Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases

Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12–C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.     

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.     

Intersection of RNA processing and the type II fatty acid synthesis pathway in yeast mitochondria

Distinct metabolic pathways can intersect in ways that allow hierarchical or reciprocal regulation. In a screen of respiration-deficient Saccharomyces cerevisiae gene deletion strains for defects in mitochondrial RNA processing, we found that lack of any enzyme in the mitochondrial fatty acid type II biosynthetic pathway (FAS II) led to inefficient 5′ processing of mitochondrial precursor tRNAs by RNase P. In particular, the precursor containing both RNase P RNA (RPM1) and tRNA^Pro accumulated dramatically. Subsequent Pet127-driven 5′ processing of RPM1 was blocked. The FAS II pathway defects resulted in the loss of lipoic acid attachment to subunits of three key mitochondrial enzymes, which suggests that the octanoic acid produced by the pathway is the sole precursor for lipoic acid synthesis and attachment. The protein component of yeast mitochondrial RNase P, Rpm2, is not modified by lipoic acid in the wild-type strain, and it is imported in FAS II mutant strains. Thus, a product of the FAS II pathway is required for RNase P RNA maturation, which positively affects RNase P activity. In addition, a product is required for lipoic acid production, which is needed for the activity of pyruvate dehydrogenase, which feeds acetyl-coenzyme A into the FAS II pathway. These two positive feedback cycles may provide switch-like control of mitochondrial gene expression in response to the metabolic state of the cell.     

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

Short-chain fatty acid (SCFA) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we conducted in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioesterase enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including 23 from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoA molecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated (trans-2-pentenoate), and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. In this study, we introduce novel thioesterases with improved specificity for saturated, branched, and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent.     

Metabolic engineering of fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli

Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75 g/L fatty alcohols (0.02 g fatty alcohol/g glycerol) with a productivity of up to 0.06 g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.     

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP⁺ dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169 mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989 mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.     

Microbial production of fatty alcohols

Fatty alcohols have numerous commercial applications, including their use as lubricants, surfactants, solvents, emulsifiers, plasticizers, emollients, thickeners, and even fuels. Fatty alcohols are currently produced by catalytic hydrogenation of fatty acids from plant oils or animal fats. Microbial production of fatty alcohols may be a more direct and environmentally-friendly strategy since production is carried out by heterologous enzymes, called fatty acyl-CoA reductases, able to reduce different acyl-CoA molecules to their corresponding primary alcohols. Successful examples of metabolic engineering have been reported in Saccharomyces cerevisiae and Escherichia coli in which the production of fatty alcohols ranged from 1.2 to 1.9 g/L, respectively. Due to their metabolic advantages, oleaginous yeasts are considered the best hosts for production of fatty acid-derived chemicals. Some of these species can naturally produce, under specific growth conditions, lipids at high titers (>50 g/L) and therefore provide large amounts of fatty acyl-CoAs or fatty acids as precursors. Very recently, taking advantage of such features, over 8 g/L of C₁₆–C₁₈ fatty alcohols have been produced in Rhodosporidium toruloides. In this review we summarize the different metabolic engineering strategies, hosts and cultivation conditions used to date. We also point out some future trends and challenges for the microbial production of fatty alcohols.     

Metabolic engineering of<Emphasis Type="Italic"> Escherichia coli</Emphasis> for improved 6-deoxyerythronolide B production

Escherichia coli is an attractive candidate as a host for polyketide production and has been engineered to produce the erythromycin precursor polyketide 6-deoxyerythronolide B (6dEB). In order to identify and optimize parameters that affect polyketide production in engineered E. coli, we first investigated the supply of the extender unit (2S)-methylmalonyl-CoA via three independent pathways. Expression of the Streptomyces coelicolor malonyl/methylmalonyl-CoA ligase (matB) pathway in E. coli together with methylmalonate feeding resulted in the accumulation of intracellular methylmalonyl-CoA to as much as 90% of the acyl-CoA pool. Surprisingly, the methylmalonyl-CoA generated from the matB pathway was not converted into 6dEB. In strains expressing either the S. coelicolor propionyl-CoA carboxylase (PCC) pathway or the Propionibacteria shermanii methylmalonyl-CoA mutase/epimerase pathway, methylmalonyl-CoA accumulated up to 30% of the total acyl-CoA pools, and 6dEB was produced; titers were fivefold higher when strains contained the PCC pathway rather than the mutase pathway. When the PCC and mutase pathways were expressed simultaneously, the PCC pathway predominated, as indicated by greater flux of ¹³C-propionate into 6dEB through the PCC pathway. To further optimize the E. coli production strain, we improved 6dEB titers by integrating the PCC and mutase pathways into the E. coli chromosome and by expressing the 6-deoxyerythronolide B synthase (DEBS) genes from a stable plasmid system.S. Murli and J. Kennedy contributed equally to this work     

Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives

A synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coli. An L-phenylalanine-overproducing E. coli strain was engineered to enhance the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds in microbes. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E.coli as a base strain, we tested our system by carrying out the production of salicylate, a high-demand aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 2-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this test case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. In order to demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1–3 g/L in test tube-scale culture.     

Engineering Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose

Demand for sustainable materials motivates the development of microorganisms capable of synthesizing products from renewable substrates. A challenge to commercial production of polyhydroxyalkanoates (PHA), microbially derived polyesters, is engineering metabolic pathways to produce a polymer with the desired monomer composition from an unrelated and renewable source. Here, we demonstrate a metabolic pathway for converting glucose into medium-chain-length (mcl)-PHA composed primarily of 3-hydroxydodecanoate monomers. This pathway combines fatty acid biosynthesis, an acyl-ACP thioesterase to generate desired C₁₂ and C₁₄ fatty acids, β-oxidation for conversion of fatty acids to (R)-3-hydroxyacyl-CoAs, and a PHA polymerase. A key finding is that Escherichia coli expresses multiple copies of enzymes involved in β-oxidation under aerobic conditions. To produce polyhydroxydodecanoate, an acyl-ACP thioesterase (BTE), an enoyl-CoA hydratase (phaJ3), and mcl-PHA polymerase (phaC2) were overexpressed in E. coli ΔfadRABIJ. Yields were improved through expression of an acyl-CoA synthetase resulting in production over 15% CDW – the highest reported production of mcl-PHA of a defined composition from an unrelated carbon source.     

Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15

Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-d-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735 mg cytidine/l using glucose as substrate, which was approx. 128 % higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2 %) slightly and increased glucose uptake (72 %). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli.     

Metabolic transistor strategy for controlling electron transfer chain activity in Escherichia coli

A novel strategy to finely control a large metabolic flux by using a “metabolic transistor” approach was established. In this approach a small change in the level or availability of an essential component for the process is controlled by adding a competitive reaction that affects a precursor or an intermediate in its biosynthetic pathway. The change of the basal level of the essential component, considered as a base current in a transistor, has a large effect on the flux through the major pathway. In this way, the fine-tuning of a large flux can be accomplished. The “metabolic transistor” strategy was applied to control electron transfer chain function by manipulation of the quinone synthesis pathway in Escherichia coli. The achievement of a theoretical yield of lactate production under aerobic conditions via this strategy upon manipulation of the biosynthetic pathway of the key participant, ubiquinone-8 (Q8), in an E. coli strain provides an in vivo, genetically tunable means to control the activity of the electron transfer chain and manipulate the production of reduced products while limiting consumption of oxygen to a defined amount.     

Structure-guided reshaping of the acyl binding pocket of ‘TesA thioesterase enhances octanoic acid production in E. coli

