Strategic nutrient sourcing for biomanufacturing intensification.

The successful design of economically viable bioprocesses can help to abate global dependence on petroleum, increase supply chain resilience, and add value to agriculture. Specifically, bioprocessing provides the opportunity to replace petrochemical production methods with biological methods and to develop novel bioproducts. Even though a vast range of chemicals can be biomanufactured, the constraints on economic viability, especially while competing with petrochemicals, are severe. There have been extensive gains in our ability to engineer microbes for improved production metrics and utilization of target carbon sources. The impact of growth medium composition on process cost and organism performance receives less attention in the literature than organism engineering efforts, with media optimization often being performed in proprietary settings. The widespread use of corn steep liquor as a nutrient source demonstrates the viability and importance of "waste" streams in biomanufacturing. There are other promising waste streams that can be used to increase the sustainability of biomanufacturing, such as the use of urea instead of fossil fuel-intensive ammonia and the use of struvite instead of contributing to the depletion of phosphate reserves. In this review, we discuss several process-specific optimizations of micronutrients that increased product titers by twofold or more. This practice of deliberate and thoughtful sourcing and adjustment of nutrients can substantially impact process metrics. Yet the mechanisms are rarely explored, making it difficult to generalize the results to other processes. In this review, we will discuss examples of nutrient sourcing and adjustment as a means of process improvement. ONE-SENTENCE SUMMARY: The potential impact of nutrient adjustments on bioprocess performance, economics, and waste valorization is undervalued and largely undercharacterized.

Production of cholesterol-like molecules impacts Escherichia coli robustness, production capacity, and vesicle trafficking.

The economic viability of bioprocesses is constrained by the limited range of operating conditions that can be tolerated by the cell factory. Engineering of the microbial cell membrane is one strategy that can increase robustness and thus alter this range. In this work, we targeted cellular components that contribute to maintenance of appropriate membrane function, such as: flotillin-like proteins, membrane structural proteins, and membrane lipids. Specifically, we exploited the promiscuity of squalene hopene cyclase (SHC) to produce polycyclic terpenoids with properties analogous to cholesterol. Strains producing these cholesterol-like molecules were visualized by AFM and height features were observed. Production of these cholesterol-like molecules was associated with increased tolerance towards a diversity of chemicals, particularly alcohols, and membrane trafficking processes such as lipid droplet accumulation and production of extracellular vesicles. This engineering approach improved the production titers for wax-esters and ethanol by 80- and 10-fold, respectively. Expression of SHC resulted in the production of steroids. Strains engineered to also express truncated squalene synthase (tERG9) produced diplopterol and generally did not perform as well. Increased expression of several membrane-associated proteins, such as YqiK, was observed to impact vesicle trafficking and further improve tolerance relative to SHC alone, but did not improve bio-production. Deletion of YbbJ increased lipid droplet accumulation as well as production of intracellular wax esters. This work serves as a proof of concept for engineering strategies targeting membrane physiology and trafficking to expand the production capacity of microbial cell factories.

Extrapolation of design strategies for lignocellulosic biomass conversion to the challenge of plastic waste.

The goal of cost-effective production of fuels and chemicals from biomass has been a substantial driver of the development of the field of metabolic engineering. The resulting design principles and procedures provide a guide for the development of cost-effective methods for degradation, and possibly even valorization, of plastic wastes. Here, we highlight these parallels, using the creative work of Lonnie O'Neal (Neal) Ingram in enabling production of fuels and chemicals from lignocellulosic biomass, with a focus on ethanol production as an exemplar process.

Survey of nonconventional yeasts for lipid and hydrocarbon biotechnology.

