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Chapter 3
Engineering the Microbial Cell Membrane To Improve Bioproduction Laura R. Jarboe,*,1 Jeffery B. Klauda,2 Yingxi Chen,1 Kirsten M. Davis,1 and Miguel C. Santoscoy1 1Department
of Chemical and Biological Engineering, Iowa State University, 2114 Sweeney Hall, 618 Bissell Road, Ames, Iowa 50011, United States 2Department of Chemical and Biomolecular Engineering, University of Maryland, 4418 Stadium Drive, College Park, Maryland 20742, United States *E-mail:
[email protected].
Inhibition of the microbial biocatalyst often limits the attainment of the yields, titers, and rates needed for economic viability production of biorenewable fuels and chemicals. In many cases, this toxicity can be attributed to damage of the lipid-rich microbial membrane. Just as the composition of a reaction vessel can be altered to improve its resistance to corrosion, the composition of the microbial cell membrane can be altered in order to decrease its vulnerability to this damage. Contrastingly, in some cases the membrane can be weakened in order to increase the space available for intracellular accumulation of a product, or the overall abundance of the membrane can be increased in order to serve as a sink for a membrane-associated product molecule. This chapter reviews efforts to engineer the microbial cell membrane, with a focus on engineering strategies that improve bioproduction of fuels and chemicals.
Introduction Microbial Inhibition Often Limits Bioproduction Inhibition of the microbial biocatalyst, either by the product or by compounds in the biomass-derived sugar stream, often limits the attainment of economic viability for bioproduction processes (1–6). This toxicity is often attributed © 2018 American Chemical Society Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
to damage of the cell membrane (7–18). For the macro scale, if a reaction vessel is corroded by its contents, a typical strategy would be to change the vessel composition. The feasibility of membrane engineering for improving fermentative production of biochemicals has been established and is reviewed here. This membrane engineering is broken into broad membrane components of the lipid tails, the lipid heads, and the membrane-associated proteins and sugars. A variety of attractive biobased products, such as butanol, fatty acids, and styrene, are toxic to the microbial biocatalyst (19–21). Hydrolysis and pyrolysis are attractive means of depolymerizing lignocellulosic biomass into fermentable monomers, but the utilization of these sugars is hindered by the presence of compounds that are inhibitory to the biocatalyst (3, 22–24). This feedstock-associated inhibition means that the growth and metabolic activity of the biocatalyst are also limited. Thus, both ends of biocatalyst metabolism are impacted by inhibition: the utilization of cheap, biomass-derived monomers is limited by inhibitory compounds in the feedstock, while commercially viable production of biorenewable fuels and chemicals is limited by product toxicity. Multiple studies have demonstrated that improving microbial tolerance of such inhibitors can improve bioproduction (4–6). However, the development of resistant organisms often relies on evolutionary or library-based methods (10, 25–31). The use of evolutionary or library-based methods for increasing tolerance is most effective when production of an inhibitory compound is the only means for the microbial biocatalyst to maintain redox balance and/or adenosine triphosphate (ATP) production, and thus growth serves as a selection marker for production and tolerance (25, 32–34). However, evolving a strain for increased tolerance of an exogenously-provided inhibitor with the goal of improving production of this compound has been met with mixed results. Thus, as we strive to improve production of inhibitory compounds that are not coupled with microbial growth, methods of strain improvement that do not rely on evolution or selection-based methods are needed. Inhibition Is Often Due To Membrane Damage The microbial cell membrane plays a vital role in many cellular processes. Membrane damage is often evident as decreased integrity and/or perturbed fluidity. Decreased integrity of the membrane can be measured via the release of metabolites, such as Mg2+ (7, 8) or lipoproteins (35) from the cell, or as the entry of membrane-impermeable molecules, such as the SYTOX nucleic acid dye, into the cell (9, 36). Integrity of the inner and outer membranes can also be quantified individually, as well as visualization of membrane deformation in the presence of membrane-damaging molecules, such as butanol (35). Fluidity is measured via the membrane polarization, which can be quantified with an amphiphilic fluorescent probe such as 1,6-diphenyl-1,3,5-hexatriene (DPH) (8, 37, 38). An increase in membrane polarization indicates increased membrane rigidity and decreased polarization indicates increased membrane fluidity (39). Too large of a change in either direction can be problematic. These types of membrane damage have been noted during characterization of bioproduction (8, 36, 40, 41). For example, membrane fluidity was measured 26 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
during fatty acid challenge and production (8, 37) and membrane integrity was measured during the challenge with a variety of organic acids (16) and alcohols (17). Finally, a titer-dependent decrease in membrane integrity was observed during styrene production (9). Membrane Engineering Can Combat Membrane Damage If the microbial biocatalyst is envisioned as a miniature reaction vessel, then the cell membrane corresponds to the walls of this reaction vessel. Corrosion of a reaction vessel by its contents is problematic and typically, an alternative vessel whose walls are resistant to this corrosion would be sought. The conclusion of this analogy is that the composition of the microbial cell membrane should be altered in order to increase resistance to membrane damage. The alterations required for increasing resistance are expected to vary according to the chemical properties of the inhibitor; they are not an expectation of a single strategy for addressing all membrane-damaging inhibitors. An extreme version of this approach is to provide cells with an exogenous membrane via encapsulation in a polymeric shell (42). This chapter focuses on changes to the cell membrane in order to improve the structural properties related to bioproduction. We consider three distinct aspects of the membrane structure (Figure 1): the membrane-associated lipids, including but not limited to the lipid tails; the head of the lipid molecule; and the proteins and sugars within and associated with the membrane. The discussion focuses on engineering strategies that have been demonstrated to improve bioproduction, with key examples summarized in Table 1.
