TY - JOUR
AU - Robinson, Colin
AB - Abstract Microalgae are a diverse group of single-cell photosynthetic organisms that include cyanobacteria and a wide range of eukaryotic algae. A number of microalgae contain high-value compounds such as oils, colorants, and polysaccharides, which are used by the food additive, oil, and cosmetic industries, among others. They offer the potential for rapid growth under photoautotrophic conditions, and they can grow in a wide range of habitats. More recently, the development of genetic tools means that a number of species can be transformed and hence used as cell factories for the production of high-value chemicals or recombinant proteins. In this article, we review exploitation use of microalgae with a special emphasis on genetic engineering approaches to develop cell factories, and the use of synthetic ecology approaches to maximize productivity. We discuss the success stories in these areas, the hurdles that need to be overcome, and the potential for expanding the industry in general. Chlamydomonas, downstream processing, metabolic engineering, microalgae, recombinant proteins, transformation. Introduction Microalgae are a large and diverse group of photosynthetic organisms ranging from prokaryotic cyanobacteria to eukaryotic algae spread across many phyla (Guiry, 2012). This diversity offers great potential that has yet to be exploited to any great extent. Microalgae are found in freshwater and marine habitats and produce half of the atmospheric oxygen on earth. The ability to grow autotrophically makes their cultivation potentially simple and cost-effective, and microalgae have attracted increasing interest as sources of natural food additives, cosmetics, animal feed additives, pigments, polysaccharides, fatty acids, and biomass (Hallmann, 2007; Borowitzka, 2013; Leu and Boussiba, 2014). An important aspect of using microalgae for industrial purposes is the GRAS (generally regarded as safe) status of numerous algae. This is essential for products intended for animal or human consumption and could significantly reduce downstream processing costs. Recently, major advances in developing microalgae as biotech platforms have been made; this is especially the case from a genetic engineering perspective. Here we give an overview of state-of-the-art engineering tools, previous successes with recombinant protein expression, and advances made in engineering cyanobacteria for high-value compound production. Furthermore, the largely untapped potential of algae grown in synthetic communities and the challenges associated with downstream processing of microalgae are discussed. High-value natural products in microalgae Microalgae are the source of several forms of high-value compounds such as carotenoids, polyunsaturated fatty acids (PUFAs), proteins, antioxidants, and pigments. Characterized by high protein and nutrient contents, some species such as Arthrospira platensis (a cyanobacterium, also known as Spirulina platensis) and Chlorella vulgaris (a green alga) are used as feed, food additives, and diet supplements (Yaakob et al., 2014). In other cases, specific high-value compounds are isolated from appropriate strains. Relatively few proteins have been purified from microalgae for commercial use, but Spirulina is a rich source of phycocyanin, a protein that constitutes 14% of the dry weight of this cyanobacterium (McCarty, 2007). The US Food and Drug Administration (FDA) has approved phycocyanin from Spirulina as a blue food colorant. Moreover, phycocyanobilin, the tetrapyrrole chromophore of phycocyanin, manifests fluorescent properties that have been exploited for labelling of antibodies in immunofluorescence and flow cytometry. In mammalian tissues, it can be enzymatically reduced to phycocyanorubin (a close homologue of bilirubin) and inhibits the activity of NADPH oxidase, thus reducing the generation of reactive oxygen species. It has been suggested that a regular intake of phycocyanobilin may provide protection against cancer and other diseases (Sørensen et al., 2013). Moreover, recent studies have proven the beneficial health effect of microalgae (Chlorella, Spirulina) by increasing natural killer cell levels and stimulating immune and anti-inflammatory system response in humans (Nielsen et al., 2010; Kwak et al., 2012). Carotenoids are important products that are extracted from microalgae, and indeed the first commercialized product derived from algae was β-carotene. It is produced in very high amounts by Dunaliella salina, a halophilic alga growing in saline habitats, which makes the cultures less susceptible to contamination. What differentiates Dunaliella β-carotene from the synthetic product (present only in the form of all-trans isomer) is that it is rich in the 9-cis isomer, and a negative effect of the use of all-trans isomer, such an effect on plasma cholesterol levels and atherogenesis, has been reported from mice studies (Harari et al., 2008; Borowitzka, 2013). Another example of a carotenoid with a well-established and growing market in the nutraceutical area is astaxanthin from the freshwater green alga Haematococcus pluvialis. Astaxanthin is mainly used as a feed supplement and pigmentation source for salmon and shrimp farming, but due to its high antioxidant properties (10-fold greater than other carotenoids) and protective activities, it has also many applications in the pharmaceutical and cosmetic industries. Astaxanthin has also been shown to prevent bacterial infection, vascular failure and cancer (Ambati et al., 2014). Fatty acids are the other natural components produced commercially from microalgae. Several marine algal species are rich in omega-3 long chain polyunsaturated fatty acids (LC-PUFAs), such as docosahexaenoic acid (DHA; e.g. from Isochrysis strain T-iso and Pavlova lutheri), eicosapentaenoic acid (EPA; e.g. Nannochloropsis gaditana, Nannochloropsis oculata), and alpha-linolenic acid (e.g. Rhodomonas salina, Tetraselmis uecica). Oils from Nannochloropsis, Rhodomonas, and Tetraselmis have higher antioxidant properties than fish oils, partly because of a high content of valuable carotenoids (fucoxanthin, lutein, neoxanthin, alloxantin) and polyphenols. Given the fact that intensive fishing endangers many fish species, algal oils may provide an alternative to fish oils in diets in the future (Ryckebosch et al., 2014). Other algal species such as D. salina and Botryococcus braunii can accumulate up to 60% of storage lipids as triacylglycerides (TAGs), potentially making them a valuable source of oil for biodiesel production (Scott et al., 2010). Compared with plants, algae exhibit higher productivities and theoretically could give 10- to 100-fold higher yields of oil per acre, although such capacities have not yet been achieved on a commercial scale (Greenwell et al., 2010). Nevertheless, algae appear to be a potential solution to the controversial food vs fuel problem that is associated with the use of fertile land to produce plant-derived biofuels. TAGs are not the only way in which microalgae could be exploited for biofuels. Under anaerobic conditions and sulphur depletion, some microalgae produce hydrogen gas, which could be used as an alternative to fossil fuels in the future (Melis et al., 2000; Zhang et al., 2002). Recently, Scoma et al. (2012) have attempted to produce hydrogen from Chlamydomonas reinhardtii using natural sunlight. Although successful, the overall yields did not exceed those obtained in lab-scale settings (Scoma et al., 2012) and so this technology is at an early stage. While the above compounds are well-established microalgal products with known potential, other microalgae are being studied for new compounds with useful properties. Some cyanobacteria have a poor reputation in the popular press for causing toxic blooms, because they are able to produce hepatotoxins and neurotoxins (e.g. anatoxin, jamaicadine, l-beta-N-methylamino-l-alanine) (Aráoz et al., 2010). However, research on cyanobacteria is undergoing a renaissance, because some identified metabolites and their derivatives have been shown to have potential as next generation antiviral (Huheihel et al., 2002), anticancer (Leão et al., 2013), and antibacterial drugs. Several of these drugs have even successfully reached phase II and III clinical trials (Dixit and Suseela, 2013). A large number of promising natural compounds are derived from filamentous marine genera such as Lyngbya, Symploca, and Oscillatoria. Usually they are short peptides built from non-canonical amino acids by the hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) (Tan, 2013). Some of the most promising candidate anticancer agents