Nonconventional yeasts have an untapped potential to expand biotechnology and enable process development necessary for a circular economy. They are especially convenient for the field of lipid and hydrocarbon biotechnology because they offer faster growth than plants and easier scalability than microalgae and exhibit increased tolerance relative to some bacteria. The ability of industrial organisms to import and metabolically transform lipids and hydrocarbons is crucial in such applications. Here, we assessed the ability of 14 yeasts to utilize 18 model lipids and hydrocarbons from six functional groups and three carbon chain lengths. The studied strains covered 12 genera from nine families. Nine nonconventional yeasts performed better than Saccharomyces cerevisiae, the most common industrial yeast. Rhodotorula toruloides, Candida maltosa, Scheffersomyces stipitis, and Yarrowia lipolytica were observed to grow significantly better and on more types of lipids and lipid molecules than other strains. They were all able to utilize mid- to long-chain fatty acids, fatty alcohols, alkanes, alkenes, and dicarboxylic acids, including 28 previously unreported substrates across the four yeasts. Interestingly, a phylogenetic analysis showed a short evolutionary distance between the R. toruloides, C. maltosa, and S. stipitis, even though R. toruloides is classified under a different phylum. This work provides valuable insight into the lipid substrate range of nonconventional yeasts that can inform species selection decisions and viability of lipid feedstocks.

A systematic framework for using membrane metrics for strain engineering.

The cell membrane plays a central role in the fitness and performance of microbial cell factories and therefore it is an attractive engineering target. The goal of this work is to develop a systematic framework for identifying membrane features for use as engineering targets. The metrics that describe the composition of the membrane can be visualized as "knobs" that modulate various "outcomes", such as physical properties of the membrane and metabolic activity in the form of growth and productivity, with these relationships varying depending on the condition. We generated a set of strains with altered membrane lipid composition via expression of des, fabA and fabB and performed a rigorous characterization of these knobs and outcomes across several individual inhibitory conditions. Here, the knobs are the relative abundance of unsaturated lipids and lipids containing cyclic rings; the average lipid length, and the ratio of linear and non-linear lipids (L/nL ratio). The outcomes are membrane permeability, hydrophobicity, fluidity, and specific growth rate. This characterization identified significant correlations between knobs and outcomes that were specific to individual inhibitors, but also were significant across all tested conditions. For example, across all conditions, the L/nL ratio is positively correlated with the cell surface hydrophobicity, and the average lipid length is positively correlated with specific growth rate. A subsequent analysis of the data with the individual inhibitors identified pairs of lipid metrics and membrane properties that were predicted to impact cell growth in seven modeled scenarios with two or more inhibitors. The L/nL ratio and the membrane hydrophobicity were predicted to impact cell growth with the highest frequency. We experimentally validated this prediction in the combined condition of 42 °C, 2.5 mM furfural and 2% v/v ethanol in minimal media. Membrane hydrophobicity was confirmed to be a significant predictor of ethanol production. This work demonstrates that membrane physical properties can be used to predict the performance of biocatalysts in single and multiple inhibitory conditions, and possibly as an engineering target. In this manner, membrane properties can possibly be used as screening or selection metrics for library- or evolution-based strain engineering.

Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories.

Adaptive laboratory evolution is often used to improve the performance of microbial cell factories. Reverse engineering of evolved strains enables learning and subsequent incorporation of novel design strategies via the design-build-test-learn cycle. Here, we reverse engineer a strain of Escherichia coli previously evolved for increased tolerance of octanoic acid (C8), an attractive biorenewable chemical, resulting in increased C8 production, increased butanol tolerance, and altered membrane properties. Here, evolution was determined to have occurred first through the restoration of WaaG activity, involved in the production of lipopolysaccharides, then an amino acid change in RpoC, a subunit of RNA polymerase, and finally mutation of the BasS-BasR two component system. All three mutations were required in order to reproduce the increased growth rate in the presence of 20 mM C8 and increased cell surface hydrophobicity; the WaaG and RpoC mutations both contributed to increased C8 titers, with the RpoC mutation appearing to be the major driver of this effect. Each of these mutations contributed to changes in the cell membrane. Increased membrane integrity and rigidity and decreased abundance of extracellular polymeric substances can be attributed to the restoration of WaaG. The increase in average lipid tail length can be attributed to the RpoCH419P mutation, which also confers tolerance to other industrially-relevant inhibitors, such as furfural, vanillin and n-butanol. The RpoCH419P mutation may impact binding or function of the stringent response alarmone ppGpp to RpoC site 1. Each of these mutations provides novel strategies for engineering microbial robustness, particularly at the level of the microbial cell membrane.

Streamlined assessment of membrane permeability and its application to membrane engineering of Escherichia coli for octanoic acid tolerance.

