TY - JOUR
AU1 - Jezierska, Sylwia
AU2 - Van Bogaert, Inge N A
AB - Abstract How small molecules cross cellular membranes is an often overlooked issue in an industrial microbiology and biotechnology context. This is to a large extent governed by the technical difficulties to study these transport systems or by the lack of knowledge on suitable efflux pumps. This review emphasizes the importance of microbial cellular membranes in industrial biotechnology by highlighting successful strategies of membrane engineering towards more resistant and hence better performing microorganisms, as well as transporter and other engineering strategies for increased efflux of primary and secondary metabolites. Furthermore, the benefits and limitations of eukaryotic subcellular compartmentalization are discussed, as well as the biotechnological potential of membrane vesicles. This paper is part of the Special Issue of JIMB dedicated to Arny Demain on the occasion of his 90th birthday in 2017. Introduction The use of microbial cell factories in industrial biotechnology offers some clear advantages over enzymatic or biocatalytic processes, such as the keen availability of substrates and intermediates and the concentration of the compounds in the intracellular environment. Nevertheless, use of microbial cells also implies dealing with elementary boundaries: the cellular membranes. In the past decades, themes such as synthetic biology, metabolic engineering and modeling, gained momentum and were also introduced in the field of industrial biotechnology. Indeed, metabolic pathway engineering and modeling led to increased production titers of endogenous primary and secondary metabolites, and furthermore enabled the challenging de novo synthesis of non-natural compounds, such as biofuels and medicinal plant metabolites. Accelerated and increased microbial production puts augmented pressure on the cellular membranes. Many of the accumulating compounds of interest interact with biological membranes—especially when having a hydrophobic nature—in this way causing cell damage when reaching a specific threshold concentration. In addition, the cellular membranes are important, but often overlooked last hurdles to take. Indeed, the pathway can be nicely modeled, including knock-out of competing pathways, balancing expression and regulation, introduction of novel or engineered proteins, etc., but what if the compound cannot efficiently leave the cell? Of course, one can opt for intracellular accumulation, but here, several prerequisites need to be met: non-toxic product, easy to lyse host cell, a straightforward downstream processing and purification process, and economical feasibility of low volumetric yield. Only if all these conditions are fulfilled, an industrial biotech process without considering cellular export is viable. In all other cases, efflux should be aimed for as this will not only overcome the previous mentioned drawbacks, but most importantly will create a pulling effect at the very downstream part of the biosynthetic pathway which in general enhances culture vitality and the overall production yields. This review discusses several proved approaches to overcome cellular membrane boundaries in the framework of industrial biotech applications. For the reader’s convenience, cited examples are summarized in Table 1. Overview of the mentioned strategies and their effect on product titers Product . Organism . Strategy . Original titer . Improved titer . Increase . Branched fatty acids (C4-6) [65] S. cerevisiae Avoid toxicity by endogenous transporter overexpression 238 mg/L 262 mg/L 10% Cadaverine [31] C. glutamicum Heterologous expression of E. coli transporter 2.12 g/L 52% exported 2.75 g/L 73% exported 30% 40% Cadaverine [26] C. glutamicum Overexpression endogenous transporter 364 mmol/mol glucose 405 mmol/mol glucose 11% β-carotene [33] S. cerevisiae Engineering membrane integrity: linoleic acid supplementation 3.69 mg/g CDW 4.59 mg/g CDW 24.3% Carotenoids [28] Rhodosporidium toruloides Avoid toxicity by heterologous transporter expression 1.9 mg/g CDW 16% exported 2.9 mg/g CDW 62% exported 53% 3.9-fold Carbomycin [47] Streptomyces halstedii Engineering membrane integrity: surfactant supplementation 0.375 g/L 0.825 g/L 230% Cellulose degradation [40] E. coli Decorated membrane vesicles Free enzymes: 1% relative degradation Armed yeast: 2.5% 23% 23-fold Ninefold Glutamate [53] C. glutamicum Engineering membrane integrity: Tween 60 supplementation 0.1 g/L 1.9 g/L 19-fold Glutamate [53] C. glutamicum Engineering membrane integrity: limited Biotin addition 0.1 g/L 1.8 g/L 18-fold Glutamate [38] C. glutamicum Engineering membrane integrity: penicillin supplementation 0.14 g/L 18.2 g/L 130-fold Glutamate [39] C. glutamicum Endogenous transporter overexpression 165 mM 204 mM 24% Isobutanol [7] S. cerevisiae Establishment of mitochondrial pathway in cytosol 13 mg/L 124 mg/L (final: 630 mg/L) 9.5-fold Isobutanol [2] S. cerevisiae Establishment of mitochondrial pathway in cytosol 28 mg/L 151 mg/L Fivefold Isobutanol [2] S. cerevisiae Establishment of total pathway in mitochondria 136 mg/L 491 mg/L 3.6-fold Itaconic acid [6] A. niger Establishment of total pathway in mitochondria or cytosol and combination 0.4 g/L cytosolic 0.8 g/L mitochondrial 1.1 g/L combined 38% Itaconic acid [30] A. niger Heterologous expression of A. terreus mitochondrial transporter 0.9 g/L 1.5 g/L 67% Itaconic acid [20] A. terreus Overexpression endogenous transporter 80 g/L 84 g/L 5% Limonene [13] E. coli Avoid toxicity by heterologous transporter expression 35 mg/L 56 mg/L 68% Medium-chain fatty alcohols [48] S. cerevisiae Targeting of synthetic enzyme to peroxisomes 0 g/L 1.3 g/L New product n-octane [15] E. coli Avoid toxicity by engineering of endogenous transporter Not applicable Not applicable Efflux: +47% Octanoic acid [54] E. coli Engineering membrane integrity: expression of the P. aeruginosa cis–trans isomerase 31.0 mg/L 43.7 mg/L 41% PHA [42] Starmerella bombicola Targeting of synthetic enzyme to peroxisomes 0% wt/dwt 2.0% wt/dwt New product PHA [27] S. cerevisiae Targeting of synthetic enzyme to peroxisomes 0% wt/dwt 0.5% wt/dwt New product Phenylalanine [17] E. coli Heterologous expression of mutated C. glutamicum transporter 1 g/L 1.9 g/L 90% Pigments [62] Monascus purpereus Engineering membrane integrity: Triton X-100 supplementation 161.5 U/mL out/in = 0.43 304.3 U/mL out/in = 1.46 88.4% 240% α-pinene [15] E. coli Avoid toxicity by engineering of endogenous transporter Not applicable Not applicable Efflux: fourfold Styrene [61] E. coli Capture toxic product in surfactant micelles 1.49 mM 5.5 mM 3.7-fold Succinic acid [60] S. cerevisiae Establishment of mitochondrial pathway in cytosol 0.3 g/L 3.9 g/L 13-fold Succinic acid [60] S. cerevisiae Heterologous expression of S. pombe transporter 2.7 g/L 4.6 g/L 70% Threonine [12] C. glutamicum Heterologous expression of E. coli transporter 2.5 g/L 6.4 g/L 2.6-fold Product . Organism . Strategy . Original titer . Improved titer . Increase . Branched fatty