are derivatives of dolastatin 10, a peptide originally isolated from sea hare Dolabella auricularia. The first one, TZT-1027 (soblidotin) is a microtubule polymerization inhibitor that exhibits antitumour activity in preclinical models, manifesting stronger activity than paclitaxel (Watanabe et al., 2006; Akashi et al., 2007). Another analogue of dolastatin 10 (monomethyl auristatin E) linked to an antibody is already approved by the FDA and used in therapies for patients with Hodgkin lymphoma (Deng et al., 2013). In summary, algae and cyanobacteria are an apparently under-explored source of natural high-value compounds and there is a rising interest in their exploitation. Just as numerous plant secondary metabolites have been used for biotechnological and biomedical purposes, there is huge scope for the identification of correspondingly valuable compounds within the vast microalgal population. New discoveries are made regularly, and new compounds and applications for industrial purposes are to be expected in the near future. Genetic tools in microalgae, and the development of microalgal cell factories As well as containing a range of high-value compounds, microalgae offer real potential as cell factories for the production of other compounds and proteins. With the advancement and availability of algal genome data, transformation protocols have been developed for a number of microalgae and this means that they can now be used to enhance the levels of natural high-value products, or for the expression of genes in order to produce novel products, or recombinant proteins including antibodies, hormones, vaccines, and insecticidal protein at economically viable levels (Hallmann, 2007; Gong et al., 2011). To date, there have been reports of successful genetic manipulation of over 40 different microalgae species including the green algae C. reinhardtii, D. salina, Chlorella vulgaris, and H. pluvialis, and the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana (Walker et al., 2005). However, the genetic toolkits developed for C. reinhardtii and P. tricornutum are the most advanced, and both nuclear and chloroplast transformation has been achieved, so these species will be reviewed as examples. In recent years, there have been rapid developments of genetic tools for microalgae in an attempt to generate more effective microalgal cell factories. Microalgae have several distinct advantages compared with plant-based production and some other bioreactor systems. First, many microalgae can double their biomass in less than 24 hours, with some species such as Chlorella sorokiniana having a doubling time under optimum conditions of less than three hours (Sorokin, 1967). Therefore, only a short period of time is required for large-scale production compared with plants. Furthermore, the time from creation of a new transgenic line to industrial scale-up can be as short as two months (Schmidt, 2004; Mayfield et al., 2007). Second, genes for recombinant proteins can be expressed from the nuclear or chloroplast genomes (León-Bañares et al., 2004), and eukaryotic microalgae possess post-translational modification pathways which allow glycosylated proteins to be produced and secreted out of the cell (Hempel and Maier, 2012; Lauersen et al., 2013a, 2013b). Third, many microalgae can be grown either phototrophically or heterotrophically in enclosed photobioreactors, preventing transgenes from escaping into the environment. This is a particular concern for transgenic plants where transgenic DNA might spread to soil bacteria or to related plant species by means of pollen transfer (Gong et al., 2011). Prior to introducing the desired gene into an algal genome, a few fundamental factors such as DNA delivery method, selection method, and control of gene expression have to be taken into account. DNA delivery method A variety of different methods have been employed for delivering transgenes into algal cells (Table 1), and the method of choice is very much determined by the cell size and nature of the cell wall of the chosen algal species (Stevens and Purton, 1997). The first demonstrations of stable transformation of an alga involved the bombardment of C. reinhardtii cells with DNA-coated microprojectiles (so-called biolistics) and proved successful for the delivery of DNA into both the chloroplast (Boynton et al., 1988) and nucleus (Debuchy et al., 1989). Biolistics was also used to transform the mitochondrial genome of C. reinhardtii (Randolph-Anderson et al., 1993). Table 1. DNA delivery methods for C. reinhardtii and P. tricornutum Organelle  Species  Delivery methods  References  Nucleus  C. reinhardtii  Microparticle bombardment, electroporation, Agrobacterium tumefaciens, glass beads, silicon carbide whiskers, and aminoclay nanoparticles  Boynton et al., 1988; Brown et al., 1991; Dunahay, 1993; Kim et al., 2014; Kindle, 1990; Kumar et al., 2004   Nucleus  P. tricornutum  Microparticle bombardment, electroporation  Apt et al., 1996; Falciatore et al., 1999; Miyahara et al., 2013; Zhang and Hu, 2014   Chloroplast  C. reinhardtii  Microparticle bombardment, glass beads  Boynton et al., 1988; Kindle et al., 1991   Chloroplast  P. tricornutum  Electroporation  Xie et al., 2014   Mitochondria  C. reinhardtii  Microparticle bombardment  Randolph-Anderson et al., 1993   Organelle  Species  Delivery methods  References  Nucleus  C. reinhardtii  Microparticle bombardment, electroporation, Agrobacterium tumefaciens, glass beads, silicon carbide whiskers, and aminoclay nanoparticles  Boynton et al., 1988; Brown et al., 1991; Dunahay, 1993; Kim et al., 2014; Kindle, 1990; Kumar et al., 2004   Nucleus  P. tricornutum  Microparticle bombardment, electroporation  Apt et al., 1996; Falciatore et al., 1999; Miyahara et al., 2013; Zhang and Hu, 2014   Chloroplast  C. reinhardtii  Microparticle bombardment, glass beads  Boynton et al., 1988; Kindle et al., 1991   Chloroplast  P. tricornutum  Electroporation  Xie et al., 2014   Mitochondria  C. reinhardtii  Microparticle bombardment  Randolph-Anderson et al., 1993   View Large Subsequently, the nuclear genome of C. reinhardtii has been genetically transformed using other delivery methods including agitation in the presence of glass beads or silicon carbide whiskers, electroporation, Agrobacterium-mediated transformation, and, more recently, positively charged aminoclay nanoparticles (Kindle, 1990; Brown et al., 1991; Dunahay, 1993; Kumar et al., 2004; Kim et al., 2014). Unlike biolistics or electroporation, methods using glass beads, silicon carbide whiskers, or Agrobacterium do not involve any specialized equipment and can be done in any laboratory at low cost, although silicon carbide whiskers can be difficult to obtain and may potentially be a health hazard (León-Bañares et al., 2004). The glass bead method remains popular, and has also been employed for chloroplast transformation as a simple alternative to biolistics (Kindle et al., 1991; Economou et al., 2014). For nuclear transformation by electroporation, cell wall-less strains (either mutants or cells treated with autolysin) are normally used (Brown et al., 1991; Shimogawara et al., 1998). However, C. reinhardtii with an intact cell wall was recently transformed using a series of multi-electroporation pulses (Yamano et al., 2013). The cheaper Agrobacterium-mediated gene transfer technique can also be used with walled strains and has been reported to give high transformation frequency compared with glass bead transformation (Kumar et al., 2004), and this can be increased further by inducing the Agrobacterium prior to infection using acetosyringone and glycine betaine (Pratheesh et al., 2014). Nuclear transformation using aminoclay nanoparticles (positively charged nanoparticles based on 3-aminopropyl-functionalized magnesium phyllosilicate) is still new, but it has also been reported to give high transformation rates, and again this simple method can be used with wild-type strains possessing a cell wall (Kim et al., 2014). This method might have general applicability to other algal species that have traditionally been transformed using particle bombardment (Walker et al., 2005) since the aminoclay nanoparticles (45nm) are several orders of magnitude smaller than the gold particles (1 μm to 3 μm) used in the bombardment. The size of particles is an important factor, because smaller particles increase the chance of cell wall penetration and genome integration (Kim et al., 2014). The nuclear genome of the diatom P. tricornutum is most commonly transformed via particle bombardment (Apt et al., 1996; Falciatore et al., 1999). Diatoms are generally quite difficult to transform due to challenges