Citrate synthase variants improve yield of acetyl-CoA derived 3-hydroxybutyrate in Escherichia coli.

BACKGROUND: The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme. RESULTS: Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA[A267T] generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source. CONCLUSIONS: This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation.

Isolation and Characterization of Levoglucosan-Metabolizing Bacteria.

Bacteria were isolated from wastewater and soil containing charred wood remnants based on their ability to use levoglucosan as a sole carbon source and on their levoglucosan dehydrogenase (LGDH) activity. On the basis of their 16S rRNA gene sequences, these bacteria represented the diverse genera Microbacterium, Paenibacillus, Shinella, and Klebsiella. Genomic sequencing of the isolates verified that two isolates represented novel species, Paenibacillus athensensis MEC069T and Shinella sumterensis MEC087T, while the remaining isolates were closely related to Microbacterium lacusdiani or Klebsiella pneumoniae. The genetic sequence of LGDH, lgdA, was found in the genomes of these four isolates as well as Pseudarthrobacter phenanthrenivorans Sphe3. The identity of the P. phenanthrenivorans LGDH was experimentally verified following recombinant expression in Escherichia coli. Comparison of the putative genes surrounding lgdA in the isolate genomes indicated that several other gene products facilitate the bacterial catabolism of levoglucosan, including a putative sugar isomerase and several transport proteins. IMPORTANCE Levoglucosan is the most prevalent soluble carbohydrate remaining after high-temperature pyrolysis of lignocellulosic biomass, but it is not fermented by typical production microbes such as Escherichia coli and Saccharomyces cerevisiae. A few fungi metabolize levoglucosan via the enzyme levoglucosan kinase, while several bacteria metabolize levoglucosan via levoglucosan dehydrogenase. This study describes the isolation and characterization of four bacterial species that degrade levoglucosan. Each isolate is shown to contain several genes within an operon involved in levoglucosan degradation, furthering our understanding of bacteria that metabolize levoglucosan.

Pyruvate Production by Escherichia coli by Use of Pyruvate Dehydrogenase Variants.

Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli, pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase (ldhA) and pyruvate oxidase (poxB). The effect of deleting phosphoenolpyruvate synthase (ppsA) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h-1, an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. IMPORTANCE Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli. This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes.

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments.

Experimental evolution is a critical tool in many disciplines, including metabolic engineering and synthetic biology. However, current methods rely on the chance occurrence of a key step that can dramatically accelerate evolution in natural systems, namely increased gene dosage. Our studies sought to induce the targeted amplification of chromosomal segments to facilitate rapid evolution. Since increased gene dosage confers novel phenotypes and genetic redundancy, we developed a method, Evolution by Amplification and Synthetic Biology (EASy), to create tandem arrays of chromosomal regions. In Acinetobacter baylyi, EASy was demonstrated on an important bioenergy problem, the catabolism of lignin-derived aromatic compounds. The initial focus on guaiacol (2-methoxyphenol), a common lignin degradation product, led to the discovery of Amycolatopsis genes (gcoAB) encoding a cytochrome P450 enzyme that converts guaiacol to catechol. However, chromosomal integration of gcoAB in Pseudomonas putida or A. baylyi did not enable guaiacol to be used as the sole carbon source despite catechol being a growth substrate. In ∼1,000 generations, EASy yielded alleles that in single chromosomal copy confer growth on guaiacol. Different variants emerged, including fusions between GcoA and CatA (catechol 1,2-dioxygenase). This study illustrates the power of harnessing chromosomal gene amplification to accelerate the evolution of desirable traits.

Engineered citrate synthase alters Acetate Accumulation in Escherichia coli.

Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated into E. coli C ΔpoxB twenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.

Engineered citrate synthase improves citramalic acid generation in Escherichia coli.

The microbial product citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized in Escherichia coli directly by the condensation of pyruvate and acetyl-CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl-CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in the gltA gene coding citrate synthase prevents acetyl-CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study the E. coli citrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl-CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed-batch process using MEC626/pZE12-cimA harboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can be used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals.

In vivo interpretation of model predicted inhibition in acrylate pathway engineered Lactococcus lactis.