Figure 1. Membrane engineering strategies can target the lipid tails, the lipid head groups, and the membrane-associated proteins and sugars. 27 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Table 1. Examples of genetically implemented membrane modification strategies that improved bioproduction Organism
Engineering Strategy
Effect
E. coli
Decreased incorporation of medium chain fatty acids into the membrane via deletion of aas (40)
Increased titer of total free fatty acids in rich media
E. coli
Enabled production of novel lipid tails via tuned recombinant expression of cis/trans isomerase (Cti) (37)
Increased titer of octanoic acid in minimal media, increased titer of styrene
E. coli
Increased relative abundance of PE phospholipid head, decreased relative abundance of PG, via increased expression of pssA (43)
Increased titer of total free fatty acids in minimal media
E. coli
Increased expression of long-chain fatty acid importer FadL to enable recovery of fatty acids for membrane assembly (44)
Increased titer of C14 fatty acids in minimal media
E. coli (45), H. campaniensis (46)
Tuned expression of actin-like cytoskeleton protein MreB to weaken the cytoskeleton and enable enlarged bacterial size
Increased PHB titer and yield
E. coli
Decreased expression of cell wall synthesis genes, such as murE and murD, to weaken the cell wall (47)
Increased conversion efficiency of glucose to PHB
E. coli
Increased expression of membrane-bending proteins, such as MtlA and ALmgs, and membrane synthesis proteins, such as PlsB and PlsC (48)
Increased titer and specific production (mg/g) of beta-carotene in rich media
B. subtilis
Increased abundance of CL via expression tuning of pgsA and clsA and altered distribution of CL by expression tuning of ftsZ (49)
Increased titer of hyaluronic acid in rich media
Lipid Engineering The lipid tails in E. coli consist of straight-chain cis-mono-unsaturated fatty acids (C16:1 and C18:1) (Figure 2), straight-chain saturated fatty acids (C12:0, C14:0, C16:0, and C18:0), and the cyclopropane fatty acids C17cyc and C19cyc. Two commonly used metrics for summarizing the lipid tail distribution are the relative abundance of unsaturated fatty acids and the average lipid length. The relationship between membrane lipid composition and tolerance of membranedamaging compounds, such as ethanol, has been known for more than 40 years (50). However, efforts to engineer the lipid distribution are more recent. 28 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Figure 2. Structures of select molecules related to membrane engineering. (1) octanoic acid (C8), (2) styrene, (3) n-butanol, (4) beta-carotene, (5) ergosterol, (6) ubiquinone, (7) oleic acid, C18:1, cis, (8) elaidic acid, C18:1, trans, (9) linoleic acid, (10) phosphatidylethanoloamine, (11) diacylglycerol, (12) ceramide, (13) DGGGP, (14) COE1-5C.