such as controlling and manipulating their life cycles, as well as penetrating their rigid cells walls (Falciatore et al., 1999). Nonetheless, recently the P. tricornutum nuclear genome was successfully transformed by electroporation (Miyahara et al., 2013; Zhang and Hu, 2014). More remarkable, given the four membranes that surround a diatom chloroplast, is a recent demonstration that electroporation can be used to deliver DNA into the chloroplast genome (Xie et al., 2014). Chloroplast genome engineering is particularly attractive given the ability to target transgenes into specific, predetermined loci via homologous recombination, and the high levels of expression that can be achieved. To date, chloroplast transformation by biolistics has been reported for five other algal species: Dunaliella tertiolecta, Euglena gracilis, H. pluvialis, Porphyridium sp., and Tetraselmis cordiformis (Purton et al., 2013; Cui et al., 2014). Selectable markers, reporter genes, and promoters Different types of selectable marker and reporter genes can be used in identifying putative transformants from the population of untransformed cells (Table 2). Endogenous selectable markers for nuclear transformation of the haploid C. reinhardtii allow the rescue of auxotrophic or non-photosynthetic mutants with examples for nuclear transformation being ARG7, NIT1, and OEE1 that allow rescue of recessive mutants defective in arginine biosynthesis, nitrate metabolism, and photosynthesis, respectively (Stevens and Purton, 1997). A wide range of dominant markers are commonly used in C. reinhardtii and confer resistance to various antibiotics or herbicides, as detailed in Table 2. For chloroplast transformation, several dominant markers (e.g. aadA and aphA6) have been developed (Table 2). In addition, endogenous chloroplast genes have been used as selectable markers for phototrophic rescue of the corresponding chloroplast mutant: for example, the atpB gene rescues an atpB mutant defective in the ATP synthase. The use of phototrophic markers is not possible for nuclear or chloroplast transformation of wild-type P. tricornutum since this diatom is an obligate phototroph. However, an engineered strain expressing a transgene encoding a glucose transporter has been described that is capable of heterotrophic growth on the sugar (Zaslavskaia et al. 2001), and this potentially could be used to develop non-phototrophic recipient strains. Reporter genes based on fluorescent proteins, luciferases, or colorimetric enzymes have been developed for both the nucleus and chloroplast (Table 2), with the key to good expression of such transgenes being the use of synthetic genes that are codon-optimized for expression in the algal host (Fuhrmann et al., 1999; Zaslavskaia et al., 2000; Purton et al. 2013). Table 2. Common marker and reporter genes used in C. reinhardtii and P. Tricornutum Species  Marker/reporter genes   Description   Reference   C. reinhardtii  Marker genes Chloroplast  atpB  ATP synthase subunit  Boynton et al., 1988   psbH  Thylakoid membrane protein (PSII subunit)  Cullen et al., 2007   aadA  Adenylyl transferase (resistance to spectinomycin)  Goldschmidt-Clermont, 1991   Nucleus  ARG7  Argininosuccinate lyase  Debuchy et al., 1989   NIT1  Nitrate reductase  Kindle et al., 1989   ble  Protein conferring resistance to bleomycin, phleomycin and zeomycin  Stevens et al., 1996   aph7”  Streptomyces hygroscopicus aminoglycoside phosphotransferase  Berthold et al., 2002   bptII  Neomycin phosphotransferase III  Hall et al., 1993   aphVIII  Aminoglycoside 3′phosphotransferase (resistance to paromomycin, kanamycin and neomycin)  Sizova et al., 1996   als  Acetolactate synthase (resistance to sulphonylurea herbicides)  Kovar et al., 2002   cry1-1  Ribosomal protein S14  Nelson and Lefebvre, 1995   oee-1  Oxygen evolving enhance protein  Chang et al., 2003   cat  Chloramphenicol acetyltransferase (resistance to chloramphenicol)  Tang et al., 1995   Reporter genes  gus  β-Glucuronidase  Kumar et al., 2004   gfp  GFP  Kumar et al., 2004   luc  Luciferase  Fuhrmann et al., 2004   chgfp  Modified GFP  Fuhrmann et al., 1999   ars  Arylsulphatase  Davies et al., 1992   P. tricornutum  Marker genes Chloroplast  cat  Chloramphenicol acetyltransferase  Xie et al. 2014   Nuclear  ble  Resistance to bleomycin, phleomycin and zeomycin  Apt et al., 1996; Zaslavskaia et al., 2001   nptII  Neomycin phosphotransferase III    hpt  Hygromycin B phosphotransferase    nat/sat1  Nourseothricin resistance    cat  Chloroamphenicol acetyltransferase (resistance to chloroamphenicol)    Reporter genes  egfp  Modified GFP  Zaslavskaia et al., 2001, 2000   glut1  Glucose transporter    hup1  Hexose transporter    Species  Marker/reporter genes   Description   Reference   C. reinhardtii  Marker genes Chloroplast  atpB  ATP synthase subunit  Boynton et al., 1988   psbH  Thylakoid membrane protein (PSII subunit)  Cullen et al., 2007   aadA  Adenylyl transferase (resistance to spectinomycin)  Goldschmidt-Clermont, 1991   Nucleus  ARG7  Argininosuccinate lyase  Debuchy et al., 1989   NIT1  Nitrate reductase  Kindle et al., 1989   ble  Protein conferring resistance to bleomycin, phleomycin and zeomycin  Stevens et al., 1996   aph7”  Streptomyces hygroscopicus aminoglycoside phosphotransferase  Berthold et al., 2002   bptII  Neomycin phosphotransferase III  Hall et al., 1993   aphVIII  Aminoglycoside 3′phosphotransferase (resistance to paromomycin, kanamycin and neomycin)  Sizova et al., 1996   als  Acetolactate synthase (resistance to sulphonylurea herbicides)  Kovar et al., 2002   cry1-1  Ribosomal protein S14  Nelson and Lefebvre, 1995   oee-1  Oxygen evolving enhance protein  Chang et al., 2003   cat  Chloramphenicol acetyltransferase (resistance to chloramphenicol)  Tang et al., 1995   Reporter genes  gus  β-Glucuronidase  Kumar et al., 2004   gfp  GFP  Kumar et al., 2004   luc  Luciferase  Fuhrmann et al., 2004   chgfp  Modified GFP  Fuhrmann et al., 1999   ars  Arylsulphatase  Davies et al., 1992   P. tricornutum  Marker genes Chloroplast  cat  Chloramphenicol acetyltransferase  Xie et al. 2014   Nuclear  ble  Resistance to bleomycin, phleomycin and zeomycin  Apt et al., 1996; Zaslavskaia et al., 2001   nptII  Neomycin phosphotransferase III    hpt  Hygromycin B phosphotransferase    nat/sat1  Nourseothricin resistance    cat  Chloroamphenicol acetyltransferase (resistance to chloroamphenicol)    Reporter genes  egfp  Modified GFP  Zaslavskaia et al., 2001, 2000   glut1  Glucose transporter    hup1  Hexose transporter    View Large For transgene expression in the nucleus or the chloroplast, promoters and untranslated regions (UTRs) from highly expressed endogenous genes are typically used. For nuclear expression these include genes for subunits of the photosynthetic complexes, ribulose bisphosphate carboxylase, or components of the light-harvesting apparatus (Walker et al., 2005), and for chloroplast expression, genes for core photosynthetic subunits such as atpA, psbA, psaA, and psbD (Purton, 2007). However, such chloroplast genes are typically under feedback control via trans-acting factors that bind to the 5′ untranslated region (UTR). Recently, (Specht and Mayfield, 2013) have shown that re-engineering these 5′UTRs can overcome such control and result in elevated levels of transgene expression. With the advancement of microalgal biotechnology, many tools and techniques for the genetic manipulation of microalgae have been developed. However, nuclear transformation still faces problems such as poor transgene integration, codon bias, positional effects, and gene silencing which can lead to poor or unstable transgene expression. Moreover, there are still limited molecular genetic tools for many of the microalgal species that have high commercial value. Chloroplast transformation is a promising strategy for precise engineering and high-level expression of transgenes, but its application is still largely limited to C. reinhardtii, and again there is a pressing need to develop reliable methods for commercial species. Hence, more work has to be carried out so that microalgae can be exploited as light-driven bioreactors in the future. Production of recombinant proteins in microalgae Several previous studies have shown that microalgae are promising production platforms for recombinant proteins. Major successes and recent advances in recombinant protein production are summarized in the following. Monoclonal antibodies are complex molecules used to treat several human diseases. To date most antibodies are produced in mammalian cells due to their ability to carry out post-translational modifications, particularly glycosylation and