To maximize the productivity of engineered metabolic pathway, in silico model is an established means to provide features of enzyme reaction dynamics. In our previous study, Escherichia coli engineered with acrylate pathway yielded low propionic acid titer. To understand the bottleneck behind this low productivity, a kinetic model was developed that incorporates the enzymatic reactions of the acrylate pathway. The resulting model was capable of simulating the fluxes reported under in vitro studies with good agreement, suggesting repression of propionyl-CoA transferase (Pct) by carboxylate metabolites as the main limiting factor for propionate production. Furthermore, the predicted flux control coefficients of the pathway enzymes under steady state conditions revealed that the control of flux is shared between Pct and lactoyl-CoA dehydratase. Increase in lactate concentration showed gradual decrease in flux control coefficients of Pct that in turn confirmed the control exerted by the carboxylate substrate. To interpret these in silico predictions under in vivo system, an organized study was conducted with a lactic acid bacteria strain engineered with acrylate pathway. Analysis reported a decreased product formation rate on attainment of inhibitory titer by suspected metabolites and supported the model.

Glucose can be transported and utilized in Escherichia coli by an altered or overproduced N-acetylglucosamine phosphotransferase system (PTS).

Escherichia coli Δglk ΔmanZ ΔptsG glucose- strains that lack the glucose phosphotransferase system (PTS) and the mannose PTS as well as glucokinase have been widely used by researchers studying the PTS. In this study we show that both fast- and slow-growing spontaneous glucose+ revertants can be readily obtained from Δglk ΔmanZ ΔptsG glucose- strains. All of the fast-growing revertants either altered the N-acetylglucosamine PTS or caused its overproduction by inactivating the NagC repressor protein, which regulates the N-acetylglucosamine PTS, and these revertants could utilize either glucose or N-acetylglucosamine as a sole carbon source. When a ΔnagE deletion, which abolishes the N-acetylglucosamine PTS, was introduced into the Δglk ΔmanZ ΔptsG glucose- strains, fast-growing revertants could no longer be isolated. Based on our results and other studies, it is clear that the N-acetylglucosamine PTS is the most easily adaptable PTS for transporting and phosphorylating glucose, other than the glucose PTS and mannose PTS, which are the primary glucose transport systems. While the slow-growing glucose+revertants were not characterized, they were likely mutations that other researchers have observed before and affect other PTSs or sugar kinases.

Enhancement of NAD(H) pool for formation of oxidized biochemicals in Escherichia coli.

The NAD+/NADH ratio and the total NAD(H) play important roles for whole-cell biochemical redox transformations. After the carbon source is exhausted, the degradation of NAD(H) could contribute to a decline in the rate of a desired conversion. In this study, methods to slow the native rate of NAD(H) degradation were examined using whole-cell Escherichia coli with two model oxidative NAD+-dependent biotransformations. A high phosphate concentration (50 mM) was observed to slow NAD(H) degradation. We also constructed E. coli strains with deletions in genes coding several enzymes involved in NAD+ degradation. In shake-flask experiments, the total NAD(H) concentration positively correlated with conversion of xylitol to L-xylulose by xylitol 4-dehydrogenase, and the greatest conversion (80%) was observed using MG1655 nadR nudC mazG/pZE12-xdh/pCS27-nox. Controlled 1-L batch processes comparing E. coli nadR nudC mazG with a wild-type background strain demonstrated a 30% increase in final L-xylulose concentration (5.6 vs. 7.9 g/L) and a 25% increase in conversion (0.53 vs. 0.66 g/g). MG1655 nadR nudC mazG was also examined for the conversion of galactitol to L-tagatose by galactitol 2-dehydrogenase. A batch process using 15 g/L glycerol and 10 g/L galactitol generated over 9.4 g/L L-tagatose, corresponding to 90% conversion and a yield of 0.95 g L-tagatose/g galactitol consumed. The results demonstrate the value of minimizing NAD(H) degradation as a means to improve NAD+-dependent biotransformations.

Glucose consumption in carbohydrate mixtures by phosphotransferase-system mutants of Escherichia coli.