acids (C4-6) [65] S. cerevisiae Avoid toxicity by endogenous transporter overexpression 238 mg/L 262 mg/L 10% Cadaverine [31] C. glutamicum Heterologous expression of E. coli transporter 2.12 g/L 52% exported 2.75 g/L 73% exported 30% 40% Cadaverine [26] C. glutamicum Overexpression endogenous transporter 364 mmol/mol glucose 405 mmol/mol glucose 11% β-carotene [33] S. cerevisiae Engineering membrane integrity: linoleic acid supplementation 3.69 mg/g CDW 4.59 mg/g CDW 24.3% Carotenoids [28] Rhodosporidium toruloides Avoid toxicity by heterologous transporter expression 1.9 mg/g CDW 16% exported 2.9 mg/g CDW 62% exported 53% 3.9-fold Carbomycin [47] Streptomyces halstedii Engineering membrane integrity: surfactant supplementation 0.375 g/L 0.825 g/L 230% Cellulose degradation [40] E. coli Decorated membrane vesicles Free enzymes: 1% relative degradation Armed yeast: 2.5% 23% 23-fold Ninefold Glutamate [53] C. glutamicum Engineering membrane integrity: Tween 60 supplementation 0.1 g/L 1.9 g/L 19-fold Glutamate [53] C. glutamicum Engineering membrane integrity: limited Biotin addition 0.1 g/L 1.8 g/L 18-fold Glutamate [38] C. glutamicum Engineering membrane integrity: penicillin supplementation 0.14 g/L 18.2 g/L 130-fold Glutamate [39] C. glutamicum Endogenous transporter overexpression 165 mM 204 mM 24% Isobutanol [7] S. cerevisiae Establishment of mitochondrial pathway in cytosol 13 mg/L 124 mg/L (final: 630 mg/L) 9.5-fold Isobutanol [2] S. cerevisiae Establishment of mitochondrial pathway in cytosol 28 mg/L 151 mg/L Fivefold Isobutanol [2] S. cerevisiae Establishment of total pathway in mitochondria 136 mg/L 491 mg/L 3.6-fold Itaconic acid [6] A. niger Establishment of total pathway in mitochondria or cytosol and combination 0.4 g/L cytosolic 0.8 g/L mitochondrial 1.1 g/L combined 38% Itaconic acid [30] A. niger Heterologous expression of A. terreus mitochondrial transporter 0.9 g/L 1.5 g/L 67% Itaconic acid [20] A. terreus Overexpression endogenous transporter 80 g/L 84 g/L 5% Limonene [13] E. coli Avoid toxicity by heterologous transporter expression 35 mg/L 56 mg/L 68% Medium-chain fatty alcohols [48] S. cerevisiae Targeting of synthetic enzyme to peroxisomes 0 g/L 1.3 g/L New product n-octane [15] E. coli Avoid toxicity by engineering of endogenous transporter Not applicable Not applicable Efflux: +47% Octanoic acid [54] E. coli Engineering membrane integrity: expression of the P. aeruginosa cis–trans isomerase 31.0 mg/L 43.7 mg/L 41% PHA [42] Starmerella bombicola Targeting of synthetic enzyme to peroxisomes 0% wt/dwt 2.0% wt/dwt New product PHA [27] S. cerevisiae Targeting of synthetic enzyme to peroxisomes 0% wt/dwt 0.5% wt/dwt New product Phenylalanine [17] E. coli Heterologous expression of mutated C. glutamicum transporter 1 g/L 1.9 g/L 90% Pigments [62] Monascus purpereus Engineering membrane integrity: Triton X-100 supplementation 161.5 U/mL out/in = 0.43 304.3 U/mL out/in = 1.46 88.4% 240% α-pinene [15] E. coli Avoid toxicity by engineering of endogenous transporter Not applicable Not applicable Efflux: fourfold Styrene [61] E. coli Capture toxic product in surfactant micelles 1.49 mM 5.5 mM 3.7-fold Succinic acid [60] S. cerevisiae Establishment of mitochondrial pathway in cytosol 0.3 g/L 3.9 g/L 13-fold Succinic acid [60] S. cerevisiae Heterologous expression of S. pombe transporter 2.7 g/L 4.6 g/L 70% Threonine [12] C. glutamicum Heterologous expression of E. coli transporter 2.5 g/L 6.4 g/L 2.6-fold Often other engineering steps are applied as well, but we focused on the membrane related improvements and hereto related reference strains Open in new tab Overview of the mentioned strategies and their effect on product titers Product . Organism . Strategy . Original titer . Improved titer . Increase . Branched fatty acids (C4-6) [65] S. cerevisiae Avoid toxicity by endogenous transporter overexpression 238 mg/L 262 mg/L 10% Cadaverine [31] C. glutamicum Heterologous expression of E. coli transporter 2.12 g/L 52% exported 2.75 g/L 73% exported 30% 40% Cadaverine [26] C. glutamicum Overexpression endogenous transporter 364 mmol/mol glucose 405 mmol/mol glucose 11% β-carotene [33] S. cerevisiae Engineering membrane integrity: linoleic acid supplementation 3.69 mg/g CDW 4.59 mg/g CDW 24.3% Carotenoids [28] Rhodosporidium toruloides Avoid toxicity by heterologous transporter expression 1.9 mg/g CDW 16% exported 2.9 mg/g CDW 62% exported 53% 3.9-fold Carbomycin [47] Streptomyces halstedii Engineering membrane integrity: surfactant supplementation 0.375 g/L 0.825 g/L 230% Cellulose degradation [40] E. coli Decorated membrane vesicles Free enzymes: 1% relative degradation Armed yeast: 2.5% 23% 23-fold Ninefold Glutamate [53] C. glutamicum Engineering membrane integrity: Tween 60 supplementation 0.1 g/L 1.9 g/L 19-fold Glutamate [53] C. glutamicum Engineering membrane integrity: limited Biotin addition 0.1 g/L 1.8 g/L 18-fold Glutamate [38] C. glutamicum Engineering membrane integrity: penicillin supplementation 0.14 g/L 18.2 g/L 130-fold Glutamate [39] C. glutamicum Endogenous transporter overexpression 165 mM 204 mM 24% Isobutanol [7] S. cerevisiae Establishment of mitochondrial pathway in cytosol 13 mg/L 124 mg/L (final: 630 mg/L) 9.5-fold Isobutanol [2] S. cerevisiae Establishment of mitochondrial pathway in cytosol 28 mg/L 151 mg/L Fivefold Isobutanol [2] S. cerevisiae Establishment of total pathway in mitochondria 136 mg/L 491 mg/L 3.6-fold Itaconic acid [6] A. niger Establishment of total pathway in mitochondria or cytosol and combination 0.4 g/L cytosolic 0.8 g/L mitochondrial 1.1 g/L combined 38% Itaconic acid [30] A. niger Heterologous expression of A. terreus mitochondrial transporter 0.9 g/L 1.5 g/L 67% Itaconic acid [20] A. terreus Overexpression endogenous transporter 80 g/L 84 g/L 5% Limonene [13] E. coli Avoid toxicity by heterologous transporter expression 35 mg/L 56 mg/L 68% Medium-chain fatty alcohols [48] S. cerevisiae Targeting of synthetic enzyme to peroxisomes 0 g/L 1.3 g/L New product n-octane [15] E. coli Avoid toxicity by engineering of endogenous transporter Not applicable Not applicable Efflux: +47% Octanoic acid [54] E. coli Engineering membrane integrity: expression of the P. aeruginosa cis–trans isomerase 31.0 mg/L 43.7 mg/L 41% PHA [42] Starmerella bombicola Targeting of synthetic enzyme to peroxisomes 0% wt/dwt 2.0% wt/dwt New product PHA [27] S. cerevisiae Targeting of synthetic enzyme to peroxisomes 0% wt/dwt 0.5% wt/dwt New product Phenylalanine [17] E. coli Heterologous expression of mutated C. glutamicum transporter 1 g/L 1.9 g/L 90% Pigments [62] Monascus purpereus Engineering membrane integrity: Triton X-100 supplementation 161.5 U/mL out/in = 0.43 304.3 U/mL out/in = 1.46 88.4% 240% α-pinene [15] E. coli Avoid toxicity by engineering of endogenous transporter Not applicable Not applicable Efflux: fourfold Styrene [61] E. coli Capture toxic product in surfactant micelles 1.49 mM 5.5 mM 3.7-fold Succinic acid [60] S. cerevisiae Establishment of mitochondrial pathway in cytosol 0.3 g/L 3.9 g/L 13-fold Succinic acid [60] S. cerevisiae Heterologous expression of S. pombe transporter 2.7 g/L 4.6 g/L 70% Threonine [12] C. glutamicum Heterologous expression of E. coli transporter 2.5 g/L 6.4 g/L 2.6-fold Product . Organism . Strategy . Original titer . Improved titer . Increase . Branched fatty acids (C4-6) [65] S. cerevisiae Avoid toxicity by endogenous transporter overexpression 238 mg/L 262 mg/L 10% Cadaverine [31] C. glutamicum Heterologous expression of E. coli transporter 2.12 g/L 52% exported 2.75 g/L 73% exported 30% 40% Cadaverine [26] C. glutamicum Overexpression endogenous transporter 364 mmol/mol glucose 405 mmol/mol glucose 11% β-carotene [33] S. cerevisiae Engineering membrane integrity: linoleic acid supplementation 3.69 mg/g CDW 4.59 mg/g CDW 24.3% Carotenoids [28] Rhodosporidium toruloides Avoid toxicity by heterologous transporter expression 1.9 mg/g CDW 16% exported 2.9 mg/g CDW 62% exported 53% 3.9-fold Carbomycin [47] Streptomyces halstedii Engineering membrane integrity: surfactant supplementation 0.375 g/L 0.825 g/L 230% Cellulose degradation [40] E. coli Decorated membrane vesicles Free enzymes: 1% relative degradation Armed yeast: 2.5% 23% 23-fold Ninefold Glutamate [53] C. glutamicum Engineering membrane integrity: Tween 60 supplementation 0.1 g/L 1.9 g/L 19-fold Glutamate [53] C. glutamicum Engineering membrane integrity: limited Biotin addition 0.1 g/L 1.8 g/L 18-fold Glutamate [38] C. glutamicum Engineering membrane integrity: penicillin supplementation 0.14 g/L 18.2 g/L 130-fold Glutamate [39] C. glutamicum Endogenous transporter overexpression 165 mM 204 mM 24% Isobutanol [7] S. cerevisiae Establishment of mitochondrial pathway in cytosol 13 mg/L 124 mg/L (final: 630 mg/L) 9.5-fold Isobutanol [2] S. cerevisiae Establishment of mitochondrial pathway in cytosol 28 mg/L 151 mg/L Fivefold Isobutanol [2] S. cerevisiae Establishment of total pathway in mitochondria 136 mg/L 491 mg/L 3.6-fold Itaconic acid [6] A. niger Establishment of total pathway in mitochondria or cytosol and combination 0.4 g/L cytosolic 0.8 g/L mitochondrial 1.1 g/L combined 38% Itaconic acid [30] A. niger Heterologous expression of A. terreus mitochondrial transporter 0.9 g/L 1.5 g/L 67% Itaconic acid [20] A. terreus Overexpression endogenous transporter 80 g/L 84 g/L 5% Limonene [13] E. coli Avoid toxicity by heterologous transporter expression 35 mg/L 56 mg/L 68% Medium-chain fatty alcohols [48] S. cerevisiae Targeting of synthetic enzyme to peroxisomes 0 g/L 1.3 g/L New product n-octane [15] E. coli Avoid toxicity by engineering of endogenous transporter Not applicable Not applicable Efflux: +47% Octanoic acid [54] E. coli Engineering membrane integrity: expression of the P. aeruginosa cis–trans isomerase 31.0 mg/L 43.7 mg/L 41% PHA [42] Starmerella bombicola Targeting of synthetic enzyme to peroxisomes 0% wt/dwt 2.0% wt/dwt New product PHA [27] S. cerevisiae Targeting of synthetic enzyme to peroxisomes 0% wt/dwt 0.5% wt/dwt New product Phenylalanine [17] E. coli Heterologous expression of mutated C. glutamicum transporter 1 g/L 1.9 g/L 90% Pigments [62] Monascus purpereus Engineering membrane integrity: Triton X-100 supplementation 161.5 U/mL out/in = 0.43 304.3 U/mL out/in = 1.46 88.4% 240% α-pinene [15] E. coli Avoid toxicity by engineering of endogenous transporter Not applicable Not applicable Efflux: fourfold Styrene [61] E. coli Capture toxic product in surfactant micelles 1.49 mM 5.5 mM 3.7-fold Succinic acid [60] S. cerevisiae Establishment of mitochondrial pathway in cytosol 0.3 g/L 3.9 g/L 13-fold Succinic acid [60] S. cerevisiae Heterologous expression of S. pombe transporter 2.7 g/L 4.6 g/L 70% Threonine [12] C. glutamicum Heterologous expression of E. coli transporter 2.5 g/L 6.4 g/L 2.6-fold Often other engineering steps are applied as well, but we focused on the membrane related improvements and hereto related reference strains Open in new tab Dealing with toxic compounds Toxicity of endogenous or exogenous compounds against a microbial cell can have several causes. For instance, intracellular accumulation might interfere with metabolic processes at the transcriptional level by gene inhibition or activation, and can cause different types of enzyme inhibition or hamper correct organelle functioning. Moreover, with the recent interest in biofuels, such as linear and aromatic hydrocarbons, alcohols and fatty acid derivatives, also membranes can be affected. Most of these fuel compounds display a log P ow value between 1.5 and 4, meaning they partition within biological membranes with often detrimental effects [22]. Yet, several bacteria occurring in environments enriched for the above molecules developed mechanisms to counter the toxic effects and these can be implemented in strain engineering. In the next sections strategies, such as modified membrane integrity, increased efflux and induction of general resistance mechanisms will be discussed. Modifying membrane integrity as a strategy for enhanced production There are several clear examples demonstrating the importance of membrane integrity as a microorganism’s strategy to withstand solubilizing compounds. Most bacteria lyse upon exposure to a few percentage of ethanol, and are hence not the most auspicious biofuel producers. However, exceptions exist. Zymomonas mobilis for instance is used in the production of alcoholic beverages in certain tropical regions [52] and ethanol percentages similar to yeast of 13–16% can be achieved due to the increased concentration of hopanoids in the plasmamembrane. These hopanoids are pentacyclic compounds similar to eukaryotic sterols rendering the membrane more resistant [19, 46]. The high membrane rigidity combined with other beneficial biochemical characteristics render Zymomonas mobilis an attractive host for biofuel production. In a similar way, the high ethanol tolerance of Saccharomyces cerevisiae is among others mediated by increased ergosterol and monosaturated fatty acid concentrations [43], and altering the membrane properties by strain engineering (e.g., by introducing desaturase genes) can be a strategy to obtain yeasts displaying increased tolerance [41]. The two above examples demonstrate the natural adaptive power of microorganisms towards toxic compounds. In some cases, however, this power is absent or insufficient to reach the targeted production volumes. In this event, several engineering strategies can be applied. For instance, Escherichia coli often is the organism of choice to many researchers and companies for synthesis of heterologous compounds as extensive knowledge and molecular tools are available. Yet, the organism does by nature not encounter toxic compounds or solvents and is hence not well prepared to face these membrane disruptors. Other organisms are more adjusted to hostile environments. One such example is Pseudomonas aeruginosa, and one of its defense mechanisms is the ability to change the cis double bond of its unsaturated fatty acids to a trans double bond by means of a cis–trans isomerase. This subtitle change removes the 30° nick at