disulphide bond formation. Recently, efforts have also been directed towards producing them in bacteria and yeast (Spadiut et al., 2014). The first expression of a functional antibody in microalgae was achieved in the chloroplast of C. reinhardtii. A large single chain antibody directed against the herpes simplex virus (HSV) glycoprotein D was successfully expressed in this green alga. The genetic construct was integrated into the chloroplast genome via homologous recombination and expression was driven by atpA or rbcL promoters and 5′ UTRs. The antibodies were shown to accumulate as soluble proteins in the chloroplast and could bind to HSV proteins in ELISA experiments. Heavy and light chains assembled by forming disulphide bonds, however, no other post-translational modifications were detected (Mayfield et al., 2003). In another study, a fully functional monoclonal antibody against anthrax protective antigen 83 (PA83) was expressed in the C. reinhardtii chloroplast. Separate constructs for the heavy and light chain were introduced and the antibody was shown to assemble spontaneously by forming the necessary 16 disulphide bonds (Tran et al., 2009). The production of antibodies in microalgae is, however, not restricted to Chlamydomonas. A monoclonal antibody was also successfully expressed in the endoplasmic reticulum (ER) of the diatom P. tricornutum. It was directed against the Hepatitis B virus surface protein and accumulated to 8.7 % of total soluble protein. Heavy and light chains, retained in the ER by DDEL retention peptides, were shown to assemble into complete antibodies and were glycosylated (Hempel et al., 2011). When the same group omitted the ER retention signal from their constructs, functional antibodies were efficiently secreted and accumulated in the culture medium in an active form (Hempel and Maier, 2012). Another group of valuable therapeutics are immunotoxins, which are most commonly used in the treatment of cancer. Immunotoxins are antibodies coupled to eukaryotic toxins and are difficult to produce in both prokaryotic and eukaryotic hosts. Tran et al. (2012) were able to show that fully functional immunotoxins can be expressed in algal chloroplasts. An antibody directed against the B-cell surface epitope CD22 was genetically linked to Exotoxin A from the human pathogenic bacterium Pseudomonas aeruginosa and expressed in the chloroplast of C. reinhardtii. It was shown that the immunotoxins were enzymatically active, bound specifically to CD22 on B-cells, and inhibited cell proliferation in vitro. Tests in mouse xenograft models showed reduced tumour progression (Tran et al., 2012). In another study the successful expression of an immunotoxin comprising an antibody recognizing B-cell CD22 coupled to gelonine—a ribosome inhibiting protein—in the C. reinhardtii chloroplast was demonstrated. The immunotoxin was shown to inhibit the survival of two B-cell lymphoma lines (Ramos and CA-46) while leaving Jurkat T-cells intact, thereby proving their specificity (Tran et al., 2013). Several vaccine antigens have been produced in the chloroplast of C. reinhardtii (Sun et al., 2003; He et al., 2007; Demurtas et al., 2013). The GRAS status of the alga makes the production of antigens without costly purification from residual toxins or impurities attractive and could even be exploited for oral delivery of vaccines. An algal-produced Staphylococcus aureus vaccine was shown to protect orally vaccinated mice against sub-lethal and lethal doses of S. aureus infection (Dreesen et al., 2010). Vaccines blocking malaria transmission were also successfully expressed in the C. reinhardtii chloroplast (Gregory et al., 2012; Jones et al., 2013). In addition, Bayne et al. (2013) showed that it is possible to produce influenza haemagglutinin (HA) in the heterotrophic alga Schizochytrium sp. thereby presenting an alternative to current egg-based vaccine production platforms (Bayne et al., 2013). Other proteins expressed in microalgae include mammary-associated serum amyloid (M-SAA), which could be used as viral and bacterial prophylaxis in newborn mammals (Manuell et al., 2007); domains 10 and 14 of human fibronectin, which have potential as antibody mimics; proinsulin and vascular endothelial growth factor, as well as the high mobility group protein B1 involved in wound healing (Rasala et al., 2010). Furthermore, a human selenoprotein, Sep15 (Hou et al., 2013) and antitoxins against botulinum neurotoxin have been successfully produced (Barrera et al., 2014). Further efforts to develop microalgae as a production platform have been made. Other than the therapeutic proteins mentioned, several enzymes have been successfully produced in C. reinhardtii. The industrially relevant Xylanase 1 was expressed in the nucleus of C. reinhardtii both in a soluble and secreted form (Rasala et al., 2012). The first membrane-bound enzyme expressed in the C. reinhardtii chloroplast was CYP79A1 from Sorghum bicolor. It was shown to be active and produced p-hydroxyphenylacetaldoxime, the precursor for a plant natural product, from endogenous tyrosine (Gangl et al., 2015). In another study, a bifunctional diterpene synthase was expressed in the chloroplast of C. reinhardtii, and is the largest recombinant protein expressed to date. This synthase is of interest for exploring the potential of terpenoid production in microalgae (Zedler et al., 2014; see next section for details). Despite the huge variety of recombinants successfully produced in algae to date, only one report exists of transferring recombinant protein production to a larger scale. A milk amyloid A-producing strain of C. reinhardtii was grown in three 100 l bags in a greenhouse setting and the maximum rate of MAA production achieved was 0.051±0.016g/l/d (Gimpel et al., 2014). The lack of other examples shows that the knowledge transfer from the lab scale to industrially relevant growth conditions for recombinant protein production in microalgae is still not established. Metabolic engineering of microalgae to produce other high-value compounds Microalgae offer additional potential as light-driven cell factories for the production of novel metabolites. Plant secondary products are one of the most important forms of target compound. Plants are a source of a broad spectrum of diverse bioactive compounds, also known as secondary or specialized metabolites. Often their biosynthesis is restricted to specific developmental stages, tissues or even cells and they can be involved in many processes important for plant growth and survival, such as protection from pathogens or herbivores, attraction of pollinators, and adaptation to environmental stress. A large number of specialized metabolites are now used by the pharmaceutical, chemical, and food industries, and approximately 50% of all approved medicines including anticancer drugs are of natural origin (Dai et al., 2014; Lassen et al., 2014b). Many of these plant-derived compounds can be produced by chemical synthesis, but in some cases due to complexity of their structure they require difficult multistep regio- and stereospecific reactions, therefore overall yields can be very low. This applies particularly to the synthesis of terpenoids—a particularly large, complex, and important family of plant secondary metabolites. One solution to this problem can be extraction from the plant source, but if a product is accumulated in scarce amounts, the result may not be economically viable. An example is paclitaxel, a cytostatic drug known under the commercial name Taxol. Its accumulation in Taxus baccata depends on the maturity of the tree and ranges from 0.064g (27-year-old tree) to 8.038g (136-year-old tree) per tree (Nadeem et al., 2002), whereas chemical synthesis is complicated and requires 40 different steps (Nicolaou et al., 1994). Towards engineering metabolic pathways An alternative method for production of high-value chemicals is to use synthetic biology or metabolic engineering tools. Both terms are often used interchangeably, however, there are significant differences between the two fields. Synthetic biology is more about designing and using defined synthetic DNA parts (biobricks), constructing genetic circuits and molecular switches to control the expression and metabolism, whereas metabolic engineering is a broader term involving protein engineering and pathway optimization in order to improve production yields of the desired product (Stephanopoulos, 2012). Nonetheless, both are understood as transferring biosynthetic pathways from one organism to another (or combining pathways from different organisms). In order to produce some specialized high-value compounds such as