However, due to their important role in plant growth, development and survival (and the pervasive transcription of essentially all plastid genes), NEP and PEP are poorly suited as stringent controllers of synthetic genetic circuits in plastids. As an alternative to transgene control by the endogenous transcription machineries, plastid transgene expression has been controlled through nucleus-encoded and plastid-targeted bacteriophage RNA polymerases or processing factors that are responsive to chemical inducers such as salicylic acid (Magee et al., 2004), ethanol (Lössl et al., 2005), copper (Surzycki et al., 2007) or thiamine (Ramundo et al., 2013). To avoid (pollen-transmissible) nuclear transgenes and increase transgene containment, inducible expression systems encoded solely in the plastid genome are particularly desirable. Plastid-only inducible circuits responsive to isopropyl β-d-1-thiogalactopyranoside (IPTG; Mühlbauer and Koop, 2005) or theophylline (Verhounig et al., 2010; Emadpour et al., 2015) have been shown to be functional, yet fall short of binary behavior due to the pronounced transcriptional leakiness present in plastids (see Gene Expression Devices). To achieve a signal-to-noise ratio sufficient for the implementation of more complex logic gates, future plastid-based genetic circuits may employ synthetic RNA-binding proteins of the PPR class (see Gene Expression Devices; Coquille et al., 2014; Gully et al., 2015) to selectively control the maturation of target mRNAs in the chloroplast (Fig. 2B; Stern et al., 2010; Barkan and Small, 2014). Systems Beyond hard-wired logic gates, synthetic biologists have explored dynamic feedback mechanisms to enhance the efficiency of engineered metabolic pathways in bacteria and yeast (Venayak et al., 2015; Del Vecchio et al., 2016). Translation of this approach to plastids is currently hampered by our limited quantitative understanding of chloroplast gene expression, though new tools for analysis of the metabolic network shared between the chloroplast and its host cell are emerging (Gloaguen et al., 2017). Metabolic engineering in plastids may further be supported by expression of synthetic subcompartments for substrate concentration, metabolite channeling and the prevention of unwanted reactions between subcompartmentalized and endogenous plastid metabolites and enzymes (Winkel, 2004; Ort et al., 2015; Hanson et al., 2016). Synthetic subcompartments have already been introduced in bacteria and yeast (Bonacci et al., 2012; Lau et al., 2018), and carboxysomal shell proteins transiently expressed in leaves of Nicotiana benthamiana have been shown to be capable of assembling into carboxysome-like structures within chloroplasts (Lin et al., 2014), encouraging further efforts in this area. Open in new tabDownload slide Open in new tabDownload slide Among the most complex systems proposed for implementation in plastids are entire synthetic genomes, inspired by recent successes in microbial synthetic genomics (Hutchison et al., 2016; Richardson et al., 2017). A minimum-size plastid genome composed of the smallest possible number of components will be of great value for two reasons: it will advance our understanding of the regulatory network underlying plastid function, and it will serve as a template for engineering synthetic plastomes to be used in biotechnological applications. We have previously proposed a design for a synthetic minimal plastome of N. tabacum that is free of all genes nonessential under heterotrophic growth conditions (Fig. 3), intergenic spacers, introns, and isoaccepting tRNA genes that are dispensable or become dispensable after genome-wide modification of codon usage (Scharff and Bock, 2014). Such a synthetic chloroplast genome can be assembled from linear DNA fragments in yeast (O’Neill et al., 2012) and, prior to plant transformation, can be amplified in vitro using rolling circle amplification (Jansen et al., 2005). The major hurdle to the successful implementation of fully synthetic plastomes in planta is the high probability of homologous recombination between the (largely nonrecodeable) rRNA and tRNA genes and their counterparts in the resident plastid genome, leading to chimeric genomes of unpredictable structure and function (O’Neill et al., 2012). In addition, the effects of synthetic lethality (i.e. the combined knock-out of two nonessential genes being lethal; e.g. Ehrnthaler et al., 2014) cannot currently be excluded to occur in a synthetic minimal plastome. Figure 3. Open in new tabDownload slide Physical map of the N. tabacum chloroplast genome with all genes classified by essentiality. Genes shown in blue are essential for both heterotrophic and autotrophic growth. Genes shown in green are essential for autotrophic growth only. Light green indicates borderline cases where knock-out plants survive under carefully controlled growth conditions. Genes shown in gray are nonessential under both heterotrophic and autotrophic growth conditions, in that their knock-out causes no or only a mild phenotype (Scharff and Bock, 2014). Origins of replication are highlighted in red. Gray arrows indicate the direction of transcription for the two DNA strands. The map was drawn using the OrganellarGenomeDRAW (OGDRAW) software (Lohse et al., 2013) based on the complete plastome sequence of N. tabacum (Shinozaki et al., 1986; GenBank accession number Z00044.2). LSC, large single-copy region; IRA, inverted repeat A; IRB, inverted repeat B; SSC, small single-copy region. Figure 3. Open in new tabDownload slide Physical map of the N. tabacum chloroplast genome with all genes classified by essentiality. Genes