Transport-controlled growth decoupling for self-induced protein expression with a glycerol-repressible genetic circuit.

Decoupling cell formation from recombinant protein synthesis is a potent strategy to intensify bioprocesses. Escherichia coli strains with mutations in the glucose uptake components lack catabolite repression, display low growth rate, no overflow metabolism, and high recombinant protein yields. Fast growth rates were promoted by the simultaneous consumption of glucose and glycerol, and this was followed by a phase of slow growth, when only glucose remained in the medium. A glycerol-repressible genetic circuit was designed to autonomously induce recombinant protein expression. The engineered strain bearing the genetic circuit was cultured in 3.9 g L-1 glycerol + 18 g L-1 glucose in microbioreactors with online oxygen transfer rate monitoring. The growth was fast during the simultaneous consumption of both carbon sources (C-sources), while expression of the recombinant protein was low. When glycerol was depleted, the growth rate decreased, and the specific fluorescence reached values 17% higher than those obtained with a strong constitutive promoter. Despite the relatively high amount of C-source used, no oxygen limitation was observed. The proposed approach eliminates the need for the substrate feeding or inducers addition and is set as a simple batch culture while mimicking fed-batch performance.

Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered E. coli.

The demand of plasmid DNA (pDNA) as a key element for gene therapy products, as well as mRNA and DNA vaccines, is increasing together with the need for more efficient production processes. An engineered E. coli strain lacking the phosphotransferase system and the pyruvate kinase A gene has been shown to produce more pDNA than its parental strain. With the aim of improving pDNA production in the engineered strain, several strategies to increase the flux to the pentose phosphate pathway (PPP) were evaluated. The simultaneous consumption of glucose and glycerol was a simple way to increase the growth rate, pDNA production rate, and supercoiled fraction (SCF). The overexpression of key genes from the PPP also improved pDNA production in glucose, but not in mixtures of glucose and glycerol. Particularly, the gene coding for the glucose 6-phosphate dehydrogenase (G6PDH) strongly improved the SCF, growth rate, and pDNA production rate. A linear relationship between the G6PDH activity and pDNA yield was found. A higher flux through the PPP was confirmed by flux balance analysis, which also estimates relevant differences in fluxes of the tricarboxylic acid cycle. These results are useful for developing further cell engineering strategies to increase pDNA production and quality.

Recombinant protein expression in proteome-reduced cells under aerobic and oxygen-limited regimes.

Industrial cultures are hindered by the physiological complexity of the host and the limited mass transfer capacity of conventional bioreactors. In this study, a minimal cell approach was combined with genetic devices to overcome such issues. A flavin mononucleotide-based fluorescent protein (FbFP) was expressed in a proteome-reduced Escherichia coli (PR). When FbFP was expressed from a constitutive protein generator (CPG), the PR strain produced 47% and 35% more FbFP than its wild type (WT), in aerobic or oxygen-limited regimes, respectively. Metabolic and expression models predicted more efficient biomass formation at higher fluxes to FbFP, in agreement with these results. A microaerobic protein generator (MPG) and a microaerobic transcriptional cascade (MTC) were designed to induce FbFP expression upon oxygen depletion. The FbFP fluorescence using the MTC in the PR strain was 9% higher than that of the WT bearing the CPG under oxygen limitation. To further improve the PR strain, the pyruvate dehydrogenase complex regulator gene was deleted, and the Vitreoscilla hemoglobin was expressed. Compared to oxygen-limited cultures of the WT, the engineered strains increased the FbFP expression more than 50% using the MTC. Therefore, the designed expression systems can be a valuable alternative for industrial cultivations.

Design of microaerobically inducible miniR1 plasmids.

Plasmid DNA manufacture is an essential step to produce gene therapy agents and next-generation vaccines. However, little attention has been paid toward developing alternative replicons that can be coupled with large-scale production conditions. Our results demonstrate that the miniR1 replicon can be efficiently induced by oxygen limitation when a copy of the regulatory protein RepA under control of a microaerobic promoter is used. The results are potentially attractive for industrial applications.

Glucose transport engineering allows mimicking fed-batch performance in batch mode and selection of superior producer strains.