terpenoids, it is often necessary to perform highly specific and complex enzymatic reactions, which can be catalysed by cytochrome P450s (P450s). For example, biosynthesis of both artemisinin and paclitaxel requires the involvement of P450s. Unfortunately the expression of plant P450s in Escherichia coli and Saccharamoyces cerevisiae is not always simple due to special requirements for post-translational modification and protein localization, and in consequence plant P450s are often inactive upon expression in these hosts (Chemler and Koffas, 2008). P450s are monooxygenases performing stereospecific hydroxylations and they are anchored in the plant ER. In order to function they need to be powered by single electron transfers from NADPH-dependent cytochrome P450 reductase (CPR). The expression level of these proteins is relatively low and the activity is often limited by NADPH and the substrate pool (Jensen et al., 2011). It has been demonstrated that these energy-consuming reactions can be bypassed by relocating P450-dependent metabolic pathways to the thylakoid membranes of tobacco chloroplasts, where the reducing power generated by photosystem I (PSI) in the form of reduced ferredoxin (Fd) is a cheap and essentially unlimited source of electrons for the P450s (Nielsen et al., 2013) (Fig. 1). More recently, it has been shown that a P450 can be expressed in the chloroplasts of transgenic C. reinhardtii and it was furthermore shown that the enzyme was targeted into the chloroplast membranes and was highly active (Gangl et al., 2015). This strongly suggests that it will be possible to produce pharmacologically important terpenoids not only in plant chloroplasts (Bock and Warzecha, 2010), but also in microalgal chloroplasts. Fig. 1. View largeDownload slide Relocation of cytochrome P450s from the ER into the thylakoid membranes for coupling with photosynthetic electron flow. Most cytochromes P450 function in the ER where they interact with specific enzymes (cytochrome P450 reductases) that provide the reducing power to drive complex substrate modifications. It has now been shown that these key enzymes can be expressed in the chloroplasts of plants, algae, and cyanobacteria, where the P450s are active and able to use reducing power from ferredoxin. This provides a novel route for the formation of complex metabolites involving P450 enzymes. Fig. 1. View largeDownload slide Relocation of cytochrome P450s from the ER into the thylakoid membranes for coupling with photosynthetic electron flow. Most cytochromes P450 function in the ER where they interact with specific enzymes (cytochrome P450 reductases) that provide the reducing power to drive complex substrate modifications. It has now been shown that these key enzymes can be expressed in the chloroplasts of plants, algae, and cyanobacteria, where the P450s are active and able to use reducing power from ferredoxin. This provides a novel route for the formation of complex metabolites involving P450 enzymes. Cyanobacteria as a tool for synthetic biology During the last decade, cyanobacteria have been gaining renewed interest as a chassis for metabolic engineering and synthetic biology approaches. Currently many efforts are being undertaken to use genetically modified cyanobacteria as a production platform for biofuels such as isoprene (Zhou and Li, 2010), bioplastics like polyhydroxybutyrate (Wang et al., 2013) and polylactic acid (Angermayr et al., 2014), fatty acids (Ruffing, 2014), ethylene (Guerrero et al., 2012; Ungerer et al, 2012), and sugars (Jacobsen and Frigaard, 2014). In many cases reported yields are not yet satisfactory, however, the ability to perform photosynthesis and the idea of converting sunlight and carbon dioxide into high-value chemicals are undoubtedly attractive. As indicated above, expression of foreign pathways involving P450s in E. coli often causes problems and is ineffective, due to difficulties with proper folding, post-translational modifications, and targeting to the membranes. Unlike most native bacterial P450 enzymes that are soluble, cyanobacterial P450s are membrane bound, exactly like plant P450s (Robert et al., 2010). All the above considerations indicate that cyanobacteria and eukaryotic microalgae can be suitable candidates for expression of foreign P450s. Expression of p-coumarate-3-hydroxylase from Arabidopsis thaliana in the cyanobacterium Synechococcus sp. PCC 6803 for the production of caffeic acid was the first reported successful expression of a plant cytochrome P450 in cyanobacteria (Xue et al., 2014). Further genetic strain modifications resulted in a 25-fold increase of production rate of p-coumaric acid (a precursor for caffeic acid) giving yields of 82.6mg/l secreted to the media (Xue et al., 2014). Another example of stable expression of a P450 enzyme (CYP79A1 from the dhurrin pathway) in Synechococcus sp. PCC 7002, and direction of the protein to the thylakoid membranes by fusing the protein with the PsaM subunit of PSI, has recently been reported (Lassen et al., 2014a). Successful heterologous expression of the mevalonic acid (MVA) pathway in Synechocystis sp. PCC 6803 (Bentley et al., 2014) and production of β-phellandrene (Bentley et al., 2013) also opens up the opportunity to express terpenoid biosynthetic pathways in cyanobacteria. Moreover, excretion of produced metabolites into the growth media (Lassen et al., 2014a; Xue et al., 2014) suggests that cyanobacteria have an unexplored potential as good platforms for metabolic engineering. Overall, it is clear that these organisms have potential as production hosts but it is fair to say that improvements are required before they can attain mainstream status as production chassis. Techniques to engineer algal communities In their natural environment, algae live intimately with many other organisms. These communities exhibit complex interactions including metabolite exchange, cell aggregation, and biofilm formation (Jagmann and Philipp, 2014). For example, at least half of all algal species require an exogenous supply of vitamin B12, and bacteria can be a source for this co-factor in return for a carbon source, thus forming a tightly regulated symbiosis (Croft et al., 2005; Kazamia et al., 2012). Further examples include the symbiosis between a unicellular prymnesiophyte alga and a cyanobacterium (UCYN-A) with the former providing a carbon source in return for fixed nitrogen (Thompson et al., 2012), and the dynamic symbiosis between the haptophyte alga Emiliania huxleyi and the bacterium Phaeobacter gallaeciensis BS107, which can be mutualistic or parasitic according to environmental conditions (Seyedsayamdost et al., 2011). In contrast, algal biotechnology has traditionally involved large-scale cultivation of axenic cultures (Scott et al., 2010; Day et al., 2012). This difference has been widely recognized and consequently there are many reviews outlining the potential benefits of using consortia in algal biotechnology. The main advantages proposed include an increase in algal productivity (accrued biomass) of mixed cultures compared with axenic cultures, enhanced crop protection from pathogens and pests due to the competitive exclusion principle, reduction of energy inputs, increased stability of the crop to include population control and resilience to environment perturbations, and finally, the division of labour between consortium members leading to increased ability to engineer complex and broader metabolic pathways (Table 3) (Ortiz-Marquez et al., 2013; Jagmann and Philipp, 2014; Kazamia et al., 2014; Nalley et al., 2014; Pandhal and Noirel, 2014; Smith and Crews, 2014). Table 3. Key benefits of engineering algal communities for biotechnological exploitation Benefit  Reasoning   Reference   Improved algal productivity  Resource-use complementarity is when algal species with different growth requirements are grown together; the competition for resources between the organisms is lowered allowing the individual species to cohabit, thus increasing net biomass of the culture.  Kazamia et al., 2014; Nalley et al., 2014; Smith and Crews, 2014   Enhanced crop protection  The competitive exclusion principle is when a culture contains a diverse array of organisms. Resources or niches are therefore occupied leading to an increased ability to competitively exclude invaders.  Kazamia et al., 2012; Nalley et al., 2014; Smith and Crews, 2014   Reduction of energy inputs  The use of organisms to provide scarce or expensive resources such as vitamin B12 or fixed nitrogen into the media.  