Escherichia coli lacking the glucose phosphotransferase system (PTS), mannose PTS and glucokinase are supposedly unable to grow on glucose as the sole carbon source (Curtis SJ, Epstein W. J Bacteriol 1975;122:1189-1199). We report that W ptsG manZ glk (ALS1406) grows slowly on glucose in media containing glucose with a second carbon source: ALS1406 metabolizes glucose after that other carbon source, including arabinose, fructose, glycerol, succinate or xylose, is exhausted. Galactose is an exception to this rule, as ALS1406 simultaneously consumes both galactose and glucose. The ability of ALS1406 to metabolize glucose in a xylose-glucose mixture was unchanged by an additional knockout in any single gene involved in carbohydrate transport and utilization, including agp (periplasmic glucose-1-phosphatase), galP (galactose permease), xylA (xylose isomerase), alsK (allose kinase), crr (glucose PTS enzyme IIA), galK (galactose kinase), mak (mannokinase), malE (maltose transporter), malX (maltose PTS enzyme IIBC), mglB (methyl-galactose transporter subunit), nagE (N-acetyl glucosamine PTS enzyme IICBA), nanK (N-acetyl mannosamine kinase) or pgm (phosphoglucose mutase). Glucose metabolism was only blocked by the deletion of two metabolic genes, pgi (phosphoglucose isomerase) and zwf (glucose-6-phosphate 1-dehydrogenase), which prevents the entry of glucose-6-phosphate into the pentose phosphate and Embden-Meyerhof-Parnas pathways. Carbon-limited steady-state studies demonstrated that xylose must be sub-saturating for glucose to be metabolized, while nitrogen-limited studies showed that xylose is partly converted to glucose when xylose is in excess. Under transient conditions, ALS1406 converts almost 25 % (mass) xylose into glucose as a result of reversible transketolase and transaldolase and the re-entry of carbon into the pentose phosphate pathway via glucose-6-phosphate 1-dehydrogenase.

Eliminating acetate formation improves citramalate production by metabolically engineered Escherichia coli.

BACKGROUND: Citramalate, a chemical precursor to the industrially important methacrylic acid (MAA), can be synthesized using Escherichia coli overexpressing citramalate synthase (cimA gene). Deletion of gltA encoding citrate synthase and leuC encoding 3-isopropylmalate dehydratase were critical to achieving high citramalate yields. Acetate is an undesirable by-product potentially formed from pyruvate and acetyl-CoA, the precursors of citramalate during aerobic growth of E. coli. This study investigated strategies to minimize acetate and maximize citramalate production in E. coli mutants expressing the cimA gene. RESULTS: Key knockouts that minimized acetate formation included acetate kinase (ackA), phosphotransacetylase (pta), and in particular pyruvate oxidase (poxB). Deletion of glucose 6-phosphate dehydrogenase (zwf) and ATP synthase (atpFH) aimed at improving glycolytic flux negatively impacted cell growth and citramalate accumulation in shake flasks. In a repetitive fed-batch process, E. coli gltA leuC ackA-pta poxB overexpressing cimA generated 54.1 g/L citramalate with a yield of 0.64 g/g glucose (78% of theoretical maximum yield), and only 1.4 g/L acetate in 87 h. CONCLUSIONS: This study identified the gene deletions critical to reducing acetate accumulation during aerobic growth and citramalate production in metabolically engineered E. coli strains. The citramalate yield and final titer relative to acetate at the end of the fed-batch process are the highest reported to date (a mass ratio of citramalate to acetate of nearly 40) without being detrimental to citramalate productivity, significantly improving a potential process for the production of this five-carbon chemical.

Synthesis of citramalic acid from glycerol by metabolically engineered Escherichia coli.

Citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. We compared citramalate and acetate accumulation from glycerol using Escherichia coli strains expressing a modified citramalate synthase gene cimA from Methanococcus jannaschii. These studies revealed that gltA coding citrate synthase, leuC coding 3-isopropylmalate dehydratase, and acetate pathway genes play important roles in elevating citramalate and minimizing acetate formation. Controlled 1.0 L batch experiments confirmed that deletions in all three acetate-production genes (poxB, ackA, and pta) were necessary to reduce acetate formation to less than 1 g/L during citramalate production from 30 g/L glycerol. Fed-batch processes using MEC568/pZE12-cimA (gltA leuC ackA-pta poxB) generated over 31 g/L citramalate and less than 2 g/L acetate from either purified or crude glycerol at yields exceeding 0.50 g citramalate/g glycerol in 132 h. These results hold promise for the viable formation of citramalate from unrefined glycerol.

