Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl-CoA supply

Microbial production of mevalonate from renewable feedstock is a promising and sustainable approach for the production of value-added chemicals. We describe the metabolic engineering of Escherichia coli to enhance mevalonate production from glucose and cellobiose. First, the mevalonate-producing pathway was introduced into E. coli and the expression of the gene atoB, which encodes the gene for acetoacetyl-CoA synthetase, was increased. Then, the deletion of the pgi gene, which encodes phosphoglucose isomerase, increased the NADPH/NADP⁺ ratio in the cells but did not improve mevalonate production. Alternatively, to reduce flux toward the tricarboxylic acid cycle, gltA, which encodes citrate synthetase, was disrupted. The resultant strain, MGΔgltA-MV, increased levels of intracellular acetyl-CoA up to sevenfold higher than the wild-type strain. This strain produced 8.0 g/L of mevalonate from 20 g/L of glucose. We also engineered the sugar supply by displaying β-glucosidase (BGL) on the cell surface. When cellobiose was used as carbon source, the strain lacking gnd displaying BGL efficiently consumed cellobiose and produced mevalonate at 5.7 g/L. The yield of mevalonate was 0.25 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing mevalonate from cellobiose or cellooligosaccharides using an engineered E. coli strain.     

The Entner–Doudoroff pathway empowers Pseudomonas putida KT2440 with a high tolerance to oxidative stress

Glucose catabolism of Pseudomonas putida is carried out exclusively through the Entner–Doudoroff (ED) pathway due to the absence of 6‐phosphofructokinase. In order to activate the Embden–Meyerhof–Parnas (EMP) route we transferred the pfkA gene from Escherichia coli to a P. putida wild‐type strain as well as to an eda mutant, i.e. lacking 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase. PfkA^{E. coli} failed to redirect the carbon flow from the ED route towards the EMP pathway, suggesting that ED was essential for sugar catabolism. The presence of PfkA^{E. coli} was detrimental for growth, which could be traced to the reduction of ATP and NAD(P)H pools along with alteration of the NAD(P)H/NADP⁺ ratio. Pseudomonas putida cells carrying PfkA^{E. coli} became highly sensitive to diamide and hydrogen peroxide, the response to which is very demanding of NADPH. The inhibitory effect of PfkA^{E. coli} could in part be relieved by methionine, the synthesis of which relies much on NADPH. These results expose the role of the ED pathway for generating the redox currency (NADPH) that is required for counteracting oxidative stress. It is thus likely that environmental bacteria that favour the ED pathway over the EMP pathway do so in order to gear their aerobic metabolism to endure oxidative‐related insults.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

Production of mevalonate by a metabolically-engineered Escherichia coli

Mevalonate is biosynthesized from acetyl-CoA and metabolized to isoprenoid compounds in a wide variety of organisms although certain types of prokaryotes employ another route for isoprenoid biosynthesis (the non-mevalonate pathway). To establish a fermentative process for mevalonate production, enzymes for mevalonate synthesis from Enterococcus faecalis were expressed in Escherichia coli, a non-mevalonate pathway bacterium. Mevalonate was accumulated, indicating a redirection of acetate metabolism by the expressed enzyme. The recombinant E. coli produced 47 g mevalonate l^–1 in 50 h of fed-batch cultivation in a 2 l jar fermenter; this is the highest titer ever reported demonstrating the superiority of E. coli in its ability of acetyl-CoA supply and its inability is degrade mevalonate.     

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for β-carotene production in recombinant Escherichia coli harboring β-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of β-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and β-carotene synthesis genes produced high amount of β-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains – MG1655, DH5α, S17-1, XL1-Blue and BL21 – the DH5α was found to be the best β-carotene producer. Using glycerol as the carbon source for β-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5α harboring the whole MVA pathway and β-carotene synthesis genes produced β-carotene of 465 mg/L at glycerol concentration of 2% (w/v).     

ATP limitation in a pyruvate formate lyase mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655 increases glycolytic flux to <Emphasis Type="SmallCaps">d</Emphasis>-lactate