Kazamia et al., 2014; Ortiz-Marquez et al., 2013   Increased stability and resilience  Stability of communities can be defined as the minimal population fluctuation despite a disturbance, and resilience can be defined as the ability for a community to revive after a disturbance. Increasing algal diversity and richness has been shown to improve the stability and resilience of algal cultures.  Nalley et al., 2014   Increasingly broad and complex engineering of metabolic pathways  Microbial communities engineered to produce valuable products have many advantages due to the division of labour. The ability to compartmentalize complex pathways into a number of strains could lead to optimized functionality and lowering metabolic burden on any one cell.  Jagmann and Phillip, 2014  Benefit  Reasoning   Reference   Improved algal productivity  Resource-use complementarity is when algal species with different growth requirements are grown together; the competition for resources between the organisms is lowered allowing the individual species to cohabit, thus increasing net biomass of the culture.  Kazamia et al., 2014; Nalley et al., 2014; Smith and Crews, 2014   Enhanced crop protection  The competitive exclusion principle is when a culture contains a diverse array of organisms. Resources or niches are therefore occupied leading to an increased ability to competitively exclude invaders.  Kazamia et al., 2012; Nalley et al., 2014; Smith and Crews, 2014   Reduction of energy inputs  The use of organisms to provide scarce or expensive resources such as vitamin B12 or fixed nitrogen into the media.  Kazamia et al., 2014; Ortiz-Marquez et al., 2013   Increased stability and resilience  Stability of communities can be defined as the minimal population fluctuation despite a disturbance, and resilience can be defined as the ability for a community to revive after a disturbance. Increasing algal diversity and richness has been shown to improve the stability and resilience of algal cultures.  Nalley et al., 2014   Increasingly broad and complex engineering of metabolic pathways  Microbial communities engineered to produce valuable products have many advantages due to the division of labour. The ability to compartmentalize complex pathways into a number of strains could lead to optimized functionality and lowering metabolic burden on any one cell.  Jagmann and Phillip, 2014  View Large Although the proposed benefits of creating algal communities are great, engineering the communities in practice is likely to be complex. We therefore focus on examples where engineering of efficient algal communities has been shown to be effective. Genetic engineering for symbiosis Genetic manipulation can be used to engineer organisms to interact in a clearly defined way by the trading of metabolites. Examples of this include engineering the nitrogen-fixing bacterium Azotobacter vinelandii to secrete fixed nitrogen into the growth media, replacing the need for addition of synthetic nitrogen inputs for algal growth (Ortiz-Marquez et al., 2012; Ortiz-Marquez et al., 2014), and the engineering of the cyanobacterium Synechocystis sp. PCC 6803 to secrete a carbon source for E. coli which it can utilize to produce low- to high-value compounds (Niederholtmeyer et al., 2010). The benefits of using one organism to provide nutrients for another include the potential to lower production costs (nutrient inputs and energy consumption) and reduce the carbon footprint of the process. However, creating these communities may require considerable investment of time and resources, limiting the size to a small number of interactions therefore limiting the benefit of the competitive exclusion principle (Table 3). Given the limited ability to exclude contaminants, this strategy, if applied on its own, would be best suited for contained cultures (with low risk of contamination) for production of high-value products in contrast to cultures exposed to the environment (for low-value product) where contaminants will easily occupy niches and even take advantage of the engineered community. Screening symbioses Screening of naturally developing populations can offer a simpler method of identifying mutually beneficial symbioses from a large number of organisms. For example, Do Nascimento et al. (2013) showed an increase of up to 30% in chlorophyll, biomass, and lipid accumulation in the oleaginous microalga Ankistrodesmus sp. strain SP2-15 when co-cultured with the bacterium Rhizobium strain 10II (Do Nascimento et al., 2013). The positive interaction was identified by subculturing non-axenic algal cultures (without added organic carbon) for three years, which was followed by the isolation of bacterial strains able to utilize carbon exudates from the algal strains. The bacteria were then screened for induction of positive growth effects on various algal strains via growth curves. Le Chevanton et al. (2013) used a similar approach where bacteria were isolated from algal cultures (Le Chevanton et al., 2013). However, the cultures were initially screened with a high-throughput optical technique to identify interactions that gave the highest chlorophyll a fluorescence. Le Chevanton et al. thus identified two bacterial strains enhancing biomass accumulation and nitrogen provision for Dunaliella sp. The final example given here highlights the importance of screening a diverse range of organisms for symbiosis. Lorincz et al. (2010) serendipitously created an artificial tripartite consortium based on the known bipartite symbiosis involving C. reinhardtii (alga) trading carbon to A. vinelandii (bacterium) in exchange for nitrogen. Alternaria infectoria (fungus) had spontaneously contaminated the co-culture, which led to a positive growth effect by providing amino acids (in particular cystathionine) to the consortium (Lőrincz et al., 2010). Analysis of naturally developing populations is therefore advantageous in its potential for screening of a large range of interactions. However, significant investment in time, materials, and manpower may be required for success. Environmental selection for symbiosis A novel approach to form communities is through manipulation of the environmental conditions to force organisms to form symbioses. For example, Hom and Murray (2014) created an obligate mutualism between the yeast S. cerevisiae and C. reinhardtii by testing a range of different conditions to identify those that allowed both organisms to grow. Under the successful conditions, S. cerevisiae provided carbon dioxide by metabolizing glucose and in return C. reinhardtii provided ammonia by metabolizing nitrite. The authors reproduced this mutualism with many additional algal and yeast species, which gives a sense of optimism about applying this technique to a range of other organisms (Hom and Murray, 2014). Similarly Ortiz-Marquez and colleagues created an artificial symbiosis between a strain of A. vinelandii engineered to secrete fixed nitrogen (ammonium) and the alga Chlorella sorokiniana by co-culturing the species in nitrogen- and carbon-deficient media (Ortiz-Marquez et al., 2012). An important similarity between these two examples was the requirement of a solid support for the symbiosis to occur (i.e. a lack of agitation or shaking), outlining the importance of cell-cell proximity and spatial structure. It should be noted that this approach is new and only a few positive examples have been reported, so it remains to be seen how widely the approach can be applied. It also remains to be seen how robust symbioses generated in this way will be. Trait-based engineering Trait-based engineering is the creation of communities by organisms with unique but complementary growth requirements. This is based on the principle of resource-use complementarity, which allows, in principle, individuals to cohabit and lead to an increase in productivity. Examples of trait-based complementarity include the co-culturing of algal species with varying accessory light-harvesting pigments maximizing the utility of the visible light spectrum (Kazamia et al., 2014; Nalley et al., 2014). Trait-based engineering could be applied to make use of the varying temperatures that occur during a 24-h cycle as well as seasonal fluctuations, by co-culturing organisms that optimally grow at a range of temperatures, such as Detonula confervacea with a temperature optimum of 11 °C to Chlorella pyrenoidosa, with a temperature optimum of 40 °C (Eppley, 1985; Myers, 1984). Another feature that could be considered in this context is the introduction of species that control organisms such as zooplankton, which are known for their predation on algal species. Kazamia et al. (2014) and Nalley et al. (2014) outlined the use of fish as a means to control zooplankton (Kazamia et al., 2014; Nalley et al., 2014). Trait-based