BACKGROUND: Fed-batch mode is the standard culture technology for industrial bioprocesses. Nevertheless, most of the early-stage cell and process development is carried out in batch cultures, which can bias the initial selection of expression systems. Cell engineering can provide an alternative to fed-batch cultures for high-throughput screening and host selection. We have previously reported a library of Escherichia coli strains with single and multiple deletions of genes involved in glucose transport. Compared to their wild type (W3110), the mutant strains displayed lower glucose uptake, growth and aerobic acetate production rates. Therefore, when cultured in batch mode, such mutants may perform similar to W3110 cultured in fed-batch mode. To test that hypothesis, we evaluated the constitutive expression of the green fluorescence protein (GFP) in batch cultures in microbioreactors using a semi defined medium supplemented with 10 or 20 g/L glucose + 0.4 g yeast extract/g glucose. RESULTS: The mutant strains cultured in batch mode displayed a fast-growth phase (growth rate between 0.40 and 0.60 h-1) followed by a slow-growth phase (growth rate between 0.05 and 0.15 h-1), similar to typical fed-batch cultures. The phase of slow growth is most probably caused by depletion of key amino acids. Three mutants attained the highest GFP fluorescence. Particularly, a mutant named WHIC (ΔptsHIcrr, ΔmglABC), reached a GFP fluorescence up to 14-fold greater than that of W3110. Strain WHIC was cultured in 2 L bioreactors in batch mode with 100 g/L glucose + 50 g/L yeast extract. These cultures were compared with exponentially fed-batch cultures of W3110 maintaining the same slow-growth of WHIC (0.05 h-1) and using the same total amount of glucose and yeast extract than in WHIC cultures. The WHIC strain produced approx. 450 mg/L GFP, while W3110 only 220 mg/L. CONCLUSION: The combination of cell engineering and high throughput screening allowed the selection of a particular mutant that mimics fed-batch behavior in batch cultures. Moreover, the amount of GFP produced by the strain WHIC was substantially higher than that of W3110 under both, batch and fed-batch schemes. Therefore, our results represent a valuable technology for accelerated bioprocess development.

Furfural biotransformation in Acinetobacter baylyi ADP1 and Acinetobacter schindleri ACE.

OBJECTIVES: To determine furfural biotransformation capabilities of Acinetobacter baylyi ADP1 and Acinetobacter schindleri ACE. RESULTS: Acinetobacter baylyi ADP1 and A. schindleri ACE could not use furfural as sole carbon source but when acetate was used as substrate, ADP1 and ACE biotransformed 1 g furfural/l in 5 and 9 h, respectively. In both cases, the product of this biotransformation was difurfuryl-ether as shown by FT-IR and 1H and 13C NMR spectroscopy. The presence of furfural decreased the specific growth rate in acetate by 27% in ADP1 and 53% in ACE. For both strains, the MIC of furfural was 1.25 g/l. Nonetheless, ADP1 biotransformed 2 g furfural/l at a rate of 1 g/l/h in the stationary phase of growth. A transcriptional analysis of possible dehydrogenases involved in this biotransformation, identified that the areB and frmA genes were highly overexpressed after the exposure of ADP1 to furfural. The products of these genes are a benzyl-alcohol dehydrogenase and an alcohol dehydrogenase. CONCLUSIONS: Acinetobacter baylyi ADP1 is a candidate for the biological detoxification of furfural, a fermentation inhibitor present in lignocellulosic hydrolysates, with the possible direct involvement of the AreB and FrmA enzymes in the process.

Plasmid DNA Production in Proteome-Reduced Escherichia coli.

The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L-1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

Physiological Response of Escherichia coli W3110 and BL21 to the Aerobic Expression of Vitreoscilla Hemoglobin.

The aerobic growth and metabolic performance of Escherichia coli strains BL21 and W3110 were studied when the Vitreoscilla hemoglobin (VHb) was constitutively expressed in the chromosome. When VHb was expressed, acetate production decreased in both strains and was nearly eliminated in BL21. Transcriptional levels of the glyoxylate shunt genes decreased in both strains when VHb was expressed. However, higher transcription of the α-ketoglutarate dehydrogenase genes were observed for W3110, while for BL21 transcription levels decreased. VHb expression reduced the transcription of the cytochrome bo3 genes only in BL21. These results are useful for better selecting a production host.