the Δ9 position (Fig. 1), creating a more compact and less fluid membrane with an increased survival in hostile environments [18]. Tan et al. [54] introduced the Pseudomonas a cis–trans isomerase gene in an octatonic acid producing E. coli strain, with the initial goal to increase tolerance towards this compound. Not only was the engineered strain more resistant towards exogenously added octanoic acid, it also displayed a 41% increased production. Moreover, this membrane alteration also rendered the strain more tolerant towards additional interesting bio-products, such as butanol, hexanol, styrene, toluene and other carboxylic acids. Nonetheless, for some compounds, a low increase or even decrease was observed (e.g., ethanol and isobutanol); the authors stress the importance of tuning the cis–trans isomerase activity to get the right cis over trans ratio for each individual inhibitory molecule. Fig. 1 Open in new tabDownload slide Influence on the molecule’s three-dimensional organization and compactness when the cis double bond is replaced by a trans double bound by a cis–trans isomerase (Cti) As illustrated above, E. coli benefits from decreased membrane fluidity in the presence of octanoic acid, whereas the opposite is advantageous to yeast upon high ethanol concentrations. The specific alterations of the membrane hence depend on the species and the effecting molecule. Carotenoid synthesis in S. cerevisiae, for instance, requires normal membrane fluidity and does not benefit from increased rigidity. On the contrary, reduced cell membrane fluidity seems to hinder metal ion uptake and in turn reduces carotenoid synthesis [33]. Apparently, the strain engineering itself causes this default of unwanted robustness. The introduced carotenoid pathway pulls away acetyl-CoA and farnesyl pyrophosphate, precursors for (unsaturated) fatty acid and ergosterol synthesis respectively, and the underrepresentation of these later compounds results in a more rigid cell membrane. To solve this problem, the strain itself was left untouched, and a simple supplementation of linoleic acid (C18:2) could restore membrane fluidity resulting in a 24.3% increased β-carotene production [33]. Furthermore, linoleic acid was successfully applied to improve a dodecane two-phase medium for β-carotene extraction [50]. Therefore, membrane integrity can be altered by genetic engineering or by applying medium additives. This latter approach also is a recognized strategy in glutamate production by Corynebacterium glutamicum. As discussed in the next section, several factors influence glutamate secretion, and supplementation of surfactants, such as Tween and polyoxyethylene glycol fatty acid esters favor glutamate release [37, 53]. This straightforward approach can be applied to various other compounds and organisms [47, 62]. Nevertheless, one has to carefully investigate the compatibility of the surfactants with cell viability and growth, and its effect and behavior in product recovery. If dealing with hydrophobic or lipogenic products as highlighted in this section, it can become hard to get rid of the surfactant and thus additional purification steps to remove co-purified surfactant can be required. Surfactants do not only influence membrane structure, but can also be applied for their micelle forming properties. Biocompatible micelles are ideal candidates for in situ product removal and sequestration. Wallace and Balskus [61] combined an engineered E. coli strain producing toxic amounts of styrene with vitamin E derived surfactants. During fermentation, styrene was captured in the micelle interiors, resulting in enhanced flux and product titers. Increased efflux as a strategy to withstand toxic compounds with better production as result Another important mechanism of wild-type strains to withstand potentially toxic biofuels is increased efflux. This feature can be introduced in industrial host strains in several ways. Dunlop et al. [13] were the first to set-up a large screening of environmental transporters to increase tolerance in E. coli. 43 heterologous pumps were selected in silico based on their homology to the solvent-resistance pump TtgB from P. putida. These were evaluated in E. coli using a competitive growth assay; only strains that gained increased tolerance could survive in the solvent containing medium. No hits were found for n-butanol and isopentanol, but for the five remaining compounds, geranyl acetate, geraniol, limonene, farnesyl hexanoate and α-pinene significant resistance were obtained, attributed to transporters originating from a variety of unrelated bacteria, among them several marine ones. This seemingly straightforward strategy often comes with a trade-off; overexpression of transporters can compromise cell viability, so balanced expression should be aimed for [58]. The same is true for endogenous transporters. The AcrAB-TolC efflux pump of E. coli (Fig. 2) has a broad substrate specificity including detergents, antibiotics, solvents, terpenes, and alkanes. Yet again, overexpression does not generate increased resistance. Therefore, protein engineering strategies were applied focusing on the inner membrane domain ArcB, responsible for substrate binding. This way, increased efflux of n-octane and α-pinene was achieved (47 and 400%, respectively [15]) as well as a 25% augmented growth rate in the presence of n-butanol [14]. Fig. 2 Open in new tabDownload slide Schematic representation of the E. coli AcrAB-TolC efflux pump Obviously, eukaryotes can be subjected to similar approaches. S. cerevisiae was equipped with ABC transporters of Yarrowia lipolytica; an organism renowned for its survival in alkane-enriched environments and efficient catabolization of these molecules. Both ABC2 and ABC3 were able to increase the tolerance limit against decane and undecane, with a 80-fold increase for decane with ABC2. The pumps seemed to be chain-length specific, as no resistance against octane, nonane and dodecane was observed [10]. Induction of general resistance mechanisms As elaborated above, several strategies can be followed to achieve increased tolerance. These are, however, not limited to merely act on membrane rigidity or efflux; other factors, such as upregulated general stress responses and systems to reduce reactive oxygen species, can have beneficial contributions as well. In this respect, black-box methods, such as adaptive evolution, can create more robust strains, often by alteration of less obvious genes or pathways. Yet, one has to keep in mind that such general approaches can also activate unwanted clearing pathways for the molecule of interest. A more guided approach was applied by Ling and co-workers [32] when exploiting the zinc cluster pleiotropic drug resistance transcription factors Pdr1p and Pdr3p. These transcription factors mediate general drug resistance against many cytotoxic substances. Mutated forms established the highest tolerance towards dodecane, while overexpression of wild-type Pdr3p was most effective to achieve undecane resistance. Differentially