engineering has particular benefits for open-raceway production, where algal species are exposed to fluctuating environmental conditions. However, this approach will require a thorough understanding of the surrounding environment of the raceway pond to be able to handpick organisms for optimal community growth. Directed evolution Another approach is to take advantage of artificial selection as a means of creating efficient co-cultures. Selecting for the ability of organisms to work together over many generations may be an efficient and productive means of achieving certain traits from mixed communities. This approach has been applied to fermentation, anaerobic digestion, bioremediation, and the production of polyhydroxyalkanoate and polyphosphates (Zeng et al., 2003; Serafim et al., 2008; Johnson et al., 2009; Sabra et al., 2010). Mooij et al. (2013) used this method to select for carbon storage molecules in the form of starch and lipids in algae. Algal inocula were taken from several different surface waters and cultured in a carbon dioxide-rich light period (nitrogen absent) and then a nitrogen-rich dark period. Cycling between these two conditions over many generations selected for organisms able to produce energy storage compounds (starch/lipids) to power nitrogen assimilation in the dark periods (Mooij et al., 2013). This particular example, due to the controlled environmental conditions (nitrogen cycling), may be best utilized with photobioreactors. However, directed evolution could be applied for production of algae in open-raceway ponds for the selection of resistance to extreme conditions such as high pH or salt concentrations as applied to the production of Arthrospira sp. and D. salina, respectively. In summary, metabolic engineering has the advantages of precisely engineering metabolite trading as well as the ability to fine tune population dynamics (You et al., 2004; Kerner et al., 2012). However, it is labour intensive as a way of creating symbiotic interactions and obtaining regulatory approval for genetically modified organisms may prove a barrier. The metabolic engineering approach is further limited to organisms whose genomes are readily manipulated. Screening and environmental selection for symbiosis has the advantage of using a potentially large range of organisms to form symbioses, and trait-based engineering and directed evolution approaches offer the potential to create highly complex communities with improved productivity. However, these techniques are in their infancy and further evidence of their efficacy is required. Algal community research has made great progress in recent years and a number of additional technologies will be utilized alongside the engineering approaches discussed above. Some of these emerging technologies include the monitoring of algal populations through the use of quantitative PCR; crop protection using traditional chemicals such as pesticides and herbicides (McBride et al., 2014); the highly parallel screening of beneficial and novel microbial symbiotic interactions through the use of microdroplet technology (Park et al., 2011); development of online databanks or libraries detailing functional traits of bacteria and algae so communities can be designed for optimum trait functionality (Guiry et al., 2014); and accurate modelling of communities to help predict environments that induce and stabilize microbial interactions (Kim et al., 2008; Klitgord and Segre, 2010; Grant et al., 2014). Downstream processing of microalgal products As described above, microalgae are light-driven cell factories that can produce a wide spectrum of natural products, or which can be engineered to produce diverse high-value compounds. Extraction and purification of these products are critical processes that can contribute to up to 60% of the total production cost (Molina Grima et al., 2003). Research to date has focused on developing methods for downstream processing of algal biomass and for oil extraction and biofuel production. Many have reviewed downstream processing for biofuel production from different algae including Chlorella, Dunaliella, Nannochloris, Nannochloropsis, Porphyridium, Schizochytrium, and Tetraselmis (Mata et al., 2010; Chen et al., 2011; Halim et al., 2012; Lee et al., 2012; Kim et al., 2013; Pragya et al., 2013; Rawat et al., 2013; Rios et al., 2013; Ahmad et al., 2014). In this section, we review the common steps for processing microalgae in order to prepare biofuels, recombinant proteins and other high-value products. Downstream processing methods for biofuels production Typically, downstream processing of microalgae used for the production of biodiesel consists of harvesting in a two-step operation in order to separate the biomass from the culture media using solid–liquid separation technologies. The first step is bulk harvesting where the biomass is concentrated by flocculation, flotation, or gravity sedimentation reaching up to 7% total solids; the second step is thickening where the biomass slurry is concentrated into a paste by more energy-intensive processes like centrifugation or filtration (Brennan and Owende, 2010). Such technologies are chosen according to the characteristics of the microalga species and the nature and quality of the final product (Amaro et al., 2011), however, they are limited in their abilities to separate biomass from the media and the operating cost that can fluctuate between 20% and 30% of the total production cost (Gudin and Therpenier, 1986). From this point, the harvested biomass paste can undergo two different pre-treatments depending on the final product requirements, the so-called dry and wet route. The dry route, which involves technologies such as spray drying, drum drying, freeze-drying, or sun drying, is the preferred method to obtain dry biomass as it offers high extraction yields, albeit with high costs and energy use [e.g. harvesting and drying combined with extraction cost is 50% of the total production cost (Pragya et al., 2013)]. An alternative to the dry route is the wet route, in which wet biomass needs to be disrupted first to release the intracellular products. Cell wall composition plays an important role in this route; cell walls from microalgae are typically a thick and rigid matrix of polysaccharides and glycoproteins that require costly downstream processing steps during the production of bio-products (Kim et al., 2013). For example, H. pluvialis possesses a thick cell wall that makes this alga highly resistant to chemical and physical disruption, thus significantly increasing astaxanthin production costs (Hagen et al., 2002). The unit operations for the wet route can be: mechanical (ultra-sonication, high pressure homogenization, microwave, bead beating, and electroporation); chemical (acids, alkalis, and organic solvents); biological (enzymes); or osmotic shock. Selection of methods depends again on the biomass characteristics. Assessment of the dry and wet routes has demonstrated that both have a positive energy balance for production of biofuels. This evidence also shows that the wet route has more potential for high valuable biofuels whereas the dry route seems more attractive for short-term biofuel productions (Xu et al., 2011). Whether the biomass goes into the dry or wet route, the physical state of the output is a dried biomass or disrupted concentrate; this stream is processed for lipid extraction. Different methods are available including: (i). Organic solvents: algal oil can be recovered by using solvent such as chloroform, methanol, benzene, diethyl ether, and n-hexane. Other metabolites such as β-carotene and astaxanthin are also extracted by solvents (Molina Grima et al., 2003). However, solvent extraction can result in high toxicity if the product is used for animal or human consumption. (ii). Supercritical fluid extraction: this is a green technology based on CO2 at supercritical conditions used as a non-toxic extracting solvent in order to separate the lipids from matrix (dried biomass or disrupted concentrate). It has been applied to extract lipids and other high valuable compounds from algae, however, the operational cost is high (Mendes et al., 1995; Thana et al., 2008). Strategies to facilitate downstream processing of recombinant protein produced in microalgae The potential of C. reinhardtii to produce recombinant proteins for industrial, nutritional, and medical uses has been discussed above, but it is notable that hardly any reports have described large-scale production or recovery of target proteins. Moreover, downstream processing of engineered microalgae is largely based on laboratory scale techniques. More than 