Medium-chain fatty acids (C6–C10) have attracted much attention recently for their unique properties compared to their long-chain counterparts, including low melting points and relatively higher carbon conversion yield. Thioesterase enzymes, which can catalyze the hydrolysis of acyl-ACP (acyl carrier protein) to release free fatty acids (FAs), regulate both overall FA yields and acyl chain length distributions in bacterial and yeast fermentation cultures. These enzymes typically prefer longer chain substrates. Herein, seeking to increase bacterial production of MCFAs, we conducted structure-guided mutational screening of multiple residues in the substrate-binding pocket of the E. coli thioesterase enzyme ‘TesA. Confirming our hypothesis that enhancing substrate selectivity for medium-chain acyl substrates would promote overall MCFA production, we found that replacement of residues lining the bottom of the pocket with more hydrophobic residues strongly promoted the C8 substrate selectivity of ‘TesA. Specifically, two rounds of saturation mutagenesis led to the identification of the ‘TesA^RD−2 variant that exhibited a 133-fold increase in selectivity for the C8-ACP substrate as compared to C16-ACP substrate. Moreover, the recombinant expression of this variant in an E. coli strain with a blocked β-oxidation pathway led to a 1030% increase in the in vivo octanoic acid (C8) production titer. When this strain was fermented in a 5-L fed-batch bioreactor, it produced 2.7 g/L of free C8 (45%, molar fraction) and 7.9 g/L of total free FAs, which is the highest-to-date free C8 titer to date reported using the E. coli type II fatty acid synthetic pathway. Thus, reshaping the substrate binding pocket of a bacterial thioesterase enzyme by manipulating the hydrophobicity of multiple residues altered the substrate selectivity and therefore fatty acid product distributions in cells. Our study demonstrates the relevance of this strategy for increasing titers of industrially attractive MCFAs as fermentation products.     

Microbial synthesis of wax esters

Microbial synthesis of wax esters (WE) from low-cost renewable and sustainable feedstocks is a promising path to achieve cost-effectiveness in biomanufacturing. WE are industrially high-value molecules, which are widely used for applications in chemical, pharmaceutical, and food industries. Since the natural WE resources are limited, the WE production mostly rely on chemical synthesis from rather expensive starting materials, and therefore solution are sought from development of efficient microbial cell factories. Here we report to engineer the yeast Yarrowia lipolytica and bacterium Escherichia coli to produce WE at the highest level up to date. First, the key genes encoding fatty acyl-CoA reductases and wax ester synthase from different sources were investigated, and the expression system for two different Y. lipolytica hosts were compared and optimized for enhanced WE production and the strain stability. To improve the metabolic pathway efficiency, different carbon sources including glucose, free fatty acid, soybean oil, and waste cooking oil (WCO) were compared, and the corresponding pathway engineering strategies were optimized. It was found that using a lipid substrate such as WCO to replace glucose led to a 60-fold increase in WE production. The engineered yeast was able to produce 7.6 g/L WE with a yield of 0.31 (g/g) from WCO within 120 h and the produced WE contributed to 57% of the yeast DCW. After that, E. coli BL21(DE3), with a faster growth rate than the yeast, was engineered to significantly improve the WE production rate. Optimization of the expression system and the substrate feeding strategies led to production of 3.7–4.0 g/L WE within 40 h in a 1-L bioreactor. The predominant intracellular WE produced by both Y. lipolytica and E. coli in the presence of hydrophobic substrates as sole carbon sources were C₃₆, C₃₄ and C₃₂, in an order of decreasing abundance and with a large proportion being unsaturated. This work paved the way for the biomanufacturing of WE at a large scale.     

Enhancement of free fatty acid production in Saccharomyces cerevisiae by control of fatty acyl-CoA metabolism

Production of biofuels derived from microbial fatty acids has attracted great attention in recent years owing to their potential to replace petroleum-derived fuels. To be cost competitive with current petroleum fuel, flux toward the direct precursor fatty acids needs to be enhanced to approach high yields. Herein, fatty acyl-CoA metabolism in Saccharomyces cerevisiae was engineered to accumulate more free fatty acids (FFA). For this purpose, firstly, haploid S. cerevisiae double deletion strain △faa1△faa4 was constructed, in which the genes FAA1 and FAA4 encoding two acyl-CoA synthetases were deleted. Then the truncated version of acyl-CoA thioesterase ACOT5 (Acot5s) encoding Mus musculus peroxisomal acyl-CoA thioesterase 5 was expressed in the cytoplasm of the strain △faa1△faa4. The resulting strain △faa1△faa4 [Acot5s] accumulated more extracellular FFA with higher unsaturated fatty acid (UFA) ratio as compared to the wild-type strain and double deletion strain △faa1△faa4. The extracellular total fatty acids (TFA) in the strain △faa1△faa4 [Acot5s] increased to 6.43-fold as compared to the wild-type strain during the stationary phase. UFA accounted for 42 % of TFA in the strain △faa1△faa4 [Acot5s], while no UFA was detected in the wild-type strain. In addition, the expression of Acot5s in △faa1△faa4 restored the growth, which indicates that FFA may not be the reason for growth inhibition in the strain △faa1△faa4. RT-PCR results demonstrated that the de-repression of fatty acid synthesis genes led to the increase of extracellular fatty acids. The study presented here showed that through control of the acyl-CoA metabolism by deleting acyl-CoA synthetase and expressing thioesterase, more FFA could be produced in S. cerevisiae, demonstrating great potential for exploitation in the platform of microbial fatty acid-derived biofuels.     

Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

cis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain of Escherichia coli K-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes, pobA, aroY, and catA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed in E. coli to establish the MA biosynthetic pathway. E. coli native genes ubiC, aroF^FBR, aroE, and aroL were overexpressed and the genes ptsH, ptsI, crr, and pykF were deleted from the E. coli genome in order to increase the precursors of the proposed MA pathway. The final engineered E. coli strain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars.     

Metabolic engineering of Escherichia coli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose

The Escherichia coli XL1-blue strain was metabolically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] through 2-ketobutyrate, which is generated via citramalate pathway, as a precursor for propionyl-CoA. Two different metabolic pathways were examined for the synthesis of propionyl-CoA from 2-ketobutyrate. The first pathway is composed of the Dickeya dadantii 3937 2-ketobutyrate oxidase or the E. coli pyruvate oxidase mutant (PoxB L253F V380A) for the conversion of 2-ketobutyrate into propionate and the Ralstonia eutropha propionyl-CoA synthetase (PrpE) or the E. coli acetyl-CoA:acetoacetyl-CoA transferase for further conversion of propionate into propionyl-CoA. The second pathway employs pyruvate formate lyase encoded by the E. coli tdcE gene or the Clostridium difficile pflB gene for the direct conversion of 2-ketobutyrate into propionyl-CoA. As the direct conversion of 2-ketobutyrate into propionyl-CoA could not support the efficient production of P(3HB-co-3HV) from glucose, the first metabolic pathway was further examined. When the recombinant E. coli XL1-blue strain equipped with citramalate pathway expressing the E. coli poxB L253F V380A gene and R. eutropha prpE gene together with the R. eutropha PHA biosynthesis genes was cultured in a chemically defined medium containing 20 g/L of glucose as a sole carbon source, P(3HB-co-2.3 mol% 3HV) was produced up to the polymer content of 61.7 wt.%. Moreover, the 3HV monomer fraction in P(3HB-co-3HV) could be increased up to 5.5 mol% by additional deletion of the prpC and scpC genes, which are responsible for the metabolism of propionyl-CoA in host strains.     

Metabolic engineering of Escherichia coli for the production of benzoic acid from glucose

Benzoic acid (BA) is an important platform aromatic compound in chemical industry and is widely used as food preservatives in its salt forms. Yet, current manufacture of BA is dependent on petrochemical processes under harsh conditions. Here we report the de novo production of BA from glucose using metabolically engineered Escherichia coli strains harboring a plant-like β-oxidation pathway or a newly designed synthetic pathway. First, three different natural BA biosynthetic pathways originated from plants and one synthetically designed pathway were systemically assessed for BA production from glucose by in silico flux response analyses. The selected plant-like β-oxidation pathway and the synthetic pathway were separately established in E. coli by expressing the genes encoding the necessary enzymes and screened heterologous enzymes under optimal plasmid configurations. BA production was further optimized by applying several metabolic engineering strategies to the engineered E. coli strains harboring each metabolic pathway, which included enhancement of the precursor availability, removal of competitive reactions, transporter engineering, and reduction of byproduct formation. Lastly, fed-batch fermentations of the final engineered strain harboring the β-oxidation pathway and the strain harboring the synthetic pathway were conducted, which resulted in the production of 2.37 ± 0.02 g/L and 181.0 ± 5.8 mg/L of BA from glucose, respectively; the former being the highest titer reported by microbial fermentation. The metabolic engineering strategies developed here will be useful for the production of related aromatics of high industrial interest.     

Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution

Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO₂ contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.