Physiological and transcriptional comparison of acetate catabolism between Acinetobacter schindleri ACE and Escherichia coli JM101.

Acinetobacter bacteria preferentially use gluconeogenic substrates instead of hexoses or pentoses. Accordingly, Acinetobacter schindleri ACE reaches a high growth rate on acetate but is unable to grow on glucose, xylose or arabinose. In this work, we compared the physiology of A. schindleri ACE and Escherichia coli JM101 growing on acetate as the carbon source. In contrast to JM101, ACE grew on acetate threefold faster, had a twofold higher biomass yield, and a 45% higher specific acetate consumption rate. Transcriptional analysis revealed that genes like ackA, pta, aceA, glcB, fumA, tktA and talA were overexpressed while acsA, sfcA, ppc and rpiA were underexpressed in ACE relative to JM101. This transcriptional profile together with carbon flux balance analysis indicated that ACE forms acetyl-CoA preferentially by the AckA-Pta (acetate kinase-phosphotransacetylase) pathway instead of Acs (acetyl-CoA synthetase) and that the glyoxylate shunt and tricarboxylic acid cycle are more active in ACE than in JM101. Moreover, in ACE, ribose 5-phosphate and erythrose 4-phosphate are formed from trioses, and NADPH is mainly produced by isocitrate dehydrogenase. This knowledge will contribute to an understanding of the carbon metabolism of Acinetobacter species of medical, biotechnological and microbiological relevance.

Design of a microaerobically inducible replicon for high-yield plasmid DNA production.

A pUC-derived replicon inducible by oxygen limitation was designed and tested in fed-batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive replication control element. The rnaII gene was expressed from Ptrc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (YpDNA/X ) were performed and the initial hypothesis was probed. Furthermore, in high cell-density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen-responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and YpDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The YpDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed-batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale-up of plasmid DNA production processes in which mass transfer limitations will not represent an issue.

Engineering E. coli for improved microaerobic pDNA production.

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA-, which, together with W3110recA-vgb+, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production.

Acinetobacter baylyi ADP1 growth performance and lipid accumulation on different carbon sources.

Acinetobacter baylyi ADP1 is a microorganism with the potential to produce storage lipids. Here, a systematic study was carried out to evaluate growth performance and accumulation of wax esters and triacylglycerols using glycerol, xylose, glucose, acetate, ethanol, and pyruvate as carbon sources. High specific growth rates (µ) were found in gluconeogenic carbon sources (ethanol, acetate, and pyruvate: 0.94 ± 0.18, 0.93 ± 0.06, and 0.61 ± 0.01 h-1, respectively), and low in glucose (0.25 ± 0.01 h-1). Interestingly, these µ values were sustained in a broad range of concentrations of glucose (0.5-50 g L-1), pyruvate (3-10 g L-1), and acetate (0.3-2 g L-1), suggesting a high tolerance to glucose and pyruvate. It was observed that ADP1 is not able to use glycerol or xylose as unique carbon sources. On the other hand, ADP1 showed sensitivity to osmotic upshifts, noted by the lysis at the beginning of cultivations on different carbon sources. However, ADP1 is adapted to relatively high substrate concentrations as indicated by the minimal inhibitory concentrations (MICs) determined at 24 h of cultivations: 350, 50, 80, and 15 g L-1 for glucose, ethanol, pyruvate, and acetate, respectively. Remarkably, ADP1 co-utilized glucose, acetate, ethanol, and pyruvate. Finally, the accumulation of storage lipids, wax esters (WEs), and triacylglycerols (TAGs) showed to be substrate dependent. Under nitrogen-limiting conditions, the TAGs:WEs (mol:mol) accumulation ratios were 1:4.9 in pyruvate and 1:1.6 in glucose, the WEs were mainly accumulated in acetate. In ethanol, no accumulation of lipids was detected.

Design of a synthetic miniR1 plasmid and its production by engineered Escherichia coli.