shown in blue are essential for both heterotrophic and autotrophic growth. Genes shown in green are essential for autotrophic growth only. Light green indicates borderline cases where knock-out plants survive under carefully controlled growth conditions. Genes shown in gray are nonessential under both heterotrophic and autotrophic growth conditions, in that their knock-out causes no or only a mild phenotype (Scharff and Bock, 2014). Origins of replication are highlighted in red. Gray arrows indicate the direction of transcription for the two DNA strands. The map was drawn using the OrganellarGenomeDRAW (OGDRAW) software (Lohse et al., 2013) based on the complete plastome sequence of N. tabacum (Shinozaki et al., 1986; GenBank accession number Z00044.2). LSC, large single-copy region; IRA, inverted repeat A; IRB, inverted repeat B; SSC, small single-copy region. Despite numerous technical advances made over the past 30 years, the number of algal and plant species whose plastids can reliably be transformed has remained small (Bock, 2015). Transplantation of transgenic plastids from a species amenable to transformation to a species recalcitrant to transformation represents an attractive alternative to painstakingly developing specialized transformation protocols for the latter. Plastomes can be horizontally transferred across graft junctions with relative ease (Stegemann and Bock, 2009; Stegemann et al., 2012; Thyssen et al., 2012; for review, see Bock, 2017) and this process has been exploited for transplanting a plastid-encoded synthetic metabolic device into a currently nontransformable species (Lu et al., 2017). The graft-mediated horizontal transfer of transgenic plastid genomes may not be feasible between distantly related species due to the close coevolution of nuclear and plastid genomes, and the probability of nuclear-cytoplasmic incompatibilities that increases with phylogenetic distance and can cause deleterious phenotypes (Schmitz-Linneweber et al., 2005; Greiner and Bock, 2013). However, the transfer will certainly facilitate the expansion of transplastomic technologies from model species and cultivars used in research to related species and elite cultivars grown commercially. 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All rights reserved. © The Author(s) 2019. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Update: Improving the Efficiency of Photosynthetic Carbon ReactionsWeber, Andreas P.M.; Bar-Even, Arren
doi: 10.1104/pp.18.01521pmid: 30610109
Photosynthetic carbon assimilation (PCA) by plants has provided the basis for human civilization since the origin of agriculture. Transformative changes in agriculture, such as mechanization in the 19th century and the development of synthetic fertilizers, hybrid breeding, and the Green Revolution in the 20th century, have helped to support an almost 10-fold growth in the human population since 1800. Yield increases continued to grow in a linear manner since the 1960s, at an average annual rate of approximately 1.7% (Long et al., 2015). However, several recent studies predict that crop yield will have to double by 2050 to keep pace with the demand of population growth while accounting for changes in consumption patterns resulting from increased wealth and urbanization as well as increased use of agricultural resources for biorefineries (Tilman et al., 2011; Walker et al., 2016). Doubling agricultural yield within the next 30 years would require an annual yield increase of 2.2%, which exceeds the average annual increase witnessed over the past 50 years. Such yield increase cannot come from expanding agricultural land use since reserves in arable land are limited and further utilization of land for agricultural use come at the expense of natural habitats and therefore erodes biodiversity (Foley et al., 2011; Clark and Tilman, 2017). Hence, yield must be increased per unit area land, either by increasing planting density or by increasing the performance of individuals within a crop canopy. Planting density in intensely managed cropping systems is approaching a ceiling (Mansfield and Mumm, 2014), which defines increased performance of individual plants within a canopy as a prime target (Long et al., 2006). Yield is a complex trait to which many intrinsic and extrinsic factors contribute, such as biotic and abiotic stresses, the efficiency of light energy capture by the photosynthetic light reactions, the efficiency of the conversion of light energy into biomass, and the harvest index (fraction of total energy in plant biomass contained in the harvestable organs). As the efficiency of light energy capture and the harvest index seem to reach their practical limit, improvement of the currently low conversion efficiency of light to biomass (∼2%) has received considerable attention (Zhu et al., 2010). This efficiency can be increased by relieving constraints imposed by individual components, as demonstrated by overexpression of SBPase and/or Gly decarboxylase, the activity of which limits carbon fixation and related processes (Timm et al., 2012; Simkin et al., 2015, 2017). Instead of modulating individual components, a synthetic biology approach can be used, where entire processes are redesigned and engineered to overcome key barriers that cannot be easily addressed by tinkering with existing systems. This Update focuses on recent progress made by synthetic biology undertakings that aim to breach the boundaries of plant carbon fixation. We discuss in detail the concepts behind these projects rather than the specific technologies used for their implementation, for which we refer the readers to other recently published reviews (Engler et al., 2014; Patron et al., 2015). Open in new tabDownload slide Open in new tabDownload slide LIMITATIONS IN PHOTOSYNTHETIC CARBON USE EFFICIENCY The vast majority of organic carbon in the biosphere is derived from inorganic carbon captured by the Calvin-Benson cycle (CBC) in plants. The CBC consumes NADPH and ATP, which are regenerated by the light reactions. Hence, the light and carbon reactions of photosynthesis are tightly coupled via the production and consumption of the key cellular redox and energy carriers. In the CBC, CO2 is first attached to the five-carbon acceptor molecule ribulose 1,5-bisphosphate (RuBP) by the enzyme Rubisco, yielding an unstable C6 intermediate that spontaneously and irreversibly hydrolyzes to yield two molecules of 3-phosphoglyceric acid (3PGA). 