Production of citramalate by metabolically engineered Escherichia coli.

Citramalic acid (citramalate) is a five carbon hydroxy-dicarboxylic acid and potential precursor for the production of methacrylic acid from renewable resources. We examined citramalate production in Escherichia coli expressing the citramalate synthase gene cimA. Although, knockouts in ldhA coding lactate dehydrogenase and glcB/aceB coding malate synthase did not benefit citramalate accumulation, knockouts in gltA coding citrate synthase, and ackA coding acetate kinase significantly increased citramalate accumulation compared to the control strain. A fed-batch process in a controlled fermenter using a glucose feed resulted in 46.5 g/L citramalate in 132 h with a yield of 0.63 g/g, over 75% of the theoretical maximum yield from glucose of 0.82 g/g. Biotechnol. Bioeng. 2016;113: 2670-2675. © 2016 Wiley Periodicals, Inc.

Transcriptional analysis and adaptive evolution of Escherichia coli strains growing on acetate.

Eighteen strains of Escherichia coli were compared for maximum specific growth rate (µ MAX) on 85 mM acetate as the sole carbon source. The C strain ATCC8739 had the greatest growth rate (0.41 h(-1)) while SCS-1 had the slowest growth rate (0.15 h(-1)). Transcriptional analysis of three of the strains (ATCC8739, BL21, SMS-3-5) was conducted to elucidate why ATCC8739 had the greatest maximum growth rate. Seventy-one genes were upregulated 2-fold or greater in ATCC8739, while 128 genes were downregulated 2-fold or greater in ATCC8739 compared to BL21 and SMS-3-5. To generate a strain that could grow more quickly on acetate, ATCC8739 was cultured in a chemostat using a progressively increasing dilution rate. When the dilution rate reached 0.50 h(-1), three isolated colonies each grew faster than ATCC8739 on 85 mM acetate, with MEC136 growing the fastest with a growth rate of 0.51 h(-1), about 25 % greater than ATCC8739. Transcriptional analysis of MEC136 showed that eight genes were downregulated 2-fold or greater and one gene was upregulated 2-fold or greater compared to ATCC8739. Genomic sequencing revealed that MEC136 contained a single mutation, causing a serine to proline change in amino acid 266 of RpoA, the α subunit of the RNA polymerase core enzyme. The 260-270 amino acid region of RpoA has been shown to be a key region of the protein that affects the interaction of the α subunit of the RNA polymerase core enzyme with several global transcriptional activators, such as CRP and FNR.

Accumulation of d-glucose from pentoses by metabolically engineered Escherichia coli.

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.

Microbial production of lactic acid.

Lactic acid is an important commodity chemical having a wide range of applications. Microbial production effectively competes with chemical synthesis methods because biochemical synthesis permits the generation of either one of the two enantiomers with high optical purity at high yield and titer, a result which is particularly beneficial for the production of poly(lactic acid) polymers having specific properties. The commercial viability of microbial lactic acid production relies on utilization of inexpensive carbon substrates derived from agricultural or waste resources. Therefore, optimal lactic acid formation requires an understanding and engineering of both the competing pathways involved in carbohydrate metabolism, as well as pathways leading to potential by-products which both affect product yield. Recent research leverages those biochemical pathways, while researchers also continue to seek strains with improved tolerance and ability to perform under desirable industrial conditions, for example, of pH and temperature.

Adaptation of Escherichia coli to elevated sodium concentrations increases cation tolerance and enables greater lactic acid production.