The economic viability of bio-production processes is often limited by damage to the microbial cell membrane and thus there is a demand for strategies to increase the robustness of the cell membrane. Damage to the microbial membrane is also a common mode of action by antibiotics. Membrane-impermeable DNA-binding dyes are often used to assess membrane integrity in conjunction with flow cytometry. We demonstrate that in situ assessment of the membrane permeability of E. coli to SYTOX Green is consistent with flow cytometry, with the benefit of lower experimental intensity, lower cost, and no need for a priori selection of sampling times. This method is demonstrated by the characterization of four membrane engineering strategies (deletion of aas, deletion of cfa, increased expression of cfa, and deletion of bhsA) for their effect on octanoic acid tolerance, with the finding that deletion of bhsA increased tolerance and substantially decreased membrane leakage.

Promoting microbial utilization of phenolic substrates from bio-oil.

The economic viability of the biorefinery concept is limited by the valorization of lignin. One possible method of lignin valorization is biological upgrading with aromatic-catabolic microbes. In conjunction, lignin monomers can be produced by fast pyrolysis and fractionation. However, biological upgrading of these lignin monomers is limited by low water solubility. Here, we address the problem of low water solubility with an emulsifier blend containing approximately 70 wt% Tween® 20 and 30 wt% Span® 80. Pseudomonas putida KT2440 grew to an optical density (OD600) of 1.0 ± 0.2 when supplied with 1.6 wt% emulsified phenolic monomer-rich product produced by fast pyrolysis of red oak using an emulsifier dose of 0.076 ± 0.002 g emulsifier blend per g of phenolic monomer-rich product. This approach partially mitigated the toxicity of the model phenolic monomer p-coumarate to the microbe, but not benzoate or vanillin. This study provides a proof of concept that processing of biomass-derived phenolics to increase aqueous availability can enhance microbial utilization.

Lessons in Membrane Engineering for Octanoic Acid Production from Environmental Escherichia coli Isolates.

Fermentative production of many attractive biorenewable fuels and chemicals is limited by product toxicity in the form of damage to the microbial cell membrane. Metabolic engineering of the production organism can help mitigate this problem, but there is a need for identification and prioritization of the most effective engineering targets. Here, we use a set of previously characterized environmental Escherichia coli isolates with high tolerance and production of octanoic acid, a model membrane-damaging biorenewable product, as a case study for identifying and prioritizing membrane engineering strategies. This characterization identified differences in the membrane lipid composition, fluidity, integrity, and cell surface hydrophobicity from those of the lab strain MG1655. Consistent with previous publications, decreased membrane fluidity was associated with increased fatty acid production ability. Maintenance of high membrane integrity or longer membrane lipids seemed to be of less importance than fluidity. Cell surface hydrophobicity was also directly associated with fatty acid production titers, with the strength of this association demonstrated by plasmid-based expression of the multiple stress resistance outer membrane protein BhsA. This expression of bhsA was effective in altering hydrophobicity, but the direction and magnitude of the change differed between strains. Thus, additional strategies are needed to reliably engineer cell surface hydrophobicity. This work demonstrates the ability of environmental microbiological studies to impact the metabolic engineering design-build-test-learn cycle and possibly increase the economic viability of fermentative bioprocesses.IMPORTANCE The production of bulk fuels and chemicals in a bio-based fermentation process requires high product titers. This is often difficult to achieve, because many of the target molecules damage the membrane of the microbial cell factory. Engineering the composition of the membrane in order to decrease its vulnerability to this damage has proven to be an effective strategy for improving bioproduction, but additional strategies and engineering targets are needed. Here, we studied a small set of environmental Escherichia coli isolates that have higher production titers of octanoic acid, a model biorenewable chemical, than those of the lab strain MG1655. We found that membrane fluidity and cell surface hydrophobicity are strongly associated with improved octanoic acid production. Fewer genetic modification strategies have been demonstrated for tuning hydrophobicity relative to fluidity, leading to the conclusion that there is a need for expanding hydrophobicity engineering strategies in E. coli.

Engineering Escherichia coli membrane phospholipid head distribution improves tolerance and production of biorenewables.