3PGA is then activated by phosphoglycerate kinase to yield 1,3-bisphosphoglycerate while consuming ATP. The latter intermediate is reduced, using NADPH as an electron donor, to give glyceraldehyde 3-phosphate (GAP), as catalyzed by glyceraldehyde phosphate dehydrogenase. ATP is also consumed during regeneration of the CO2 acceptor RuBP from GAP in the regenerative phase of the CBC. Overall, three molecules of ATP and two molecules of NADPH are consumed per assimilated CO2, without further considering the cost involved in making, maintaining, and degrading pathway enzymes as well as plant structures, such as chloroplasts, cell walls, etc. (see Amthor [2010] for a comprehensive review on the conversion of solar energy to plant phytomass and Williams [2016] for a general introduction to PCA). The above calculation holds true only under the assumption that all reactions catalyzed by Rubisco are carboxylation reactions. In reality, however, under current atmospheric conditions in C3 plants at a leaf temperature of 25°C, approximately 25% of the reactions of Rubisco use O2 instead of CO2, which leads to the oxygenation of RuBP and the production of one molecule of 3PGA and one molecule of 2-phosphoglycolic acid (2PG). 2PG is a dead-end metabolite that must be recycled to GAP by an elaborate series of enzymic reactions and transport steps that are distributed over the chloroplasts, peroxisomes, mitochondria, and cytoplasm of the plant cell. This metabolic recycling process is called photorespiration (PR; Bauwe et al., 2010; Walker et al., 2016). PR is one of the major factors contributing to inefficiency of photosynthetic energy conversion (Fig. 1). First and most importantly, PR recovers only 75% of the carbon contained in 2PG, whereas 25% is lost as CO2, directly counteracting carbon fixation by the CBC. Further, ammonia is released during this process (one molecule per two 2PG molecules assimilated), which needs to be reassimilated at the expense of one molecule of ATP and two reduced ferredoxins per molecule of ammonia. Moreover, redox power is dissipated in the oxidation of glycolate using molecular oxygen. Finally, one ATP is required to convert glycerate into 3PGA by glycerate kinase, the final step in the PR pathway. This brings the tally to 3.5 ATP and two NADPH for the recycling of two molecules of 2PG into one 3PGA. Hence, at 25°C and current atmospheric conditions, approximately one-third of the total ATP and NADPH consumed by a photosynthesizing leaf of a C3 plant is dedicated to the process of PR (see Walker et al. [2016] for details and tools to model energy consumption by PR under different environmental conditions). Figure 1. Open in new tabDownload slide Schematic of native PR and of five synthetic bypasses to this pathway. Native PR is shown by black arrows, where brown arrows highlight especially wasteful reactions. The glycerate pathway (chloroplastic version shown in purple and peroxisomal version in pink) and the glycolate complete oxidation pathways (green arrows) have already been tested in plants, but all result in the release of CO2. The arabinose 5-phospate pathway (blue arrows) is a carbon-conserving route, assimilating glycolate to the CBC without the loss of CO2. Red arrows show a potential pathway that harnesses the low reduction potential in the chloroplast to reduce CO2 to formate, which is then assimilated into the photorespiratory pathway, transforming it into a carbon-fixing route. Figure 1. Open in new tabDownload slide Schematic of native PR and of five synthetic bypasses to this pathway. Native PR is shown by black arrows, where brown arrows highlight especially wasteful reactions. The glycerate pathway (chloroplastic version shown in purple and peroxisomal version in pink) and the glycolate complete oxidation pathways (green arrows) have already been tested in plants, but all result in the release of CO2. The arabinose 5-phospate pathway (blue arrows) is a carbon-conserving route, assimilating glycolate to the CBC without the loss of CO2. Red arrows show a potential pathway that harnesses the low reduction potential in the chloroplast to reduce CO2 to formate, which is then assimilated into the photorespiratory pathway, transforming it into a carbon-fixing route. Under conditions of low CO2 availability (e.g. when stomates close in the light due to low water potential) or at high temperatures, oxygenation rates of Rubisco and hence energy consumption by PR can be much higher. Even in conditions in which energy is not limiting, the oxygenation reaction and the release of CO2 during PR substantially limits the rate of carbon fixation. Thus, although PR performs important functions, such as provision of the amino acid Ser (Benstein et al., 2013) and dissipation of excess excitation energy from chloroplasts to