Altering the relative distribution of saturated and unsaturated lipid tails in the E. coli membrane has been shown to increase tolerance toward ethanol (51), fatty acids (36), and hexane (52). This strategy was effective in decreasing the membrane leakage caused by fatty acids, but fatty acid production was not increased (36). Tuning of the relative abundance of saturated and unsaturated lipid tails has also been demonstrated in S. cerevisiae (38), ranging from 20% of acyl chains being unsaturated to 80% being unsaturated. The increase in the relative abundance of unsaturated lipids was demonstrated to increase the membrane fluidity (38), similar to the familiar tendency of cooking oils to trend to higher fluidity as the degree of unsaturation increases. 29 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Studies of fatty acid production have concluded that the incorporation of shorter fatty acids as phospholipid tails is detrimental to membrane function. Elimination of the pathway responsible for this incorporation improved fatty acid tolerance and production in rich media (40). S. cerevisiae cells challenged with exogenous octanoic acid (C8) (Figure 2) were observed to show a dose-dependent increase in the average length of membrane lipids (7). Media supplementation with oleic acid (C18:1) (Figure 2) was shown to increase growth in the presence of C8 and to decrease the associated loss of membrane integrity, though attempts to engineer S. cerevisiae for sufficiently increased membrane oleic acid content without this exogenous supplement were not successful (7). A strain of S. cerevisiae expressing a mutant form of the Acc1 acetyl-CoA carboxylase was found to have altered membrane lipid composition, both in the form of increased average length and an increase in saturated lipid tails (53). This strain was found to have increased tolerance of exogenous hexanoic acid, C8, 2-propanol, and n-butanol, as well as increased membrane integrity during C8 challenge (53). Other genetic manipulation targets for altering the degree of lipid unsaturation and average lipid length have been demonstrated in S. cerevisiae (54). Cyclopropane fatty acids are often implicated in characterization of inhibited or evolved E. coli strains (55–58). A variety of studies have described engineering of the cyclopropane fatty acid content, including fatty acid tolerance in E. coli (59) and enabling the production of cyclopropane fatty acids in yeast (7). Strains deficient in cyclopropane fatty acid production have increased sensitivity to stressors such as heat, pressure, and inorganic acid stress (60, 61), but increasing the production of cyclopropane fatty acids usually does not improve resistance to these stressors (7, 8). It has been demonstrated that enabling E. coli to isomerize some of the native cis unsaturated fatty acid (CUFA) lipid tails to trans unsaturated fatty acids (TUFA) (Figure 2) increased tolerance to several membrane-damaging compounds and conditions (62). TUFA production was enabled by expression of the Pseudomonas putida cis/trans isomerase enzyme (Cti). Strains with a TUFA/CUFA ratio of approximately 0.085 had the largest increase in specific growth rate in the presence of octanoic acid and also showed an approximate 40% increase in fatty acid titers in minimal media. This membrane engineering strategy was found to primarily impact membrane fluidity. Specifically, the membrane polarization increased, indicating an increase in membrane rigidity, which presumably mitigates the fluidizing effect of the fatty acids. Further characterization showed that this strain also had increased specific growth rate relative to the non-TUFA producing control when challenged with butanol, styrene, and 42 °C, as well as a significant increase in styrene production titers. Some organisms also produce sphingolipids, which contain an amine group and an alcohol group (63). Ceramides are a subset of sphingolipids (Figure 2). Strains of Zygosaccharomyces bailii that were treated with myriocin, known to decrease the production of sphingolipids, showed increased sensitivity to acetic acid, formic acid, and lactic acid (64), and ceramides were found to have a dramatic effect on the properties of in silico membranes (65). Genetic engineering strategies for reducing the relative abundance of sphingolipids in S. cereivisae have been demonstrated, with a negative impact on cell viability (54). 30 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
In addition to the fatty acid tails of the phospholipid molecules, microbial membranes may also contain other types of lipid molecules. E. coli was observed to have a drastic increase in abundance of ubiquinone (Figure 2) in the cell membrane during osmotic challenge, and in vitro characterization demonstrated that increased ubiquinone content was associated with increased liposome stability (66). Ubiquionone is a 1,4-benzoquinone with a side chain of isoprenoid subunits and thus does not fit the standard conception of a lipid, but it is membrane-associated hydrocarbon. Sterols are steroids with a hydroxyl group at position three of the A ring, one of the four rings that define the steroid group. Strains of S. cerevisiae evolved for thermotolerance or butanol tolerance (67) had altered distribution and/or abundance of certain sterols, such as ergosterol (Figure 2). In addition to the genetic modification strategies described above, supplementation with exogenous lipid-type molecules have provided further proof of concept for engineering of the membrane at the lipid composition level. Media supplementation with the polyunsaturated omega-6 18-carbon fatty acid linoleic acid increased the fluidity of S. cerevisiae during beta-carotene production, resulting in an increase in beta-carotene titers (68). Provision of exogenous COE1-5C (Figure 2), an oligo-polyphenylene-vinylene conjugated oligoelectrolyte, increased the specific growth rate of E. coli during challenge with relatively high (3.5 vol%) concentrations