Economically competitive microbial production of biorenewable fuels and chemicals is often impeded by toxicity of the product to the microbe. Membrane damage is often identified as a major mechanism of this toxicity. Prior efforts to strengthen the microbial membrane by changing the phospholipid distribution have largely focused on the fatty acid tails. Herein, a novel strategy of phospholipid head engineering is demonstrated in Escherichia coli. Specifically, increasing the expression of phosphatidylserine synthase (+pssA) was found to significantly increase both the tolerance and production of octanoic acid, a representative membrane-damaging solvent. Tolerance of other industrially-relevant inhibitors, such as furfural, acetate, toluene, ethanol and low pH was also increased. In addition to the increase in the relative abundance of the phosphoethanolamine (PE) head group in the +pssA strain, there were also changes in the fatty acid tail composition, resulting in an increase in average length, percent unsaturation and decreased abundance of cyclic rings. This +pssA strain had significant changes in: membrane integrity, surface potential, electrochemical potential and hydrophobicity; sensitivity to intracellular acidification; and distribution of the phospholipid tails, including an increase in average length and percent unsaturation and decreased abundance of cyclic rings. Molecular dynamics simulations demonstrated that the +PE membrane had increased resistance to penetration of ethanol into the hydrophobic core and also the membrane thickness. Further hybrid models in which only the head group distribution or fatty acid tail distribution was altered showed that the increase in PE content is responsible for the increase in bilayer thickness, but the increased hydrophobic core thickness is due to altered distribution of both the head groups and fatty acid tails. This work demonstrates the importance of consideration of the membrane head groups, as well as a modeling approach, in membrane engineering efforts.

Improving Escherichia coli membrane integrity and fatty acid production by expression tuning of FadL and OmpF.

BACKGROUND: Construction of microbial biocatalysts for the production of biorenewables at economically viable yields and titers is frequently hampered by product toxicity. Membrane damage is often deemed as the principal mechanism of this toxicity, particularly in regards to decreased membrane integrity. Previous studies have attempted to engineer the membrane with the goal of increasing membrane integrity. However, most of these works focused on engineering of phospholipids and efforts to identify membrane proteins that can be targeted to improve fatty acid production have been unsuccessful. RESULTS: Here we show that deletion of outer membrane protein ompF significantly increased membrane integrity, fatty acid tolerance and fatty acid production, possibly due to prevention of re-entry of short chain fatty acids. In contrast, deletion of fadL resulted in significantly decreased membrane integrity and fatty acid production. Consistently, increased expression of fadL remarkably increased membrane integrity and fatty acid tolerance while also increasing the final fatty acid titer. This 34% increase in the final fatty acid titer was possibly due to increased membrane lipid biosynthesis. Tuning of fadL expression showed that there is a positive relationship between fadL abundance and fatty acid production. Combinatorial deletion of ompF and increased expression of fadL were found to have an additive role in increasing membrane integrity, and was associated with a 53% increase the fatty acid titer, to 2.3 g/L. CONCLUSIONS: These results emphasize the importance of membrane proteins for maintaining membrane integrity and production of biorenewables, such as fatty acids, which expands the targets for membrane engineering.

Damage to the microbial cell membrane during pyrolytic sugar utilization and strategies for increasing resistance.

Lignocellulosic biomass is an appealing feedstock for the production of biorenewable fuels and chemicals, and thermochemical processing is a promising method for depolymerizing it into sugars. However, trace compounds in this pyrolytic sugar syrup are inhibitory to microbial biocatalysts. This study demonstrates that hydrophobic inhibitors damage the cell membrane of ethanologenic Escherichia coli KO11+lgk. Adaptive evolution was employed to identify design strategies for improving pyrolytic sugar tolerance and utilization. Characterization of the resulting evolved strain indicates that increased resistance to the membrane-damaging effects of the pyrolytic sugars can be attributed to a glutamine to leucine mutation at position 29 of carbon storage regulator CsrA. This single amino acid change is sufficient for decreasing EPS protein production and increasing membrane integrity when exposed to pyrolytic sugars.

Producing glucose 6-phosphate from cellulosic biomass: structural insights into levoglucosan bioconversion.

The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-ß-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.

Bioconversion of anhydrosugars: Emerging concepts and strategies.