mitochondria (Eisenhut et al., 2017), it also represents a major source of inefficiency that substantially reduces the yield potential of C3 crops (Betti et al., 2016; Walker et al., 2016). It is hence not surprising that many research groups and consortia worldwide set out to improve the efficiency of photosynthetic carbon conversion by attempting to reduce the occurrence of the Rubisco oxygenation reaction or bypass the inefficiencies of PR. Multiple strategies are followed to this end: some are inspired by the diversity of naturally existing carbon concentration mechanisms (CCMs) in plants, algae, and cyanobacteria, while others aim to implement synthetic metabolic routes to supplement or replace the canonical PR pathway (Bar-Even, 2018; South et al., 2018). INCREASING THE CO2 CONCENTRATION AT THE SITE OF RUBISCO Oxygen competes for the enediolate carbanion of RuBP bound to Rubisco. Hence, increasing CO2 concentration at the site of Rubisco would effectively reduce the rate of oxygenation by increasing the rate of carboxylation of the enediolate carbanion (Tcherkez et al., 2006; Savir et al., 2010). Indeed, cyanobacteria, algae, and land plants have independently evolved mechanisms to concentrate CO2 at the site of Rubisco and thereby reduce oxygenation. Cyanobacteria and green algae have evolved a combination of bicarbonate pumps and physical confinement of Rubisco with carbonic anhydrase in close proximity. In angiosperms, a biochemical CO2 pump, so-called C4 photosynthesis, has evolved independently at least 66 times from the ancestral C3 state (Sage et al., 2012). To reduce the rate of the Rubisco oxygenation reaction and thereby the inefficiencies associated with PR, it would be desirable to engineer such CCMs into crops to increase their yield potential. C4 PHOTOSYNTHESIS In C4 photosynthesis, the photosynthetic pathway loses cell autonomy (with few exceptions) and becomes distributed over two distinct cell types, one functioning as a PCA unit and the other as a photosynthetic carbon reduction (PCR) unit. For C4 photosynthesis to function, the PCA and PCR cells are arranged as concentric layers, with each PCA unit having direct cell-to-cell contact with a PCR unit. Together, these two cell types form functional photosynthetic units. The mandatory interaction between PCA and PCR cells requires a characteristic leaf morphology, called Kranz anatomy, in which the vascular bundles (VBs) in leaves are surrounded by concentric layers of bundle sheath and mesophyll cells. Because each PCA cell requires contact with a PCR cell, only four cell layers fit between two VBs, leading to a stereotypic VB-PCR-PCA-PCA-PCR-VB pattern. PCA cells contain little Rubisco activity. Instead, they exhibit high activity of phosphoenolpyruvate carboxylase (PEPC), an enzyme that condenses phosphoenolpyruvate with inorganic carbon to give the C4 acid oxaloacetate, the name-giving initial product of carbon fixation in C4 plants. PEPC reacts with the anion HCO3 −, not CO2. In contrast to Rubisco, this reaction is not sensitive to oxygen. Oxaloacetate is then converted to malate and/or Asp, which diffuse along their concentration gradients to PCR cells, where they are decarboxylated. The decarboxylation of C4 acids in PCR cells increases the local CO2 concentration by approximately 10-fold over the concentration in PCA cells, which is sufficiently high to strongly reduce the rate of PR. Rubisco, which is mostly confined to PCR cells in C4 plants, hence operates under high CO2 concentration, which increases its efficiency and allows for reduced investment into this key protein. The C3 decarboxylation products pyruvate and/or Ala diffuse back to PCA cells, where they are converted to phosphoenolpyruvate, and a new cycle of CO2 transport to PCR cells by C4 acids can begin. The C4 CCM is associated with an increased cost of two ATP per molecule of CO2 assimilated (five ATP and two NADPH per CO2). Hence, the pathway only pays off if the energy costs of PR exceed the extra ATP requirement for running the C4 pump. Further, evolving C4 photosynthesis, as outlined above, requires a special leaf anatomy. Some plant species, for example, the BEP-clade of grasses that contains many important crop species, such as rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare), may lack the genetic prerequisites to evolve the corresponding leaf anatomy (Christin et al., 2013). Thus, introduction of the C4 trait into plant species that may lack the ability to naturally evolve it is a challenging task (Denton et al., 2013; Schuler et al., 2016). Efforts to engineer C4 photosynthesis into C3 plant species were hampered by an incomplete list of genes and gene functions required to support the trait. The prospects massively improved with the advent of next-generation sequencing technologies, which enabled the comparison of gene expression patterns between related C3, C3-C4 intermediate, and C4 species, thereby generating lists of candidate genes involved in setting up C4 leaf anatomy and completing the inventory of genes encoding the metabolic enzymes and solute transporters required to run C4 biochemistry (Wang et al., 2013, 2016; Fouracre et al., 2014; Burgess and Hibberd, 2015; Weber, 2015). Also, computational modeling of the evolutionary trajectory from C3 to C4 photosynthesis provided guidance for the implementation of the C4 trait in C3 backgrounds (Heckmann et al., 2013; Williams et al., 2013; Li et al., 2017). This recent work has indicated that achieving a C3-C4 intermediate state in which PR serves as a mechanism that increases the CO2 concentration in bundle sheath cells (Fig. 2) represents