regulated genes were associated with multi-drug resistance, stress responses, and membrane modifications. Specifically related to membranes, ABC efflux pump genes yor1, snq2, pdr5, and pdr15 were clearly upregulated, as well as the lysophosphatidic acid acyltransferase gene ict1, responsible for enhanced phospholipid synthesis. Pleiotropic drug resistance seems to be an important response of host cells when designed for heterologous compound synthesis, and no extreme toxicity is required to trigger these effects, as illustrated by carotenoid synthesis in S. cerevisiae [59]. Here, genes related to detoxification and transport were found be upregulated, among them six transporter genes of which pdr10 turned out to be the most important one [59]. The above examples illustrate that genes either directly or indirectly involved in pleiotropic drug resistance could be attractive targets for host improvement. In addition, this is exactly what Lee et al. implemented when engineering Rhodosporidium toruloides for improved carotenoid production. By simply introducing the S. cerevisiae pdr10 gene, yield raised from 1.9 to 2.9 mg/g cell dry weight and carotenoid export increased from 16 to 62% [28]. Finally, one must realize that efforts to reduce product toxicity do not necessarily translate into improved production. For instance, in the metabolic engineering of S. cerevisiae for the synthesis of short branched-chain fatty acids (C4-6), overexpression of the ABC transporter PDR12 only marginally increased secretion; the 30-fold improvement was attributed to other modifications [65]. Indeed, the ability to tolerate a high titer of the final product is not necessarily equivalent to the ability to synthesize more. The old adagio ‘go get what you screen for’ is certainly also true in this case. Higher tolerance by increased efflux can also affect product precursors when more promiscuous transporters are involved, leading to undesired accumulation of intermediates in the extracellular space, and obviously, reduced yields. Alterations in membrane integrity can certainly increase product resistance, but as illustrated by the carotenoid example, this can result in suboptimal uptake of essential compounds, again with a negative effect on the overall performance. Moreover, when applying more general approaches, such as adaptive evolution or modifying transcription factors, chances of activating undesired mechanisms, such as shuttling the compound or its intermediates to alternative biosynthetic pathways or catabolic routes, are higher. The challenges of extracellular primary metabolite accumulation: the amino-acid case study Primary metabolites, such as amino acids or organic acid intermediates from the Krebs cycle, should in normal physiological conditions not accumulate intracellularly nor be secreted in the extracellular environment. Metabolic engineering allowed the creation of several strains overcoming these limitations, such as E. coli and yeast strains secreting succinate (see further in this review). Yet, sometimes natural isolates are doing the job and it is interesting to find out what the mechanisms behind these capabilities are and how they can be activated in an industrial context. In this respect, the well-established microbial production processes of amino acids form an interesting case study. The first industrial fermentation process of glutamate was developed in 1956, and somewhat later, C. glutamicum became the organism of choice [44]. Strikingly, glutamate accumulation in the medium only occurred under biotin-limiting conditions, restricting the use of cheap substrates, such as sugar molasses. It was suggested that biotin limitation increased the permeability of the membranes. Indeed, lack of biotin decreases fatty acid synthase activity, resulting in a limited available fatty acid pool, which in turn leads to reduced phospholipid concentrations in the membrane. Moreover, substrates, such as glucose or glycerol, are converted to acetyl-CoA, but this intermediate cannot be shuttled towards fatty acid synthesis due to the biotin limitation and metabolism is driven towards glutamate synthesis. Hence, the double effect of biotin restriction leads to continuous synthesis and secretion. To allow the utilization of the standard raw materials, such as molasses, other membrane permeabilizing strategies were developed. As mentioned in the previous section, surfactants, such as polyoxyethylene glycol fatty acid esters, can be applied to destabilize the membrane; they cause a decrease in the membrane phospholipid levels as well as in the degree of unsaturation. Another way of reducing phospholipid levels is the use of penicillin [38]. This antibiotic triggers the excretion of N-acetylglucosamine derivatives and phospholipids. A final approach to control phospholipid levels is the use of mutants auxotrophic for oleic acid or glycerol. This will also free acetyl-CoA pools for glutamate synthesis [56]. The above examples all support the so-called leaky membrane model. Yet, this model does not explain why leakage is specific to glutamate and occurs against the concentration gradient. Only in 2007, the small conductance mechanosensitive channel MscCG was found to play a key role glutamate export [39]. The transporter responds to membrane stress, either at the transcriptional level (use of detergents) or by activation (biotin limitation) [9], and offers opportunities for engineering: overexpression in C. glutamicum ATCC 13869 results in a 1.05–1.24 fold increase of glutamate titers [39]. Moreover, as MscCG belongs to a group of osmoregulated ion channels of importance to maintain correct cell turgor, it is not surprising that this pump can also export other ions in response to osmotic stress. For example, Hashimoto et al. [17] demonstrated that the constitutively active MscCG A111V point mutant can be applied in an engineered E. coli strain to increase l-phenylalanine secretion. Glutamate is clearly the amino acid produced in the largest quantities (over 2 million ton per year). In addition, fermentative production of lysine, yet again by C. glutamicum, accounts for almost 1.5 million ton per year [66]. Again, secretion can be stimulated by increased membrane permeability, and this time generated by limited aeration conditions which influence the membrane fatty acid composition. Lysine secretion was already until a large extend characterized in the 1980s and 1990s [8, 35] and differs in several aspects from the glutamate system. A specific energy-dependent secondary carrier system was suggested, and the efflux of the cationic lysine is co-facilitated by two OH− ions. Hence, secretion depends on membrane potential, pH gradient, and the chemical gradient of lysine. Despite high intracellular glutamate concentrations, this molecule is not transporter by the lysine system [8]. The exporter LysE was identified in 2000 and turned out to be only 236 amino acids in size. LysE is induced by intracellular l-lysine and l-arginine, which both can be exported [5]. Escherichia coli is