As methods for the use of anhydrosugars in chemical and biofuel production continue to develop, our collective knowledge of anhydrosugar processing enzymes continues to improve, including their mechanistic details, structural dynamics and modes of substrate binding. Of particular interest, anhydrosugar kinases, such as levoglucosan kinase (LGK) and 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), utilize an unusual mechanism whereby the sugar substrate is both cleaved and phosphorylated. The phosphorylated sugar can then be routed to other metabolic pathways, thereby allowing its further bioconversion. Advanced engineering efforts to improve the catalytic efficiency and stability of LGK have been steadily progressing. Other enzymes that cleave the glycosidic bond of disaccharide sugars containing an anhydrosugar component are also being identified and characterized. Accordingly, the potential future use of these enzymes in large-scale production strategies is becoming increasingly viable. Here, a mini-review of the observed characteristics of anhydrosugar processing enzymes is presented along with recent developments in the bioconversion of these sugars. © 2016 IUBMB Life 68(9):700-708, 2016.

Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables.

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.

Production of biorenewable styrene: utilization of biomass-derived sugars and insights into toxicity.

Fermentative production of styrene from glucose has been previously demonstrated in Escherichia coli. Here, we demonstrate the production of styrene from the sugars derived from lignocellulosic biomass depolymerized by fast pyrolysis. A previously engineered styrene-producing strain was further engineered for utilization of the anhydrosugar levoglucosan via expression of levoglucosan kinase. The resulting strain produced 240 ± 3 mg L(-1) styrene from pure levoglucosan, similar to the 251 ± 3 mg L(-1) produced from glucose. When provided at a concentration of 5 g L(-1), pyrolytic sugars supported styrene production at titers similar to those from pure sugars, demonstrating the feasibility of producing this important industrial chemical from biomass-derived sugars. However, the toxicity of contaminant compounds in the biomass-derived sugars and styrene itself limit further gains in production. Styrene toxicity is generally believed to be due to membrane damage. Contrary to this prevailing wisdom, our quantitative assessment during challenge with up to 200 mg L(-1) of exogenously provided styrene showed little change in membrane integrity; membrane disruption was observed only during styrene production. Membrane fluidity was also quantified during styrene production, but no changes were observed relative to the non-producing control strain. This observation that styrene production is much more damaging to the membrane integrity than challenge with exogenously supplied styrene provides insight into the mechanism of styrene toxicity and emphasizes the importance of verifying proposed toxicity mechanisms during production instead of relying upon results obtained during exogenous challenge.

Quantifying Attachment and Antibiotic Resistance of from Conventional and Organic Swine Manure.

Broad-spectrum antibiotics are often administered to swine, contributing to the occurrence of antibiotic-resistant bacteria in their manure. During land application, the bacteria in swine manure preferentially attach to particles in the soil, affecting their transport in overland flow. However, a quantitative understanding of these attachment mechanisms is lacking, and their relationship to antibiotic resistance is unknown. The objective of this study is to examine the relationships between antibiotic resistance and attachment to very fine silica sand in collected from swine manure. A total of 556 isolates were collected from six farms, two organic and four conventional (antibiotics fed prophylactically). Antibiotic resistance was quantified using 13 antibiotics at three minimum inhibitory concentrations: resistant, intermediate, and susceptible. Of the 556 isolates used in the antibiotic resistance assays, 491 were subjected to an attachment assay. Results show that isolates from conventional systems were significantly more resistant to amoxicillin, ampicillin, chlortetracycline, erythromycin, kanamycin, neomycin, streptomycin, tetracycline, and tylosin ( < 0.001). Results also indicate that isolated from conventional systems attached to very fine silica sand at significantly higher levels than those from organic systems ( < 0.001). Statistical analysis showed that a significant relationship did not exist between antibiotic resistance levels and attachment in from conventional systems but did for organic systems ( < 0.001). Better quantification of these relationships is critical to understanding the behavior of in the environment and preventing exposure of human populations to antibiotic-resistant bacteria.

Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity.