an important stepping stone during C4 evolution, which informs efforts aimed at synthetic experimental evolution of the pathway (Schuler et al., 2016). Figure 2. Open in new tabDownload slide C3-C4 intermediate photosynthesis as a CCM. In C3-C4 intermediate photosynthesis, leaf mesophyll cells lose the activity of the mitochondrial glycine decarboxylase complex (GDC) while this activity is maintained in the bundle sheath cells. Hence, Gly produced as a consequence of PR in mesophyll cells is transported to bundle sheath cells, where it is decarboxylated by GDC in mitochondria. This locally increases the CO2 concentration in bundle sheath cells and promotes efficient fixation by Rubisco in bundle sheath chloroplasts. Ser and/or hydroxypyruvate are transported back to mesophyll cells for completion of the photorespiratory pathway. Figure 2. Open in new tabDownload slide C3-C4 intermediate photosynthesis as a CCM. In C3-C4 intermediate photosynthesis, leaf mesophyll cells lose the activity of the mitochondrial glycine decarboxylase complex (GDC) while this activity is maintained in the bundle sheath cells. Hence, Gly produced as a consequence of PR in mesophyll cells is transported to bundle sheath cells, where it is decarboxylated by GDC in mitochondria. This locally increases the CO2 concentration in bundle sheath cells and promotes efficient fixation by Rubisco in bundle sheath chloroplasts. Ser and/or hydroxypyruvate are transported back to mesophyll cells for completion of the photorespiratory pathway. A recent breakthrough en route to establishing C4 photosynthesis in the C3 crop rice was achieved by ectopic expression of Golden-like (GLK) transcription factor genes from maize (Zea mays; Wang et al., 2017). Transgenic rice lines expressing either ZmGLK1 or ZmG2 displayed increased numbers of differentiated chloroplasts in bundle sheath and mestome sheath cells of rice leaf VBs. Excitingly, increased chloroplast volume was accompanied by increased mitochondrial volume and increased amounts of plasmodesmata in these cell types. This indicates that proliferation of organelle volume and plasmodesmata numbers are emergent properties, triggered by the promotion of chloroplast differentiation through GLK transcription factors. Interestingly, transgenic rice calli that ectopically express ZmGLK1 or ZmG2 accumulate chlorophyll without the addition of cytokinin, indicating that the role of the GATA transcription factors GNC and GNL in regulating chlorophyll biosynthesis and plastid differentiation (Behringer and Schwechheimer, 2015; Cortleven and Schmülling, 2015) could be bypassed by expression of GLK1 or G2. Increased organelle volume in cells surrounding the vasculature is characteristic of proto-Kranz anatomy, which is observed in several plant families that have evolved C3-C4 intermediate and C4 photosynthesis, such as Flaveria or Heliotropium, and is considered an important stepping stone on the evolutionary trajectory from C3 to C4 photosynthesis via intermediary states (Sage et al., 2012; Schlüter et al., 2017). However, to date, proto-Kranz anatomy has not been observed in BEP-clade grasses (Khoshravesh et al., 2016), hence the proto-Kranz-like anatomy observed in transgenic rice lines expressing ZmGLK1 or ZmG2 can be considered an important foundation and anatomical enabler for further engineering of rice toward C4. The next challenges on the way to C4 rice are now the implementation of a C4-like venation pattern (see Sedelnikova et al. [2018] for a recent review) and increasing the activity of enzymes and transporters mediating the C4 biochemistry in a cell-specific manner. CARBOXYSOMES AND PYRENOIDS Cyanobacteria and algae have evolved CCMs that rely on bringing Rubisco in close physical proximity with carbonic anhydrase and on increasing the cellular bicarbonate concentration through bicarbonate and/or CO2 transporters (Kerfeld and Melnicki, 2016; Meyer et al., 2016). In contrast to the C4 photosynthetic CCM outlined above, carboxysomes and pyrenoids do not depend on a specialized leaf anatomy or distribution of the CCM over specialized cell types and should hence be more straightforward to engineer into plant chloroplasts (Long et al., 2016). The understanding of the molecular architecture of the pyrenoid from Chlamydomonas reinhardtii has seen dramatic progress during the past 3 years. Using high-throughput fluorescent protein tagging and affinity purification approaches, together with earlier proteomics work, 86 components of the C. reinhardtii pyrenoid have been identified (Mackinder et al., 2016, 2017). One key finding of this work was the discovery of the protein Essential Pyrenoid Component1, which occurs in equal abundance with Rubisco in the pyrenoid and likely serves to bind Rubisco via four interaction domains, therefore providing a scaffold for the assembly of Rubisco into the pyrenoid lattice. The C. reinhardtii pyrenoid does not behave like a crystalline structure, but is rather like a lipid and is a highly dynamic structure that de novo assembles and disassembles during cell cycle progression (Freeman Rosenzweig et al., 2017). While expression of individual components of the C. reinhardtii CCM did not enhance the growth of Arabidopsis (Arabidopsis thaliana) plants, these recent findings now set the stage for attempts to engineer a functional pyrenoid in plant cells (Atkinson et al., 2016). Cyanobacterial CO2 and bicarbonate transporters increase the concentration of inorganic carbon inside the cytoplasm by 3 orders of magnitude over the external concentration (Mangan and Brenner, 2014). Bicarbonate then diffuses into carboxysomes, self-assembling