the organism of choice when fermentative production of l-threonine is intended. This is mainly attributed to its superior secretion of threonine when compared to C. glutamicum. Certainly, C. glutamicum possesses the metabolic capacities as threonine synthesis derives from the lysine biosynthetic pathway and as its outstanding glutamate and lysine producing capabilities illustrate the power of the pathways. Therefore, it would be interesting to see if industrial relevant threonine synthesis is possible in a transport engineered C. glutamicum strain. Overexpression of the endogenous ThrE carrier resulted in a 40% extracellular increase, but extracellular l-lysine was also observed, as well as the threonine degradation product glycine [49]. Hence, four E. coli threonine transports RhtA, RhtB, RhtC and YeaS were evaluated in C. glutamicum. The first one belonging to the major facilitator superfamily (MSF) and the later ones being RhtB paralogues. Furthermore, the transporters differ in their specificities regarding inhibition by and export of different amino acids. RhtA and RhtC both contributed to threonine accumulation, but RhtA also mediated undesired homoserine and serine export, rendering this exporter less suitable. Although titers could be increased from 2.5 until 6.4 g/L [12], there is still a long way to go commercial implementation becomes feasible as with E. coli product titers of 100 g/L can be obtained [29]. The lysine-derived diamine cadaverine or 1,5-diaminopentane recently came into the picture as bio-based platform chemical. Availability of microbial derived diaminopentane would open up sustainable routes for the production of novel nylons: copolymerization with succinic acid or sebacic acid from castor oil leads to polymers with desirable properties [26]. The clear link with lysine biosynthesis—strictly spoken, only a suitable lysine decarboxylase needs to be introduced—again puts C. glutamicum forward as the ideal host. Nevertheless, until 2011, no diaminopentane exporter was described for this organism, so to improve secretion heterologous exporters needed to be introduced. CadB from E. coli encodes a cadaverine-lysine antiporter involved in acid stress adaptation and turned out to be able to increase diaminopentane production and secretion in C. glutamicum upon overexpression in a pH independent way [31]. Yet, large progress was made when several engineering steps were combined with the overexpression of cg2893; an endogenous C. glutamicum major facilitator permease discovered by genome wide transcription profiling [25]. One of the other noteworthy modifications is the disabling of lysE, in this way avoiding extracellular loss of the lysine precursor. With the final C. glutamicum DAP-16 strain commercial relevant titers of 88 g/L and a productivity of 2.2 g/L/h were achieved [26]. The power of secondary metabolites Secondary metabolites are omnipresent among kingdoms, but mainly plants and microorganism are notorious for the synthesis of diverse biological active compounds, such as antibiotics, polyketides, and terpenoids. Often these molecules need to be transported to execute their functions. In plants, this means accumulation in cell compartments or even specific sink organs. In microorganisms, this means export to the outside environment. In many cases, the microbial transporters of secondary metabolites are easier to identify compared to the ones of primary metabolites; in the latter case, specific transporters are often even completely absent. Prerequisite to transporter identification is access to the genome sequence and some basic knowledge on the biosynthesis of the metabolite to screen for the right gene cluster or operon; in most cases, secondary metabolite genes are co-localized in gene clusters also harboring the specific transporter. With this knowledge, one can start classic strain (metabolic) engineering, including transporter expression tuning, modification, and protein engineering. During the last years, interest has shifted towards plant secondary metabolites, such as terpenoids, polyketides, alkaloids and flavonoids, as they display many beneficial medical properties. Due to the low productivity and complex recovery in plants, major efforts have been put in establishing engineered microbial cell factories for their synthesis. In addition, as stated in the introduction, an efficient production process for these heterologous compounds requires appropriate efflux. Yet, plant transporters are hard to identify as knowledge on these systems is still largely unknown; genomes are not sequenced, large and complex, and display complicated intron–exon architectures. Furthermore, biological screens are laborious and lengthy. However, unlocking these plant transporters could boost heterologous synthesis in microorganisms. Comparative transcriptomics or proteomics can be of assistance here as illustrated by the discovery of the ABC transporter NpPDR1, proposed to participate in export of the antifungal diterpene sclareol in Nicotinia species [21]. Meanwhile, orthologues have been discovered in other plant species [64]. This is of particular interest for the industrial biotech sector as sclareol synthesis is being developed both in yeast and E. coli [45, 55]; introduction of a plant transporter might enhance production in microbial eukaryotes, such as S. cerevisiae. The benefits and limitations of eukaryotic subcellular compartmentalization Compartmentalization of biochemical process is of marginal importance in prokaryotic cells, is clearly present in lower eukaryotes, and can become an important bottleneck in plant biotechnology, often impeding overproduction of recombinant proteins and secondary metabolites. Cell compartments are separated from thy cytosol by specific membranes. In the ER-Golgi-vacuole system, traffic is mediated by specific vesicles and tethering, and the cargo are mainly proteins, either in the unfolded, folded or post-translational modified state. Hence, for industrial biotechnology of non-protein compounds, these compartments are of less importance. On the other hand, mitochondria and peroxisomes host specific biochemical pathways as part of the global cell metabolism. Compartmentalization is beneficial to the wild-type eukaryotic cell, as co-acting enzymes are efficiently combined and shielded from interfering processes or inhibitors. Yet, in extended metabolic engineering strategies, one often wants to link different pathways or direct intermediates from the one to the other (synthetic) pathway, and in these cases, the organelle membranes can get in the way. Mitochondria Mitochondria are primarily occupied with respiration and synthesis of ATP, but also fulfill some other specific biochemical reactions. As part of their ATP and electron generating function, also the Krebs cycle is hosted here. This cycle generates several intermediates or even final target molecules of great interest to industrial biotechnology. Yet, having them cross the double mitochondrial and outer cell membrane is not always an easy