Carboxylic acids are an attractive biorenewable chemical, but as with many biorenewables, their toxicity to microbial biocatalysts limits their fermentative production. While it is generally accepted that membrane damage is the main mechanism of fatty acid toxicity, previous metabolic engineering efforts that increased membrane integrity did not enable increased carboxylic acid production. Here we used an evolutionary approach to improve tolerance to exogenous octanoic acid, with the goal of learning design strategies from this evolved strain. This evolution of an Escherichia coli MG1655 derivative at neutral pH in minimal media produced a strain with increased tolerance not only to octanoic acid, but also to hexanoic acid, decanoic acid, n-butanol and isobutanol. This evolved strain also produced carboxylic acids at a 5-fold higher titer than its parent strain when expressing the Anaerococcus tetradius thioesterase. While it has been previously suggested that intracellular acidification may contribute to carboxylic acid toxicity, we saw no evidence that the evolved strain has increased resistance to this acidification. Characterization of the evolved strain membrane showed that it had significantly altered membrane polarization (fluidity), integrity (leakage) and composition relative to its parent. The changes in membrane composition included a significant increase in average lipid length in a variety of growth conditions, including 30°C, 42°C, carboxylic acid challenge and ethanol challenge. The evolved strain has a more dynamic membrane composition, showing both a larger number of significant changes and larger fold changes in the relative abundance of membrane lipids. These results highlight the importance of the cell membrane in increasing microbial tolerance and production of biorenewable fuels and chemicals.

Adaptation and tolerance of bacteria against acetic acid.

Acetic acid is a weak organic acid exerting a toxic effect to most microorganisms at concentrations as low as 0.5 wt%. This toxic effect results mostly from acetic acid dissociation inside microbial cells, causing a decrease of intracellular pH and metabolic disturbance by the anion, among other deleterious effects. These microbial inhibition mechanisms enable acetic acid to be used as a preservative, although its usefulness is limited by the emergence of highly tolerant spoilage strains. Several biotechnological processes are also inhibited by the accumulation of acetic acid in the growth medium including production of bioethanol from lignocellulosics, wine making, and microbe-based production of acetic acid itself. To design better preservation strategies based on acetic acid and to improve the robustness of industrial biotechnological processes limited by this acid's toxicity, it is essential to deepen the understanding of the underlying toxicity mechanisms. In this sense, adaptive responses that improve tolerance to acetic acid have been well studied in Escherichia coli and Saccharomyces cerevisiae. Strains highly tolerant to acetic acid, either isolated from natural environments or specifically engineered for this effect, represent a unique reservoir of information that could increase our understanding of acetic acid tolerance and contribute to the design of additional tolerance mechanisms. In this article, the mechanisms underlying the acetic acid tolerance exhibited by several bacterial strains are reviewed, with emphasis on the knowledge gathered in acetic acid bacteria and E. coli. A comparison of how these bacterial adaptive responses to acetic acid stress fit to those described in the yeast Saccharomyces cerevisiae is also performed. A systematic comparison of the similarities and dissimilarities of the ways by which different microbial systems surpass the deleterious effects of acetic acid toxicity has not been performed so far, although such exchange of knowledge can open the door to the design of novel approaches aiming the development of acetic acid-tolerant strains with increased industrial robustness in a synthetic biology perspective.

Technoeconomic evaluation of bio-based styrene production by engineered Escherichia coli.

Styrene is an important commodity chemical used in polymers and resins, and is typically produced from the petrochemical feedstocks benzene and ethylene. Styrene has recently been produced biosynthetically for the first time using engineered Escherichia coli, and this bio-based route may represent a lower energy and renewable alternative to petroleum-derived styrene. However, the economics of such an approach has not yet been investigated. Using an early-stage technoeconomic evaluation tool, a preliminary economic analysis of bio-based styrene from C(6)-sugar feedstock has been conducted. Owing to styrene's limited water solubility, it was assumed that the resulting fermentation broth would spontaneously form two immiscible liquid phases that could subsequently be decanted. Assuming current C(6) sugar prices and industrially achievable biokinetic parameter values (e.g., product yield, specific growth rate), commercial-scale bio-based styrene has a minimum estimated selling price (MESP) of 1.90 USD kg(-1) which is in the range of current styrene prices. A Monte Carlo analysis revealed a potentially large (0.45 USD kg(-1)) standard deviation in the MESP, while a sensitivity analysis showed feedstock price and overall yield as primary drivers of MESP.