proteinaceous microcompartments packed with Rubisco and carbonic anhydrase, where it is dehydrated to generate CO2. The low permeability of carboxysomes toward CO2 and O2 further ensures high CO2 concentration and low O2 concentration at the vicinity of Rubisco, thereby reducing and almost eliminating the oxygenation reaction. Using a diffusion-reaction model, it was recently shown that implementation of a carboxysome-type CCM in plant chloroplasts could potentially increase yield by 36% to 60% (McGrath and Long, 2014). Using simulations with individual CCM components, the model predicts a minimum set of components for a carboxysome to be effective in plant chloroplasts. These include knockout of the endogenous plastid-stroma carbonic anhydrase, plus the addition of the cyanobacterial BicA or BCT1 transporters, and/or the NAD(P) dehydrogenase complex1,3. Already the addition of the transporters BicA, BCT1, or SbtA to the plastid envelope membrane alone was predicted to increase the CO2 uptake under light saturation (McGrath and Long, 2014). Targeting of BicA and SbtA to the chloroplast envelope membrane was recently achieved in transiently transformed Nicotiana benthamiana cells (Rolland et al., 2016). Furthermore, production of a Rubisco-containing carboxysome in tobacco (Nicotiana tabacum) chloroplasts that replaces the endogenous tobacco Rubisco was recently achieved (Long et al., 2018), albeit the resulting transgenic plants displayed low growth rates even at 2% CO2, as expected from the modeling approach outlined above (McGrath and Long, 2014). The next challenge will now be to combine the individual components into a functional CCM. BYPASSES TO THE CANONICAL PR PATHWAY The first step of the PR pathway inside the chloroplast stroma is the dephosphorylation of 2PG to generate glycolate. Glycolate is then exported from the chloroplast by a glycolate transporter and imported into the peroxisomes, where it is oxidized to glyoxylate by glycolate oxidase (GOX). Many autotrophic and heterotrophic bacteria possess a dedicated pathway for the metabolism of glyoxylate to 3PGA (Eisenhut et al., 2006). This glycerate pathway proceeds via two reactions, the first condensing two molecules of glyoxylate to tartronate semialdehyde (by glyoxylate carboxylase; EC 4.1.1.47) while releasing CO2, and the second reducing tartronate semialdehyde to glycerate (by tartronate reductase; EC 1.1.1.60). Glycerate is then converted to 3PGA by glycerate kinase. This bacterial glycerate pathway has inspired plant researchers to implement it as a shortcut to the canonical PR pathway. This route prevents the wasteful release of ammonia in mitochondria by the GDC and hence increases the energy efficiency of the pathway (Peterhansel and Maurino, 2011). Furthermore, CO2 is released inside the chloroplasts and not in the mitochondria, thereby increasing local CO2 concentration in proximity to Rubisco and suppressing further oxygenation. The implementation of the glycerate pathway in the chloroplast stroma requires the initial oxidation of glycolate to glyoxylate (Fig. 1, purple arrows). Hence, at least three enzymatic activities are needed inside the chloroplast stroma: GOX, glyoxylate carboxylase, and tartronate reductase. The GOX reaction releases H2O2, which needs to be detoxified, hence a fourth enzyme in the stroma is needed: catalase. Alternatively, glycolate can be converted to glyoxylate by a bacterial glycolate dehydrogenase, which does not generate H2O2. However, the bacterial enzyme (using a still unknown electron acceptor) consists of three subunits, which complicates the engineering task. In a first ground-breaking study, the bacterial glycolate dehydrogenase complex was introduced together with glyoxylate carboxylase and tartronate reductase into Arabidopsis chloroplasts. The resulting lines produced more biomass than the wild type, at least under short-day conditions (Kebeish et al., 2007). In another study, expression of the pathway in the chloroplasts of the biofuel crop Camelina sativa increased vegetative biomass and seed yields by more than 50% and supported faster development (Dalal et al., 2015). Another bypass that completely oxidizes glycolate inside the chloroplast was developed by Maier et al. (2012)(Fig. 1, green arrows). In this pathway, glycolate is oxidized to glyoxylate by GOX and the resulting H2O2 is detoxified by expression of a plastid-targeted catalase. Glyoxylate is then condensed with acetyl-CoA to give malate (by malate synthase, as in the glyoxylate cycle). Finally, malate is oxidized to regenerate acetyl-CoA by the consecutive activities of NADP-dependent malic enzyme and pyruvate dehydrogenase, both of which are present in C3 plant chloroplasts at sufficient amounts to drive this pathway. Overall, this pathway converts all the carbon atoms of glycolate to CO2, which is released inside the chloroplast and reassimilated by Rubisco. Despite being energetically less favorable than the wild-type pathway (12 ATP and eight NADPH needed to reassimilate the four molecules of CO2 released from two molecules of glycolate formed by two oxygenation reactions of Rubisco), Arabidopsis plants expressing this glycolate oxidation pathway displayed increased biomass production under short-day conditions. This is surprising since computational modeling predicted that the photosynthetic rate of plants expressing this pathway would be 31% lower than in wild-type plants (Xin et al., 2015). One explanation for the discrepancy between the model and experimental results is that increased metabolism of glycolate relieves inhibition of Rubisco by this metabolite. However, Nölke