A synthetic plasmid consisting of the minimal elements for replication control of the R1 replicon and kanamycin resistance marker, which was named pminiR1, was developed. pminiR1 production was tested at 30 °C under aerobic and microaerobic conditions in Escherichia coli W3110 recA- (W1). The plasmid DNA yields from biomass (YpDNA/X) were only 0.06 ± 0.02 and 0.22 ± 0.11 mg/g under aerobic and microaerobic conditions, respectively. As an option to increase YpDNA/X values, pminiR1 was introduced in an engineered E. coli strain expressing the Vitreoscilla hemoglobin inserted in chromosome (W12). The YpDNA/X values using strain W12 increased to 0.85 ± 0.05 and 1.53 ± 0.14 mg/g under aerobic and microaerobic conditions, respectively. pminiR1 production in both strains was compared with that of pUC57Kan at 37 °C under aerobic and microaerobic conditions. The YpDNA/X values for pminiR1 using strain W12 were 6.25 ± 0.16 and 9.27 ± 0.95 mg/g under aerobic and microaerobic conditions, respectively. Such yields were similar to those obtained for plasmid pUC57Kan using strain W12 (6.9 ± 0.64 and 10.85 ± 1.06 mg/g for aerobic and microaerobic cultures, respectively). Therefore, the synthetic minimal plasmid based on the R1 replicon is a valuable alternative to pUC plasmids for biotechnological applications.

Evaluation of microbial globin promoters for oxygen-limited processes using Escherichia coli.

Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTRmax) of 7 and 11 mmol L-1 h-1. Different FbFP fluorescence intensities were observed and the OTRmax affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor, the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli.

Genomic and physiological characterization of a laboratory-isolated Acinetobacter schindleri ACE strain that quickly and efficiently catabolizes acetate.

An Acinetobacter strain, designated ACE, was isolated in the laboratory. Phylogenetic tests and average nucleotide identity value comparisons suggested that ACE belongs to the species Acinetobacterschindleri. We report for the first time the complete genome sequence of an A. schindleri strain, which consists of a single circular chromosome of 3 001 209 bp with an overall DNA G+C content of 42.9 mol% and six plasmids that account for 266 844 bp of extrachromosomal material. The presence or absence of genes related to carbon catabolism and antibiotic resistance were in agreement with the phenotypic characterization of ACE. This strain grew faster and with a higher biomass yield on acetate than the reference strain Acinetobacter baylyi ADP1. However, ACE did not use aromatic compounds and was unable to grow on common carbon sources, such as glucose, xylose, glycerol or citrate. The gluconeogenic and the catechol pathways are complete in ACE, but compounds that are converted to protocatechuate did not sustain growth since some genes of this pathway are missing. Likewise, this strain could not grow on glucose because it lacks the genes of the Entner-Doudoroff pathway. Minimal inhibitory concentration data showed that ACE was susceptible to most of the antimicrobial agents recommended for the clinical treatment of Acinetobacter spp. Some genes related to a possible human-microbe interaction were found in the ACE genome. ACE is likely to have a low pathogenic risk, as is the case with other A. schindleri strains. These results provide a valuable reference for broadening the knowledge of the biology of Acinetobacter.

Heterogeneous oxygen availability affects the titer and topology but not the fidelity of plasmid DNA produced by Escherichia coli.

BACKGROUND: Dissolved oxygen tension (DOT) is hardly constant and homogenously distributed in a bioreactor, which can have a negative impact in the metabolism and product synthesis. However, the effects of DOT on plasmid DNA (pDNA) production and quality have not been thoroughly investigated. In the present study, the effects of aerobic (DOT ≥30% air sat.), microaerobic (constant DOT = 3% air sat.) and oscillatory DOT (from 0 to 100% air sat.) conditions on pDNA production, quality and host performance were characterized. RESULTS: Microaerobic conditions had little effect on pDNA production, supercoiled fraction and sequence fidelity. By contrast, oscillatory DOT caused a 22% decrease in pDNA production compared with aerobic cultures. Although in aerobic cultures the pDNA supercoiled fraction was 98%, it decreased to 80% under heterogeneous DOT conditions. The different oxygen availabilities had no effect on the fidelity of the produced pDNA. The estimated metabolic fluxes indicated substantial differences at the level of the pentose phosphate pathway and TCA cycle under different conditions. Cyclic changes in fermentative pathway fluxes, as well as fast shifts in the fluxes through cytochromes, were also estimated. Model-based genetic modifications that can potentially improve the process performance are suggested. CONCLUSIONS: DOT heterogeneities strongly affected cell performance, pDNA production and topology. This should be considered when operating or scaling-up a bioreactor with deficient mixing. Constant microaerobic conditions affected the bacterial metabolism but not the amount or quality of pDNA. Therefore, pDNA production in microaerobic cultures may be an alternative for bioreactor operation at higher oxygen transfer rates.

Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO-K1 cells.

Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO-K1 cell culture was investigated. For this purpose, CHO-K1 cells were transfected with plasmids carrying the vgb or gfp gene to express VHb or green fluorescence protein (GFP), respectively. VHb expression increased the specific growth rate and biomass yields on glucose and glutamine by 60 %, and reduced the amount of lactate produced per cell by 40 %, compared to the GFP-expression controls. Immunofluorescence microscopy showed that VHb is distributed in the cytoplasm and organelles, which support the hypothesis that VHb could serve as an oxygen carrier, enhancing aerobic respiration. These results are useful for the development of better producing cell lines for industrial applications.

Characterization of Endogenous and Reduced Promoters for Oxygen-Limited Processes Using Escherichia coli.

Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (Pvgb) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a nonoxygen dependent marker protein, was used as a reporter. Fluorescence and fluorescence emission rates under oxygen-limited conditions were the highest when FbFP was under transcriptional control of PadhE, Ppfl and Pvgb. The lengths of the E. coli endogenous promoters were shortened by 60%, maintaining their key regulatory elements. This resulted in improved promoter activity in most cases, particularly for PadhE, Ppfl and PnarK. Selected promoters were also evaluated using an engineered E. coli strain expressing Vitreoscilla hemoglobin (VHb). The presence of the VHb resulted in a better repression using these promoters under aerobic conditions, and increased the specific growth and fluorescence emission rates under oxygen-limited conditions. These results are useful for the selection of promoters for specific applications and for the design of modified artificial promoters.

Expression of an alkane monooxygenase (alkB) gene and methyl tert-butyl ether co-metabolic oxidation in Pseudomonas citronellolis.

Pseudomonas citronellolis UAM-Ps1 co-metabolically transforms methyl tert-butyl ether (MTBE) to tert-butyl alcohol with n-pentane (2.6 mM), n-octane (1.5 mM) or dicyclopropylketone (DCPK) (4.4 mM), a gratuitous inducer of alkane hydroxylase (AlkB) activity. The reverse transcription quantitative real-time PCR was used to quantify the alkane monooxygenase (alkB) gene expression. The alkB gene was expressed in the presence of n-alkanes and DCPK and MTBE oxidation occurred only in cultures when alkB was transcribed. A correlation between the number of alkB transcripts and MTBE consumption was found (ΜΤΒΕ consumption in µmol = 1.44e(-13) x DNA copies, R(2) = 0.99) when MTBE (0.84 mM) was added. Furthermore, alkB was cloned and expressed into Escherichia coli and the recombinant AlkB had a molecular weight of 42 kDa. This is the first report where the expression of alkB is related to the co-metabolic oxidation of MTBE.

Aerobic expression of Vitreoscilla hemoglobin efficiently reduces overflow metabolism in Escherichia coli.

Overflow metabolism is a prevalent problem for aerobic cultivations of Escherichia coli. Although several process and molecular approaches have been applied to prevent overflow metabolism, these approaches often result in reductions in growth rate, biomass yield or accumulation of other byproducts. In this report, we present an alternative approach based on increasing the efficiency of aerobic metabolism by the expression of the Vitreoscilla stercoraria hemoglobin (VHb) to avoid overflow metabolism. VHb is expected to increase the consumption of NADH in the respiratory chain, leading to increased activity of the tricarboxylic acid (TCA) cycle. This would result in a faster consumption of acetyl Co-A and a decrease in acetate production. When this strategy was tested in E. coli strains, acetate production decreased by 50% in MG1655 and more than 90% in W3110, without affecting growth rates or biomass yields. VHb expression in mutant strains with higher TCA activity and reduced acetate formation resulted in a significant increase in growth and glucose consumption rates, whereas acetate production did not increase. The results presented here show that enhancing the efficiency of aerobic metabolism is a valuable approach to avoid overflow metabolism in E. coli and to attain high cell densities in batch mode.