of butanol (69). Supplementation of E. coli with cedar wood oil significantly increased the membrane fluidity and increased production of menaquinone (18). Lipid Head Group Engineering Lipid tails make up the bulk of the membrane interior and are attached to head groups. E. coli natively produces three different types of phospholipid head molecules: phosphatidylethanoloamine (PE) (Figure 2), phosphatidylglycerol (PG), and cardiolipin (CL). Each of these three molecules has different chemical properties. Perturbing the relative abundance of these head groups in E. coli by altering the expression of the genes encoding their biosynthesis pathways can substantially change the membrane composition and properties. For example, a strain engineered for increased PE content showed a substantial increase in octanoic acid tolerance and fatty acid production (43). Further characterization of this strain showed increased resistance to the membrane permeabilization and intracellular acidification caused by exogenously provided octanoic acid and a greater than 20% change in membrane surface potential (43). Finally, this engineered strain showed increased tolerance of other inhibitors relevant to cost-effective bioproduction, such as the biomass-derived inhibitors furfural and acetic acid. Studies performed in B. subtilis have shown that altering the relative abundance of the native phospholipid head groups increased secretion of alpha-amylase (70), increased osmotic tolerance (71), and increased sensitivity to certain antimicrobial compounds (72). Alteration of the native phospholipid head distribution in S. cerevisiae increased growth and decreased the relative abundance of peroxidized lipids during growth in the presence of lactic acid at 31 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
low pH (73). E. coli was engineered for production of nonnative glycolipid head group diacylglycerol (DAG) (Figure 2) in the absence of production of the PE phospholipid head (74). The engineered strains were shown to have altered cell length and altered resistance to some membrane-damaging antibiotics (74). The membranes of Archaea differ from Bacteria and Eukarya in a variety of ways. One of these differences is the connection of the lipid tail to the glycerol-phosphate backbone with an ether linkage, as opposed to the ester linkage used by bacteria and eukaryotes. Additionally, the lipid tails of the archaeal membrane consist of isoprenoids instead of fatty acids, where the isoprenoids contain multiple unsaturated bonds and methyl groups. E. coli was engineered to express an isoprenoid ether lipid biosynthesis pathway, resulting in production of digeranylgeranylglyceryl phosphate (DGGGP) (Figure 2) (75). More recently, E. coli was engineered to produce the ether phospholipids archaetidylethanolamine (AE) and archaetidylglycerol (AG) (76, 77). E. coli strains producing the AG-containing membrane were demonstrated to have increased cell length, heat tolerance, and tolerance to freeze/thaw (76). Membrane Proteins and Lipopolysaccharide Engineering In addition to lipid molecules, the microbial cell membrane is rich in proteins and a variety of sugar-type molecules. These are also important aspects of the membrane properties and therefore, the membrane function. Proteins involved in membrane assembly, repair, and structural properties have also proven to be a very promising method for altering membrane properties and improving bioproduction. It has been demonstrated multiple times that enabling export of inhibitory products can improve production of those compounds (78, 79). Tolerance of inhibitory compounds can also be increased by preventing their entry into the cell. For example, deletion of the OmpF porin increased octanoic acid tolerance (80). This strategy represents an increase in tolerance by decreasing the intracellular concentration of the inhibitor and would not fall under what we classify here as “membrane engineering.” However, identification and engineering of product transporters becomes increasingly important as membrane robustness is increased. Products that previously exited the cell by passing through the membrane can possibly accumulate to toxic levels in strains subjected to improvement by membrane engineering. More simply, the desire to produce “stronger walls” leads to a need for “bigger doors.” In contrast to the standard example of increasing exporter abundance in order to increase production, increasing the abundance of the long-chain fatty acid importer FadL was shown to increase fatty acid production (44). This increased expression of FadL not only increased fatty acid titers, but also increased the membrane lipid mass per dry cell weight by more than 10% (44). Presumably, this increased expression of FadL allowed cells to recoup some of the long-chain fatty acids needed for membrane assembly that were being lost via the fatty acid efflux system. Unlike many of the bioproduced bulk fuels and chemicals, which are produced internally and then excreted from the microbial cell factory, some bioproducts accumulate inside the cell as inclusion bodies or accumulate in the membrane. 32 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
In these cases, membrane engineering can be used to increase the storage capacity for the product and thereby improve bioproduction. Using polyhydroxybutyrate (PHB) as a model inclusion body, it was demonstrated that tuning of the expression of the actin-like protein MreB was able to increase PHB production in both E. coli (45) (Figure 3) and Halomonas campaniensis (47). In E. coli, mreB expression was placed under control of SulA (45), where SulA inhibits cell division. The altered expression of MreB weakened the cystoskeleton, resulting in larger cells with increased sphericity (45). In H. campaniensis, mreB expression occurred from a temperature-sensitive plasmid (47). MreB has also been described as a frequent location of point mutations when E. coli was evolved for osmotolerance (81).