30 proteins with biotechnological applications have been expressed in Chlamydomonas (Rasala and Mayfield, 2014) including a number that have been expressed in the chloroplast. Although the chloroplast has been shown to be a robust platform for commercial production of proteins, it has some limitations as chloroplast-expressed proteins cannot be secreted, thus, cells must be processed after cultivation to recover and purify the product. Regardless of the nature of the bio-product produced in the microalgae, downstream processing follows the same route as in biofuels; therefore the operations to recover high-value products that require high purity (recombinant proteins, pigments, carotenoids, PUFAs, terpenoids, etc.) include harvesting, cell disruption (if the compound is intracellular), and purification. Figure 2 illustrates the recovery and purification process of recombinant proteins produced in microalgae. Fig. 2. View largeDownload slide Downstream processing of recombinant proteins extracted from transgenic algae systems. A flow-chart for the downstream processing of algal extracts by the ‘dry route’ involving freeze-drying or the ‘wet route’ involving cell disruption and fractionation. Fig. 2. View largeDownload slide Downstream processing of recombinant proteins extracted from transgenic algae systems. A flow-chart for the downstream processing of algal extracts by the ‘dry route’ involving freeze-drying or the ‘wet route’ involving cell disruption and fractionation. Since recombinant protein production in Chlamydomonas is largely limited to small-scale cultivation, the current harvesting is mainly done by conventional centrifugation. Harvested biomass can be processed using the approach of dry and wet routes. For the dry route, the drying biomass has potential in the production of edible vaccines and enzymes; research suggests that dry algal extract is an ideal vehicle for oral delivery of vaccines and enzymes as the cell wall protects the antigens and enzymes (Yoon et al., 2011). Production of vaccines that can be administrated orally is benefited by the effectiveness of freeze-drying to preserve antigen-expressing Chlamydomonas; some vaccine-producing strains have been reported to be stable for up to 20 months; therefore, the cost is reduced by eliminating extraction and purification steps (Specht and Mayfield, 2014). Algal-based recombinant vaccines administered orally as dry biomass have demonstrated effectiveness in two studies, namely protection against S. aureus infection (Dreesen et al., 2010) and a potential malaria vaccine (Gregory et al., 2013). The wet route for extraction offers the possibility of recovery of recombinant proteins with high purity. However, cell disruption techniques can be limited since the proteins of interest can be compromised during harsh conditions. Cell lysis can be accomplished by combining mechanical, physical, and chemical methods. The most common methods are freezing, sonication, and lysis with a buffer, and any combination of them will result in a disrupted concentrate that must go through more purification steps. Cell disruption methods adopted by the community vary considerably, some common approaches are: freezing followed by lysis buffer (Gregory et al., 2013), and lysis buffer coupled with sonication (Rasala et al., 2012; Demurtas et al., 2013). There is an alternative third route in the process, termed the ‘supernatant route’. This route is only possible when nuclear-expressed proteins are coupled with a signal peptide in order to be secreted into the culture media and thereby facilitate product recovery (Table 4). After cultivation, biomass is discarded as the recombinant proteins are present in the media. The proteins are then concentrated by filtration, lyophilization, or precipitation and further purified. Lauersen et al. (2015) optimized culture conditions and parameters of an engineered Chlamydomonas strain able to secrete an ice-binding protein with potential use in the food industry; they found that photomixotrophic cultivations led to accumulation of ~10mg l−1 of this protein in a small scale. Larger scale experiments (10L) conducted in plastic bags under the same conditions resulted in ~12mg l−1 of recombinant protein accumulated in the medium. Compared with other microbial systems where secretion of recombinants proteins into the culture media is well established, microalgae still needs further development and optimization in order to have comparable secretion yields. Table 4. Secretion of recombinant proteins from C. reinhardtii Signal peptide   Protein secreted   Reference   Arylsulphatase (ARS2)  Luciferase and erythropoietin  Eichler-Stahlberg et al., 2009   POXA1b (from Pleurotus ostreatus)  Laccase  Chiaiese et al., 2011   Arylsulphatase (ARS1)  Xylanase  Rasala et al., 2012   Carbonic anhydrase 1 (CAH1)  Gaussia luciferase  Lauersen et al., 2013a  Luciferase  Luciferase  Ruecker et al., 2008   Carbonic anhydrase 1 (CAH1)  Ice-binding protein  Lauersen et al., 2013b  Signal peptide   Protein secreted   Reference   Arylsulphatase (ARS2)  Luciferase and erythropoietin  Eichler-Stahlberg et al., 2009   POXA1b (from Pleurotus ostreatus)  Laccase  Chiaiese et al., 2011   Arylsulphatase (ARS1)  Xylanase  Rasala et al., 2012   Carbonic anhydrase 1 (CAH1)  Gaussia luciferase  Lauersen et al., 2013a  Luciferase  Luciferase  Ruecker et al., 2008   Carbonic anhydrase 1 (CAH1)  Ice-binding protein  Lauersen et al., 2013b  View Large Thus, although microalgae appear to be an attractive platform for biomanufacturing of high-value proteins for industrial, nutritional, and medical uses, downstream operations have technical challenges and such processes are still limited to small-scale proof of concept studies. On the other hand, the high cost of downstream processing of high-value products from transgenic algae can be avoided in applications where no product purification is required, thus resulting in less expensive processing. The next step is to explore higher scale production levels in order to make the platforms competitive with other expression platforms. The first (and so far only) example of large-scale production of an algal-expressed therapeutic protein was MAA in a volume of 100 l in a greenhouse (Gimpel et al., 2014). Conclusions In this review, we have highlighted the biotechnological potential of microalgae from several different angles. Microalgae are a valuable source of natural products, including carotenoids, antioxidants, and pigments, in addition to being used as feed stock or for the production of biodiesel. Advances in genetic engineering also mean that some species can be transformed and used as cell factories for other high-value products, including recombinant proteins. Several vaccine antigens, antibodies, and some enzymes have now been produced in the model alga C. reinhardtii. In addition, efforts have been directed towards metabolic engineering in algae and cyanobacteria. On a different note, we have also summarized the current research into engineering algal communities and the potential benefit of co-culturing bacteria and algae to increase productivity, reduce energy inputs, and protect cultures from pathogens. Considering these aspects, the great potential of microalgae in biotechnology becomes evident. However, large-scale production remains a challenge and microalgae still struggle to compete with existing platforms. It remains to be seen whether microalgae will be used for a wide range of industrial applications or only for more specialized applications, where there are severe shortcomings in competing platforms. The niche for microalgae still needs to be developed. The GRAS status of many microalgae, their inexpensive culturing, and potential for large-scale growth in bioreactors are definitely distinctive advantages of these photosynthetic microorganisms and upcoming years will reveal where the industry is headed. Funding This work was supported by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grant agreement n° 317184 and from ‘Plant Power: Light-Driven Synthesis of Complex Terpenoids Using Cytochromes P450’ (12-131834) funded by the Danish Council for Strategic Research, Programme Commission on Strategic Growth Technologies (PEJ, CR). Priscilla D. Rajakumar was funded by the UK’s Commonwealth Scholarship Commission. 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TI - Biotechnological exploitation of microalgae
JO - Journal of Experimental Botany
DO - 10.1093/jxb/erv426
DA - 2015-09-22
UR - https://www.deepdyve.com/lp/oxford-university-press/biotechnological-exploitation-of-microalgae-faLW5Wh0ZU
SP - 6975
EP - 6990
VL - 66
IS - 22
DP - DeepDyve
ER -