et al. (2014) renders this explanation unlikely because lines displaying the highest yield gains also have higher glycolate concentrations than the wild type. In this work, only the three subunits of the Escherichia coli glycolate dehydrogenase (D, E, and F) were expressed in transgenic potato (Solanum tuberosum) plants, leading to increased conversion of glycolate to glyoxylate in chloroplasts. Albeit no enzyme activities for further conversion of glyoxylate were introduced, photosynthetic capacity was enhanced (increased A max at 400 μL L−1 CO2) and tuber yield was increased by 2.3-fold (Nölke et al., 2014). The mechanism underpinning this increased yield remains to be determined. However, it seems that glycolate conversion to glyoxylate by itself suffices to enhance carbon fixation. Supporting this notion, fixation of 14CO2 was doubled in tobacco leaf discs exposed to 5 to 25 mm glyoxylate, indicating that Rubisco’s oxygenation is repressed by glyoxylate (Oliver and Zelitch, 1977). Similarly, CO2 fixation in soybean (Glycine max) mesophyll cells was increased by 150% after incubation with glyoxylate (Oliver, 1980). As an alternative explanation, the growth benefits of the PR bypasses might result from pleiotropic effects, such as a decreased need for Rubisco protein due to higher plastidial CO2 concentration, which would reduce costs for de novo nitrogen assimilation and protein maintenance. In all of these pathways, the metabolism of glycolate inside the chloroplast competes with export of glycolate from chloroplasts by the glycolate exporter PLGG1. As suggested by Weber and Bräutigam (2013), repression of this transporter should increase the flux into the bypasses and thereby increase their efficiency. Indeed, recent work by South et al. (2019) combined the above-described pathways, as well as another novel bypass, with the repression of PLGG1 expression using an antisense RNA in transgenic tobacco lines. They found that this combination reduced export of glycolate from the chloroplast and increased biomass gain as compared with plants in which a bypass was established without suppressing glycolate transport. The most efficient bypass found in this work was a variant of the glycolate oxidation pathway (Maier et al., 2012), in which the H2O2-producing GOX was replaced with a glycolate dehydrogenase from C. reinhardtii mitochondria (South et al., 2019). Hence, this pathway fully decarboxylates photorespiratory glycolate via glyoxylate, malate synthase, and NADP-malic enzyme. Importantly, as noted above, introduction of the photorespiratory bypasses suppressed the photorespiratory phenotype caused by reduced expression of PLGG1. This constitutes strong evidence for the concept that photorespiratory bypasses render the canonical photorespiratory pathway at least partially dispensable. This further opens new avenues for assessing assumptions on the roles of canonical PR beyond detoxification of 2-phosphoglycolate and salvage of CBC intermediates, such as production of Ser (Benstein et al., 2013) or dissipation of excess excitation energy (Eisenhut et al., 2017). We conclude this section by emphasizing that much is yet unknown about the mechanisms that underlie the growth benefits obtained by the PR bypasses and whether they are truly related to the more efficient metabolism of glycolate. It is worth noting that a similar alternative PR route, aimed at bypassing downstream PR by converting peroxisomal glyoxylate to tartronate semialdehyde and hydroxypyruvate (Fig. 1, pink arrows), did not enhance photosynthetic productivity (Carvalho et al., 2011). This casts doubt on the validity of traditional explanations for growth enhancement via PR bypasses. BYPASSING PR WITHOUT CO2 RELEASE A bolder approach to the problem of PR is to try to abolish CO2 release altogether. For example, it was suggested that glyoxylate could be recycled into central metabolism via the activity of the downstream cycle of the prokaryotic 3-hydroxypropionate bicycle (Zarzycki et al., 2009; Shih et al., 2014). The net reaction of this cycle is the conversion of glyoxylate to pyruvate, a key cellular intermediate that can be further metabolized to various compounds. However, pyruvate cannot be easily reassimilated to the CBC because phosphoglycerate mutase and enolase, key enzymes of glycolysis/gluconeogenesis, are usually not expressed in chloroplasts (Prabhakar et al., 2009; Fukayama et al., 2015). While pyruvate can potentially be exported to the cytosol, converted to 3PGA or triose phosphate, and imported back into the chloroplast, the complexity of this route renders it quite unlikely to be useful. Another recent study put forward the synthetic malyl-CoA-glycerate cycle as a way to convert glycolate to acetyl-CoA (Yu et al., 2018). While this pathway could be useful for the biosynthesis of chemicals that originate from acetyl-CoA, it cannot be considered a true PR bypass as acetyl-CoA cannot be easily reassimilated to the CBC. A different photorespiratory bypass was suggested to involve the reduction of 2PG to 2-phosphoglycolaldehyde, which is then condensed with dihydroxyacetone phosphate to give xylulose 1,5-bisphosphate (Ort et al., 2015). The latter intermediate is then dephosphorylated to give the CBC intermediate xylulose 5-phosphate. While the particular metabolism of this route might not be ideal, 2PG is unlikely to accumulate to high enough levels as to enable its reduction and xylulose 1,5-bisphosphate is a potent inhibitor of Rubisco (Bracher et al., 2015), the general structure it suggests could be very efficient. In particular, reduction of glycolate, the cellular