A derivative strain of Escherichia coli MG1655 for d-lactate production was constructed by deleting the pflB, adhE and frdA genes; this strain was designated “CL3.” Results show that the CL3 strain grew 44% slower than its parental strain under nonaerated (fermentative) conditions due to the inactivation of the main acetyl-CoA production pathway. In contrast to E. coli B and W3110 pflB derivatives, we found that the MG1655 pflB derivative is able to grow in mineral media with glucose as the sole carbon source under fermentative conditions. The glycolytic flux was 2.8-fold higher in CL3 when compared to the wild-type strain, and lactate yield on glucose was 95%. Although a low cell mass formed under fermentative conditions with this strain (1.2 g/L), the volumetric productivity of CL3 was 1.31 g/L h. In comparison with the parental strain, CL3 has a 22% lower ATP/ADP ratio. In contrast to wild-type E. coli, the ATP yield from glucose to lactate is 2 ATP/glucose, so CL3 has to improve its glycolytic flux in order to fulfill its ATP needs in order to grow. The aceF deletion in strains MG1655 and CL3 indicates that the pyruvate dehydrogenase (PDH) complex is functional under glucose-fermentative conditions. These results suggest that the pyruvate to acetyl-CoA flux in CL3 is dependent on PDH activity and that the decrease in the ATP/ADP ratio causes an increase in the flux of glucose to lactate.     

Improving poly-3-hydroxybutyrate production in Escherichia coli by combining the increase in the NADPH pool and acetyl-CoA availability

The biosynthesis of poly-3-hydroxybutyrate (P3HB), a biodegradable bio-plastic, requires acetyl-CoA as precursor and NADPH as cofactor. Escherichia coli has been used as a heterologous production model for P3HB, but metabolic pathway analysis shows a deficiency in maintaining high levels of NADPH and that the acetyl-CoA is mainly converted to acetic acid by native pathways. In this work the pool of NADPH was increased 1.7-fold in E. coli MG1655 through plasmid overexpression of the NADP⁺-dependent glyceraldehyde 3-phosphate dehydrogenase gene (gapN) from Streptococcus mutans (pTrcgapN). Additionally, by deleting the main acetate production pathway (ackA-pta), the acetic acid production was abolished, thus increasing the acetyl-CoA pool. The P3HB biosynthetic pathway was heterologously expressed in strain MG1655 Δack-pta/pTrcgapN, using an IPTG inducible vector with the P3HB operon from Azotobacter vinelandii (pPHB _Av ). Cultures were performed in controlled fermentors using mineral medium with glucose as the carbon source. Accordingly, the mass yield of P3HB on glucose increased to 73 % of the maximum theoretical and was 30 % higher when compared to the progenitor strain (MG1655/pPHB _Av ). In comparison with the wild type strain expressing pPHB _Av , the specific accumulation of PHB (g_PHB/g_DCW) in MG1655 Δack-pta/pTrcgapN/pPHB _Av increased twofold, indicating that as the availability of NADPH is raised and the production of acetate abolished, a P3HB intracellular accumulation of up to 84 % of the E. coli dry weight is attainable.     

Production of isoamyl acetate in<Emphasis Type="Italic"> ackA-pta</Emphasis> and/or<Emphasis Type="Italic"> ldh</Emphasis> mutants of<Emphasis Type="Italic"> Escherichia coli</Emphasis> with overexpression of yeast<Emphasis Type="Italic"> ATF2</Emphasis>

The gene coding for alcohol acetyltransferase (ATF2), which catalyzes the esterification of isoamyl alcohol and acetyl coenzyme A (acetyl-CoA), was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli. This genetically engineered strain of E. coli produced the ester isoamyl acetate when isoamyl alcohol was added externally to the cell culture medium. Various competing pathways at the acetyl-CoA node were inactivated to increase the intracellular acetyl-CoA pool and divert more carbon flux to the ester synthesis pathway. Several strains with deletions in the ackA-pta and/or ldh pathways and bearing the ATF2 on a high-copy-number plasmid were constructed and studied. Compared to the wild-type, ackA-pta and nuo mutants produced higher amounts of ester and an ackA-pta-ldh-nuo mutant lower amounts. Isoamyl acetate production correlated well with intracellular coenzyme A (CoA) and acetyl-CoA levels. The ackA-pta-nuo mutant had the highest intracellular CoA/acetyl-CoA level and hence produced the highest amount of ester (1.75 mM) during the growth phase under oxic conditions and during the production phase under anoxic conditions.     