job. Ever since succinate was mentioned in 2004 as one of the “top value added chemicals from biomass” in the report from the US department of energy [63], several companies and institutes invested in the development of engineered strains producing succinate in economical relevant amounts [1]. Some opted for E. coli or specific bacterial hosts, such as the rumen isolate Basfia succiniciproducens and here no problems regarding compartmentalization, are encountered [4]. Yet, others, such as Riverdia (JV Roquette and DSM), selected S. cerevisiae as a host and circumvented mitochondrial localization of the four key enzymes by expressing them cytoplasmatic, while at the same time removing the mitochondrial ones. Furthermore, a dicarboxylic acid transporter was introduced to accumulate succinate extracellular. Among the numerous endogenous and exogenous evaluated proteins, the malate permease MAE1 protein from Schizosaccharomyces pombe turned out to be most effective [60]. A similar approach was applied regarding the engineering of S. cerevisiae for isobutanol accumulation. However, in this case, isobutanol is only a marginal by-product resulting from valine catabolism. Moreover, biosynthesis is split up between mitochondria and the cytosol. The anabolic reactions starting from pyruvate are part of the valine biosynthetic route and are located in the mitochondrial matrix, while the remainder of the pathway starting from 2-ketoisovalerate (the Ehrlich pathway) is situated in the cytosol. Brat et al. [7] transferred all mitochondrial enzymes to the cytosol, resulting in a ninefold product increase. By further gene and pathway optimization, a yield of 0.63 g/L was obtained, at that time surpassing all previous reports on isobutanol production in yeast. Yet, Avalos et al. [2] followed the opposite strategy and compartmentalized all enzymes in the mitochondria by linking them to the N-terminal mitochondrial localization signal from the cytochrome c oxidase subunit IV. They claim that introduction of the Ehrlich pathway into the mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%). Targeting the enzymes to the mitochondria should achieve greater local enzyme concentrations and increased availability of intermediates. However, it is worth mentioning that the overall isobutanol yields were similar to the ones obtained by Brat et al. Also itaconic acid was designated by the US department of energy as a high potential biochemical building block. This unsaturated dicarboxylic acid can be microbially produced by Aspergillus sp. starting from the Krebs cycle intermediate cis-aconitate (Fig. 3). A. niger can be converted to an itanonic acid production platform by heterologous expression of the cytosolic cis-aconitate decarboxylase from A. terreus and improved productivity is obtained when aconitase, converting citric acid to cis-aconitate is introduced as well. Blumhoff et al. overexpressed the two enzymes either in the cytosol or in the mitochondria to investigate the effect of enzyme targeting [6]. In line with the findings of Avalos et al., they concluded that mitochondrial expression doubles the productivity compared to cytosolic activity. Moreover, they noticed that combined expression could further increase the product titer until 1.4 g/L. Fig. 3 Open in new tabDownload slide Key biochemical processes involved in itaconic acid production in A. terreus; citrate synthase (CS), aconitase (ACO), mitochondrial carrier protein (MTT), cis-aconitate decarboxylase (CAD), major facilitator superfamily transporter (MFS) Others focused on the natural producer A. terreus and investigated the different boundaries the intermediates and products need to conquer. The mitochondrial carrier protein gene mttA was found in the A. terreus itaconic acid gene cluster and turned out to preferentially transport cis-aconitic acid over the mitochondrial membrane, with, respectively, a reduced and no activity towards citric acid and itaconic acid. Heterologous expression in A. niger resulted in a clear accumulation of aconitic acid and 2 g/L of itaconic acid was obtained with the best mutant [30, 51]. In the same gene cluster, a putative Major Facilitator Superfamily transporter gene, mfsA was found. Overexpression of this single gene in A. terreus led to a 5% increased production as compared to the wild-type strain, resulting in about 84 g/L for the best mutant [20]. Peroxisomes As their name suggests, peroxisomes are organelles harboring enzymes dealing with reactive oxygen species, such as hydrogen peroxide. However, in lower eukaryotes, they are mainly associated with β-oxidation of fatty acids. Many β-oxidation intermediates are interesting building blocks for green chemistry, but in contrast to bacterial systems, again, boundaries need to be conquered. Certain bacteria, such as Pseudomonas sp., produce polyhydroxyalkanoates (PHA), biopolymers with industrial relevance composed of β-hydroxy fatty acids originating from β-oxidation which are polymerized by a PHA-synthase. In bacteria, the whole process occurs cytosolic and PHA accumulates in the cells [34]. Yet, higher volumetric yields could be achieved when producing PHA in organisms with bigger cells or generating more biomass. Here yeasts come into the picture. Nevertheless, when introducing PHA synthesis in yeast species, one needs to deal with the peroxisomes. An elegant way to tackle this is targeting the PHA-synthase towards these organelles by adding a C-terminal Peroxisomal Targeting Signal (PKS1) which only consists of the three amino acids SKL. This results in intracellular PHA synthesis in yeast [16, 27, 42]. In a similar fashion, also medium-chain fatty alcohols can be derived from the eukaryotic β-oxidation pathway. By targeted expression of a fatty acyl-CoA reductase in the S. cerevisiae peroxisome, the medium-chain fatty acyl-CoA molecules are hijacked and converted to medium-chain fatty alcohols [48]. Overexpression of the PEX7 protein, mediating enzyme import in the peroxisomes, could further increase the yields [48]. Membrane vesicles Many organisms use membrane vesicles to deliver cargo to the environment and among them also Gram-negative and positive bacteria and fungi. Bacterial membrane vesicles were already described 50 years ago, but are until date not really explored concerning industrial biotechnology applications. Membrane vesicles typically range in size from 20 to 400 nm and are involved in various functions, such as virulence, defense, biofilm formation, communication and horizontal gene transfer as becomes clear from their specific cargo: lipopolysaccharides (LPS), phospholipids, specific proteins or enzymes, and nucleotides [57]. It is true that membrane vesicles are mainly associated with virulence factors (e.g., LPS, toxins, such as hemolysine and aerolysine) or drug resistance mechanisms (e.g., β-lactamases), but they enclose some biotechnological potential. They can for instance be used as cell-free vaccines. Furthermore, it is well known that fungi effectively degrade environmental polymers using enzyme