concentration of which is in the millimolar range, to glycolaldehyde could be followed by aldol condensation with a phosphor-sugar of the CBC to produce a longer chain phosphor-sugar that is reassimilated to the cycle without carbon loss (Bar-Even, 2018). Recently, a pathway based on the reduction of glycolate to glycolaldehyde was demonstrated in vitro (Trudeau et al., 2018). In this study, the substrate specificities of two enzymes were engineered to enable this reduction: acetyl-CoA synthetase was engineered to efficiently accept glycolate and generate glycolyl-CoA, and propionyl-CoA reductase was engineered to accept glycolyl-CoA as well as NADPH (the native enzyme prefers NADH). These engineered enzymes, which jointly convert glycolate to glycolaldehyde, were then combined with existing enzymes: an aldolase that condenses glycolaldehyde with GAP to give arabinose 5-phosphate, an isomerase that converts arabinose 5-phosphate to ribulose 5-phosphate, and the native kinase that generates RuBP. Together, this enzymatic sequence converted glycolate to Rubisco’s substrate RuBP without the release of CO2 and ammonia (Fig. 1, blue arrows). While the function of this metabolic sequence has been demonstrated in vitro, it awaits testing in plants. RADICAL REDESIGN OF PCA WITHOUT RUBISCO AND THE CBC Instead of fixing PR, other studies suggested replacing Rubisco and the CBC. The first of these studies used a computational approach to systematically identify all pathways that can be established using mix-and-match of existing enzymes from different sources (Bar-Even et al., 2010). Multiple candidate routes were uncovered this way, and one group of pathways, utilizing the most efficient carboxylating enzyme, PEPC (Cotton et al., 2018), was analyzed in detail and predicted to support a higher carbon fixation rate than the CBC. A recent study took a more compelling step toward synthetic carbon fixation, realizing in vitro a synthetic pathway (CETCH pathway) that converts CO2 into the C2 intermediate glyoxylate (Schwander et al., 2016). This pathway was based on the reductive carboxylation of organic acids carrying unsaturated bonds (i.e. crotonyl-CoA and acrylyl-CoA). Yet, while such synthetic carbon fixation routes provide an exciting avenue for further research, it is highly unlikely that we could use one of them to replace the CBC. The CBC seems to be too central in plant metabolism to be replaced, and the establishment of a long cyclic pathway in a plant host is highly challenging. As an alternative strategy, instead of replacing the CBC, we can install a complementary route to assist its activity. This was suggested, for example, for the malyl-CoA-glycerate cycle mentioned above. A particularly interesting option is to use synthetic routes that are based on CO2 reduction rather than carboxylation (Cotton et al., 2018). Specifically, the low reduction potential that persists in the chloroplast could be harnessed to reduce CO2 into formate. Formate can be then assimilated to central metabolism via numerous routes (Bar-Even, 2016), directly contributing to carbon fixation. The advantages of this strategy are its very high energetic efficiency, its minimal overlap with central metabolism, and an implementation that requires a relatively small number of enzymes (Bar-Even, 2018; Cotton et al., 2018). An especially interesting formate assimilation strategy involves attaching this C1 intermediate to tetrahydrofolate and reducing it to 5,10-methylenetetrahydrofolate, which can be condensed with photorespiratory Gly to make Ser (Fig. 1, red arrows). This would avoid Gly decarboxylation and transform PR from being a carbon-negative route into a carbon-positive process (Bar-Even, 2018). CONCLUSION Clearly, reducing the rate of PR by increasing the concentration of CO2 at the site of Rubisco, and rerouting photorespiratory metabolism by introduction of synthetic bypasses and novel pathways that turn PR into a carbon-positive process, hold huge potential for increased yield, without increasing the need for arable land. Even higher yield gains should become possible by combining the engineering of photosynthetic carbon metabolism with recent approaches aimed at more rapid adaptation to fluctuating light conditions by reducing the loss of excitation energy due to overprotection of photosystems (Kromdijk et al., 2016; Slattery et al., 2018). Quoting Norman Borlaug (“Then I wake up and become disillusioned to find that mutation genetics programs are still engaged mostly in such minutiae as putting beards on wheat plants and taking off the hairs.”), we should leave the minutiae behind and reap the full potential of synthetic biology to overcome one of the major challenges of the 21st century, sustainably feeding a growing population without destroying the environment. Open in new tabDownload slide Open in new tabDownload slide LITERATURE CITED Amthor JS ( 2010 ) From sunlight to phytomass: On the potential efficiency of converting solar radiation to phyto-energy . 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Annu Rev Plant Biol 61 : 235 – 261 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the German Federal Ministry of Research (031B0205 and 031B0194), the EU H2020 (CSA CropBooster-P), the Deutsche Forschungsgemeinschaft (EXC 1028), and the Max Planck Society (to A.B.-E.). 2 Author for contact: [email protected]. 3 Senior author. A.B.-E. and A.P.M.W. conceived the concept and wrote the article. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.18.01521 © 2019 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2019. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.