Adaptive evolution was employed to generate sodium (Na(+))-tolerant mutants of Escherichia coli MG1655. Four mutants with elevated sodium tolerance, designated ALS1184, ALS1185, ALS1186, and ALS1187, were independently isolated after 73 days of serial transfer in medium containing progressively greater Na(+) concentrations. The isolates also showed increased tolerance of K(+), although this cation was not used for selective pressure. None of the adapted mutants showed increased tolerance to the nonionic osmolyte sucrose. Several physiological parameters of E. coli MG1655 and ALS1187, the isolate with the greatest Na(+) tolerance, were calculated and compared using glucose-limited chemostats. Genome sequencing showed that the ALS1187 isolate contained mutations in five genes, emrR, hfq, kil, rpsG, and sspA, all of which could potentially affect the ability of E. coli to tolerate Na(+). Two of these genes, hfq and sspA, are known to be involved in global regulatory processes that help cells endure a variety of cellular stresses. Pyruvate formate lyase knockouts were constructed in strains MG1655 and ALS1187 to determine whether increased Na(+) tolerance afforded increased anaerobic generation of lactate. In fed-batch fermentations, E. coli ALS1187 pflB generated 76.2 g/liter lactate compared to MG1655 pflB, which generated only 56.3 g/liter lactate.

Production of biomass and filamentous hemagglutinin by Bordetella bronchiseptica.

The mammalian pathogen Bordetella bronchiseptica was grown under controlled batch conditions with glutamate as the primary carbon and nitrogen source. First, a Box-Behnken statistical design quantified the effect of Mg, sulfate, and nicotinate on the antigen filamentous hemagglutinin (FHA) formation. Using lactic acid as a secondary carbon source for pH control, Mg, and SO4 each negatively affected antigen expression, while nicotinate positively affected antigen expression. Sulfate had a stronger negative effect than Mg with 10 mM eliminating FHA altogether; the highest FHA expression (about 1,000 ng/mL) occurred when either Mg concentration or SO4 concentration, but not both, was about 0.1 mM. Using two Mg and SO4 compositions modeled to yield the greatest antigen expression, three other organic acids were compared as the secondary carbon source: acetate, citrate, and succinate. Mixtures of acetate and glutamate resulted in the greatest organic acid consumption, OD, and FHA concentration (about 1,500 ng/mL), although significant acetate accumulated during these batch processes. The mechanism leading to elevated FHA expression when acetate is the secondary carbon source is unknown, particularly since these cultures were most prone to phase shift to Bvg(-) cultures.

Production of Esters in Escherichia coli Using Citrate Synthase Variants.

Acetate esters comprise a wide range of products including fragrances and industrial solvents. Biosynthesis of esters offers a promising alternative to chemical synthesis because such routes use renewable carbohydrate resources and minimize the generation of waste. One biochemical method for ester formation relies on the ATF1 gene from Saccharomyces cerevisiae, which encodes alcohol-O-acyltransferase (AAT) which converts acetyl-CoA and an exogenously supplied alcohol into the ester. In this study, the formation of several acetate esters via AAT was examined in Escherichia coli chromosomally expressing citrate synthase variants, which create a metabolic bottleneck at acetyl-CoA. In shake flask cultures, variant strains generated more acetate esters than the strains expressing the wild-type citrate synthase. In a controlled bioreactor, E. coli GltA[A267T] generated 3.9 g propyl acetate in 13 h, corresponding to a yield of 0.155 g propyl acetate/g glucose, which is 18% greater than that obtained by the wild-type GltA control. These results demonstrate the ability of citrate synthase variants to redistribute carbon from central metabolism into acetyl-CoA-derived biochemicals.

Differential sensitivities of the growth of Escherichia coli to acrylate under aerobic and anaerobic conditions and its effect on product formation.

The effect of acrylate on the growth of Escherichia coli was determined under aerobic and anaerobic conditions in glucose-defined medium. Growth occurred with up to 35 mM acrylate under aerobic conditions but ceased at 5 mM acrylate under anaerobic conditions. This differential sensitivity can be attributed to inhibition of pyruvate formate lyase and/or pflB gene repression, as this enzyme is necessary for anaerobic growth of E. coli. The effect of acrylate on end-product distribution was also determined by growing E. coli first aerobically, then switching to anaerobic conditions. In the absence of acrylate, E. coli generated the typical distribution of mixed-acid products, with about 12 % of pyruvate being metabolically converted to lactate. In contrast, in the presence of 5 mM acrylate, E. coli converted 83 % of pyruvate to lactate, consistent with a reduction in pyruvate formate lyase activity.