Engineering the lycopene synthetic pathway in <Emphasis Type="Italic">E. coli</Emphasis> by comparison of the carotenoid genes of <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> and <Emphasis Type="Italic">Pantoea ananatis</Emphasis>

The lycopene synthetic pathway was engineered in Escherichia coli using the carotenoid genes (crtE, crtB, and crtI) of Pantoea agglomerans and Pantoea ananatis. E. coli harboring the P. agglomerans crt genes produced 27 mg/l of lycopene in 2YT medium without isopropyl-beta-d-thiogalactopyranoside (IPTG) induction, which was twofold higher than that produced by E. coli harboring the P. ananatis crt genes (12 mg/l lycopene) with 0.1 mM IPTG induction. The crt genes of P. agglomerans proved better for lycopene production in E. coli than those of P. ananatis. The crt genes of the two bacteria were also compared in E. coli harboring the mevalonate bottom pathway, which was capable of providing sufficient carotenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with exogenous mevalonate supplementation. Lycopene production significantly increased using the mevalonate bottom pathway and 60 mg/l of lycopene was obtained with the P. agglomerans crt genes, which was higher than that obtained with the P. ananatis crt genes (35 mg/l lycopene). When crtE among the P. ananatis crt genes was replaced with P. agglomerans crtE or Archaeoglobus fulgidus gps, both lycopene production and cell growth were similar to that obtained with P. agglomerans crt genes. The crtE gene was responsible for the observed difference in lycopene production and cell growth between E. coli harboring the crt genes of P. agglomerans and P. ananatis. As there was no significant difference in lycopene production between E. coli harboring P. agglomerans crtE and A. fulgidus gps, farnesyl diphosphate (FPP) synthesis was not rate-limiting in E. coli. Sang-Hwal Yoon and Ju-Eun Kim: These authors contributed equally to this work.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

Metabolic flux distribution for γ-linolenic acid synthetic pathways in<Emphasis Type="Italic">Spirulina platensis</Emphasis>

Spirulina produces γ-linolenic acid (GLA), an important pharmaceutical substance, in a relatively low level compared with fungi and plants, prompting more research to improve its GLA yield. In this study, metabolic flux analysis was applied to determine the cellular metabolic flux distributions in the GLA synthetic pathways of twoSpirulina strains, wild type BP and a high-GLA producing mutant Z19/2. Simplified pathways involving the GLA synthesis ofS. platensis formulated comprise of photosynthesis, gluconeogenesis, the pentose phosphate pathway, the anaplerotic pathway, the tricarboxylic cycle, the GLA synthesis pathway, and the biomass synthesis pathway. A stoichiometric model reflecting these pathways contains 17 intermediates and 22 reactions. Three fluxes—the bicarbonate (C-source) uptake rate, the specific growth rate, and the GLA synthesis rate—were measured and the remaining fluxes were calculated using linear optimization. The calculation showed that the flux through the reaction converting acetyl-CoA into malonyl-CoA in the mutant strain was nearly three times higher than that in the wild-type strain. This finding implies that this reaction is rate controlling. This suggestion was supported by experiments, in which the stimulating factors for this reaction (NADPH and MgCl₂) were added into the culture medium, resulting in an increased GLA-synthesis rate in the wild type strain.     

Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5 mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD⁺-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5 mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.     

Optimal cofactor swapping can increase the theoretical yield for chemical production in Escherichia coli and Saccharomyces cerevisiae

Maintaining cofactor balance is a critical function in microorganisms, but often the native cofactor balance does not match the needs of an engineered metabolic flux state. Here, an optimization procedure is utilized to identify optimal cofactor-specificity “swaps” for oxidoreductase enzymes utilizing NAD(H) or NADP(H) in the genome-scale metabolic models of Escherichia coli and Saccharomyces cerevisiae. The theoretical yields of all native carbon-containing molecules are considered, as well as theoretical yields of twelve heterologous production pathways in E. coli. Swapping the cofactor specificity of central metabolic enzymes (especially GAPD and ALCD2x) is shown to increase NADPH production and increase theoretical yields for native products in E. coli and yeast—including l-aspartate, l-lysine, l-isoleucine, l-proline, l-serine, and putrescine—and non-native products in E. coli—including 1,3-propanediol, 3-hydroxybutyrate, 3-hydroxypropanoate, 3-hydroxyvalerate, and styrene.     

Synthese von 3-Merkapto-3-methylpentan-5-lacton

An NADPH-dependent sorbose reductase from Candida albicans was identified to catalyze the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE). The activity of the recombinant enzyme toward COBE was 6.2 U/mg. The asymmetric reduction of COBE was performed with two coexisting recombinant Escherichia coli strains, in which the recombinant E. coli expressing glucose dehydrogenase was used as an NADPH regenerator. An optical purity of 99% (e.e.) and a maximum yield of 1240 mM (S)-4-chloro-3-hydroxybutanoate were obtained under an optimal biomass ratio of 1:2. A highest turnover number of 53,900 was achieved without adding extra NADP⁺/NADPH compared with those known COBE-catalytic systems.     