cocktails, and vesicular delivery would promote concentrated enzyme release away from the cell surface [11]. This natural event can be mimicked to use membrane vesicles as nanobiocatalysts for enzyme reactions, in a way resembling compartmentalization in cell organelles and hence bringing along the same advantages of enzyme and product concentration. Directing enzymes to the vesicle interior can be achieved by linking the protein of interest with a signal peptide for Tat- or Sec-mediated periplasmatic export [24]. Nonetheless, also the exterior can be targeted by vesicle decoration. Park et al. [40] took advantage of the fact that membrane vesicles naturally contain proteins found in the outer cell membrane. Hence, they assembled a three-enzyme cascade of cellulose degrading enzymes (an endoglucanase, an exoglucanase and a β-glucosidase) on a cohesion-dockerin scaffold and linked it to the outer membrane vesicles by fusion to the ice nucleation protein (INP) anchoring motif (Fig. 4). Isolated E. coli vesicles decorated with these enzymes hydrolyzed cellulose 23-fold faster as compared to free enzymes. The authors also claim that their approach displays superior activity when compared to armed yeast cells displaying the same trivalent cellulosome structure. This most likely results from the higher enzyme-to-volume ratio and improved substrate accessibility of these nanoscale instead of microscale systems [3]. Fig. 4 Open in new tabDownload slide Example of extracellular enzyme display on outer membrane vesicles after [40]; CBM cellulose binding module, INP ice nucleation protein anchoring motive, E1, E2, and E3 different cellulose degrading enzymes For application purposes, the membrane vesicle concentration should ideally be as high as possible. The addition of chemicals, such as EDTA or antibiotics, can govern this, because they weaken the overall membrane structure resulting in vesicle release. Though this often causes undesirable changes in the content of the vesicles. Engineering proteins that affect membranes is a better strategy, and in this respect, the Tol-Pal system that bridges the inner and outer membranes in Gram-negative bacteria is a good target: mutation and disruption of the system lead to loss of outer membrane integrity and membrane vesicles overproduction [36]. Concluding remarks This review covers various aspects of how biological membrane boundaries influence industrial microbiology and biotechnology processes. On the one hand, these boundaries need to be conquered when increased efflux is considered as a strategy to avoid cell toxicity or simply to achieve higher product yields. Here, one can target membrane integrity, efflux pumps or pleiotropic factors. Key issue is, however, the identification of useful transporters, either endogenous or heterologous ones. The amino-acid case study demonstrates that this is often the last missing link in pathway reconstruction. This due to the fact that membrane proteins have been largely neglected as biochemical and analytical methods to study them are far more challenging than the ones for soluble enzymes or proteins. The growing availability of omics data can be of help here: transporters responsible for secondary metabolite secretion are often co-localized in the biosynthetic gene cluster or transcriptome analysis for conditions when efflux is induced can put forward several candidates for upregulation. Large-scale experiments on upregulation, modification or screening of (heterologous) candidate transporters are quite straightforward when the competitive growth assay can be applied, as is the case when dealing with toxic compounds. It becomes more complex when dealing with harmless compounds and no such selection procedure can be applied. A suitable high-throughput technique will for sure accelerate the research, but this depends on the specific compound of interest. Useful features are for instance a quantifiable direct or indirect color production or killing of other organisms like in the case of antibiotics. The availability of high-throughput methods will on the one hand enable screening of large environmental or metagenomic libraries. On the other hand, it will also stimulate more thorough protein engineering of transporters, allowing to find altered specificities or activity levels. Protein engineering can be done in a random way or in a rationalized way targeting specific amino acids or domains. Prerequisite for this latter option is knowledge on the three-dimensional structure and interactions, ideally supported by crystal structures. However, obtaining these for membrane embedded proteins is very difficult, so a lot of powerful input for transporter protein engineering is still lacking. Even if a potential useful transporter is identified, simple overexpression does not always result in the intended increased production. Expression of these membrane proteins should be carefully balanced as overrepresentation can disturb cell growth or normal membrane function (in contrast to the cytosol, membranes only offer a limited two-dimensional space) [58]. This is supported by the natural expression levels of transporters in yeasts when compared to non-transporter genes: 19.3 transcripts per cell versus 31.7 [23]. Instead of looking for the optimal expression level, one can also improve activity to do a better job with the same amount of proteins (as illustrated for AcrAB-TolC). The presence of membranes can also be considered advantage. Localization of enzymes, substrates and intermediates in a small microbial cell factory surrounded by membranes increases the local concentration and reaction fluxes, and safeguards access to cofactors; issues that cannot be guaranteed for biocatalytic processes. Additional subcellular localization evokes ambiguous approaches. On the one hand, one can take advantage of the intensive concentration of the pathway, where interactions with other biochemical processes are often less prominent. On the other hand, subcellular localization is considered undesired if proper transport out of the organelle cannot be secured or if interactions with cytosolic pathways are desired. Export by membrane vesicles has been known for decades, but has until recently not been developed for biotechnological applications. Yet, several strategies are possible to further explore the potential of this mode of transport and delivery. Acknowledgements Sylwia Jezierska received a doctoral (PhD) grant Strategic Basic Research from the Research Foundation Flanders (FWO), Grant Number 151610. The authors wish to thank Barbara Toch for revising the manuscript. References 1. 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TI - Crossing boundaries: the importance of cellular membranes in industrial biotechnology
JF - Journal of Industrial Microbiology and Biotechnology
DO - 10.1007/s10295-016-1858-z
DA - 2017-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/crossing-boundaries-the-importance-of-cellular-membranes-in-industrial-dezTldzUds
SP - 721
EP - 733
VL - 44
IS - 4-5
DP - DeepDyve
ER -