Figure 3. TEM morphology of PHB-producing E. coli with expression tuning of mreB to increase the available cell volume for inclusion bodies accumulation. Left, control strain. Right, engineered strain. Scale bar, 5 μm. Reproduced with permission from (45). Copyright 2015 Elsevier.
The idea of weakening the cell wall to increase the available space for inclusion bodies was expanded with additional genetic targets in E. coli (46). Increased expression of certain cell wall synthesis genes increased the Young’s Modulus and thickness of the cell membrane, resulting in a decrease in PHB content per dry mass. Decreased expression of these genes increased PHB abundance. Finally, work in H. campaniesis (47) showed that tuned expression of cell division protein FtsZ increased cell volume by increasing cell length, increasing PHB production. The work described above aimed to increase the volume available for inclusion bodies. Some metabolic products partition into the cell membrane itself, and increasing the abundance of the membrane could increase the production of these compounds. This approach was used to increase production of beta-carotene by E. coli (48). Specifically, increased expression of membrane-bending proteins and the membrane synthesis pathway resulted in an increase in both the bulk beta-carotene titer and the amount of beta-carotene produced per biomass (48). The sugars and proteins produced on or near the cell surface also contribute to the physical properties and performance of the organism (82). Strains of E. 33 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
coli containing a single amino acid mutation in the carbon storage regulator CsrA showed increased membrane integrity in the presence of biomass-derived pyrolytic sugars (83). This change in membrane integrity was associated with a twofold decrease in the abundance of extracellular proteins, but no change in the abundance of extracellular polysaccharides (83). The surface display of proteins with various physical properties, such as hydrophobicity, altered the tolerance of S. cerevisiae to various inhibitors, such as nonane (84). Perturbation of the core oligosaccharide lipopolysaccharide biosynthesis pathway in E. coli was demonstrated to alter the cell surface hydrophobicity, outer membrane permeability, and sensitivity to various antibiotics (85).
In Vitro and in Silico Characterization The work described thus far demonstrates the potential for membrane engineering to improve bioproduction and the range of membrane composition and properties that can currently be achieved. However, characterization of membranes in vitro and in silico can provide further insight for engineering strategies, particularly in terms of membrane compositions that are difficult to attain in vivo. As described above, exogenous supplementation with the oligopolyphenylene-vinylene conjugated oligoelectrolyte COE1-5C increased the growth of E. coli during challenge with butanol (69). COE1-5C was selected as an exogenous membrane insertion molecule based on molecular dynamic simulations of lipid bilayers in the presence of butanol (69). As also described above, altering the abundance of the native phospholipid head groups in E. coli increased tolerance and production of fatty acids (43). However, interpretation of the changes in the membrane composition was complicated by the fact that in addition to changes in the head group distribution, the lipid tails also had altered distribution. Molecular dynamic simulations allowed separation of these changes, leading to the conclusion that the increased PE content was responsible for increased membrane thickness, but that the changes in headgroup and lipid distribution both contributed to the increased hydrophobic core thickness (43). Molecular dynamics simulations have also led to increased insight in terms of the effect of membrane composition on interactions with various membrane-damaging compounds (64, 86–89). Experimental characterization of in vitro systems also provides insight into membrane engineering strategies. Disruption of supported bilayers of varying composition can be observed over time (90). This type of finely detailed experimental characterization of supported bilayers can lead to increased discrimination of the mode of action of different membrane-damaging compounds (91). Advances are also being made in the assembly of lipid vesicles for experimental characterization (92, 93).
34 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Concluding Remarks The work reviewed here demonstrates the potential of membrane engineering to improve bioproduction. This engineering can have a variety of intentions, such as to combat the damage imposed by a specific molecule, to weaken the membrane to increase the available space for intracellular species, or to increase the amount of available membrane for product accumulation. The design, build, test, learn cycle (26) for membrane engineering can be further strengthened by characterization of evolved strains and proteins that currently have unknown function (94). Continued efforts to combine experimental in vitro systems, whole-cell systems, and in silico modeling are expected to lead to further gains in this area.
Acknowledgments This work was supported in part by the NSF Center for Biorenewable Chemicals Engineering Research Center (EEC-0831570), Energy for Sustainability program (CBET-1604576), and the USDA National Institute of Food and Agriculture program (2017-67021-26137). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the editors, H.N. Cheng, Richard A. Gross, and Patrick B. Smith, for the invitation to contribute to this work.
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