Comparison of engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> for the production of an optically pure keto alcohol

In this study, the production of enantiomerically pure (1R,4S,6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one ((−)-2) through stereoselective bioreduction was used as a model reaction for the comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli as biocatalysts. For both microorganisms, over-expression of the gene encoding the NADPH-dependent aldo-keto reductase YPR1 resulted in high purity of the keto alcohol (−)-2 (>99% ee, 97–98% de). E. coli had three times higher initial reduction rate but S. cerevisiae continued the reduction reaction for a longer time period, thus reaching a higher conversion of the substrate (95%). S. cerevisiae was also more robust than E. coli, as demonstrated by higher viability during bioreduction. It was also investigated whether the NADPH regeneration rate was sufficient to supply the over-expressed reductase with NADPH. Five strains of each microorganism with varied carbon flux through the NADPH regenerating pentose phosphate pathway were genetically constructed and compared. S. cerevisiae required an increased NADPH regeneration rate to supply YPR1 with co-enzyme while the native NADPH regeneration rate was sufficient for E. coli. Nádia Skorupa Parachin and Magnus Carlquist have contributed equally to the paper.     

Production of isopropanol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary–secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.     

Dynamic control of the mevalonate pathway expression for improved zeaxanthin production in Escherichia coli and comparative proteome analysis

Engineered heterologous multi-gene metabolic pathways often suffer from flux imbalance and toxic metabolites, as the production host typically lacks the regulatory mechanisms for the heterologous pathway. Here, we first coordinated the expression of all genes of the mevalonate (MEV) pathway from Saccharomyces cerevisiae using the tunable intergenic regions (TIGRs), and then dynamically regulated the TIGR-mediated MEV pathway to prevent the accumulation of toxic metabolites by using IPP/FPP-responsive promoter. After introduction of the dynamically controlled TIGR-mediated MEV pathway into Escherichia coli, the content and concentration of zeaxanthin in shaker flask cultures were 2.0- and 2.1-fold higher, respectively, than those of the strain harboring the statically controlled non-TIGR-mediated MEV pathway. The content and concentration of zeaxanthin in E. coli ZEAX (pZSP_gadE-MevT_TIGR-MevB_TIGRIS-2) reached 722.46 mg/L and 23.16 mg/g dry cell weight (DCW), respectively, in 5.0 L fed-batch fermentation. We also comparatively analyzed the proteomes between E. coli ZEAX and E. coli ZEAX (pZSP_gadE-MevT_TIGR-MevB_TIGRIS-2) to understand the mechanism of zeaxanthin biosynthesis. The results of the comparative proteomes demonstrate that zeaxanthin overproduction may be associated with increased precursor availability, increased NADPH availability, increased ATP availability, oxidative stress response, and increased membrane storage capacity for zeaxanthin due to changes in both cellular shape and membrane composition.     

Re-examination of metabolic fluxes in <Emphasis Type="Italic">Escherichia coli</Emphasis> during anaerobic fermentation of glucose using <Superscript>13</Superscript>C labeling experiments and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy

Improved design of metabolic flux estimation using mixed label ¹³C labeling experiments and identifiability analysis motivated re-examination of metabolic fluxes during anaerobic fermentation in the Escherichia coli. Comprehensive metabolic flux maps were determined by using a mixture of differently labeled glucose and compared to conventional flux maps obtained using extracellular measurements and comprehensive metabolic flux maps obtained using only U-¹³C glucose as the substrate. As expected, conventional flux analysis performs poorly in comparison to ¹³C-MFA, especially in the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Identifiability analysis indicated and experiments confirmed that a mixture of 10% U-^l3C glucose, 25% 1-¹³C glucose, and 65% naturally labeled glucose significantly improved the statistical quality of all calculated fluxes in the PP pathway, the EMP pathway, the anaplerotic reactions, and the tricarboxylic acid cycle. Modifying the network topology for the presence and absence of the Entner-Doudoroff pathway and the glyoxylate shunt did not affect the value or quality of estimated fluxes significantly. Extracellular measurement of formate production was necessary for the accurate estimation of the fluxes around the formate node.     

Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering

The overexpression of acetyl-CoA (CoA) synthetase (ACS) in Escherichia coli showed significant reduction in acetate during glucose fermentation. It also greatly enhanced acetate assimilation when acetate was used as a carbon source. These features are ideal for applications in metabolic engineering. ACS overexpression can be strategically applied to reduce acetate byproduct, recover wasted carbon, and redirect carbon flux toward more favorable pathways. The native acs gene was cloned and overexpressed in E. coli. Studies showed significant effects on acetate production and assimilation in cultures grown in minimal and complex media with glucose or acetate as the carbon source.