Cell surface display of a β-glucosidase employing the type V secretion system on ethanologenic Escherichia coli for the fermentation of cellobiose to ethanol

We used the autodisplay system AIDA-I, which belongs to the type V secretion system (TVSS), to display the β-glucosidase BglC from Thermobifida fusca on the outer membrane of the ethanologenic Escherichia coli strain MS04 (MG1655 ?pflB, ?adhE, ?frdA, ?xylFGH, ?ldhA, PpflB::pdc (Zm)-adhB (Zm)). MS04 that was transformed with the plasmid pAIDABglCRHis showed cellobiase activity (171 U/g(CDW)) and fermented 40 g/l cellobiose in mineral medium in 60 h with an ethanol yield of 81 % of the theoretical maximum. Whole-cell protease treatment, SDS-PAGE, and Western-blot analysis demonstrated that BglC was attached to the external surface of the outer membrane of MS04. When attached to the cells, BglC showed 93.3 % relative activity in the presence of 40 g/l ethanol and retained 100 % of its activity following 2 days of incubation at 37 °C with the same ethanol concentration. This study shows the potential of the TVSS (AIDA-I) and BglC as tools for the production of lignocellulosic bio-commodities.     

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.     

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.     

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.     

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.     

Use of a genetically-engineered Escherichia coli strain as a sample process control for quantification of the host-specific bacterial genetic markers

Quantitative PCR (qPCR) assays targeting the host-specific Bacteroides-Prevotella 16S rRNA genetic markers have been proposed as one of the promising approaches to identify the source of fecal contamination in environmental waters. One of the concerns of qPCR assays to environmental samples is the reliability of quantified values, since DNA extraction followed by qPCR assays are usually performed without appropriate sample process control (SPC) and internal amplification controls (IACs). To check the errors in sample processing and improve the reliability of qPCR results, it is essential to evaluate the DNA recovery efficiency and PCR amplification efficiency of the target genetic markers and correct the measurement results. In this study, we constructed a genetically-engineered Escherichia coli K12 strain (designated as strain MG1655 Δlac::kan) as sample process control and evaluated the applicability to environmental water samples. The recovery efficiency of the SPC strain MG1655 Δlac::kan was similar to that of Bacteroides fragilis JCM 11019, when DNA were extracted from water samples spiked with the two bacteria. Furthermore, the SPC was included in the qPCR assays with propidium monoazide (PMA) treatment, which can exclude the genetic markers from dead cells. No significant DNA loss was observed in the PMA treatment. The inclusion of both the SPC (strain MG1655 Δlac::kan) and IAC in qPCR assays with PMA treatment gave the assurance of reliable results of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental water samples.     

Engineering of a robust Escherichia coli chassis and exploitation for large-scale production processes

In large-scale bioprocesses microbes are exposed to heterogeneous substrate availability reducing the overall process performance. A series of deletion strains was constructed from E. coli MG1655 aiming for a robust phenotype in heterogeneous fermentations with transient starvation. Deletion targets were hand-picked based on a list of genes derived from previous large-scale simulation runs. Each gene deletion was conducted on the premise of strict neutrality towards growth parameters in glucose minimal medium. The final strain of the series, named E. coli RM214, was cultivated continuously in an STR-PFR (stirred tank reactor – plug flow reactor) scale-down reactor. The scale-down reactor system simulated repeated passages through a glucose starvation zone. When exposed to nutrient gradients, E. coli RM214 had a significantly lower maintenance coefficient than E. coli MG1655 (Δm_s = 0.038 g_Glucose/g_CDW/h, p < 0.05). In an exemplary protein production scenario E. coli RM214 remained significantly more productive than E. coli MG1655 reaching 44% higher eGFP yield after 28 h of STR-PFR cultivation. This study developed E. coli RM214 as a robust chassis strain and demonstrated the feasibility of engineering microbial hosts for large-scale applications.     

Re-engineering Escherichia coli for ethanol production

A lactate producing derivative of Escherichia coli KO11, strain SZ110, was re-engineered for ethanol production by deleting genes encoding all fermentative routes for NADH and randomly inserting a promoterless mini-Tn5 cassette (transpososome) containing the complete Zymomonas mobilis ethanol pathway (pdc, adhA, and adhB) into the chromosome. By selecting for fermentative growth in mineral salts medium containing xylose, a highly productive strain was isolated in which the ethanol cassette had been integrated behind the rrlE promoter, designated strain LY160 (KO11, Δfrd::celY _Ec ΔadhE ΔldhA, ΔackA lacA::casAB _Ko rrlE::(pdc _Zm -adhA _Zm -adhB _Zm -FRT-rrlE) pflB ⁺ ). This strain fermented 9% (w/v) xylose to 4% (w/v) ethanol in 48 h in mineral salts medium, nearly equal to the performance of KO11 with Luria broth.     

Increasing reducing power output (NADH) of glucose catabolism for reduction of xylose to xylitol by genetically engineered Escherichia coli AI05

Anaerobic homofermentative production of reduced products requires additional reducing power (NADH and/or NADPH) output from glucose catabolism. Previously, with an anaerobically expressed pyruvate dehydrogenase operon (aceEF-lpd), we doubled the reducing power output to four NADH per glucose (or 1.2 xylose) catabolized anaerobically, which satisfied the NADH requirement to establish a non-transgenic homoethanol pathway (1 glucose or 1.2 xylose ? 2 acetyl-CoA + 4 NADH ? 2 ethanol) in the engineered strain, Escherichia coli SZ420 (?frdBC ?ldhA ?ackA ?focA-pflB ?pdhR::pflBp6-pflBrbs-aceEF-lpd). In this study, E. coli SZ420 was further engineered for reduction of xylose to xylitol by (1) deleting the alcohol dehydrogenase gene (adhE) to divert NADH from the ethanol pathway; (2) deleting the glucose-specific PTS permease gene (ptsG) to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose; (3) cloning the aldose reductase gene (xylI) of Candida boidinii to reduce xylose to xylitol. The resulting strain, E. coli AI05 (pAGI02), could in theory simultaneously uptake glucose and xylose, and utilize glucose as a source of reducing power for the reduction of xylose to xylitol, with an expected yield of four xylitol for each glucose consumed (Y_RPG = 4) under anaerobic conditions. In resting cell fermentation tests using glucose and xylose mixtures, E. coli AI05 (pAGI02) achieved an actual Y_RPG value of ~3.6, with xylitol as the major fermentation product and acetate as the by-product.     

2D [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C] NMR study of carbon fluxes during glucose utilization by <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells-glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)—were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655Δ(edd-eda), MG1655Δ(zwf, edd-eda), and MG1655Δ(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.     

Sequential Thermochemical Hydrolysis of Corncobs and Enzymatic Saccharification of the Whole Slurry Followed by Fermentation of Solubilized Sugars to Ethanol with the Ethanologenic Strain <Emphasis Type="Italic">Escherichia coli</Emphasis> MS04

Interest in the use of corncobs as feedstock for bioethanol production is growing. This study assesses the feasibility of sequential thermochemical diluted sulfuric acid pretreatment of corncobs at moderate temperature to hydrolyze the hemicellulosic fraction, followed by enzymatic hydrolysis of the whole slurry, and fermentation of the obtained syrup. The total sugar concentration after enzymatic hydrolysis was 85.21 g/l, i.e., 86 % of the sugars were liberated from the polymeric fractions, together with a low amount of furfural (0.26 g/l) and 4.01 g/l of acetic acid. The syrups, which contained 36.3, 40.9, 4.47, and 1.84 g/l of xylose, glucose, arabinose, and mannose, respectively, were fermented (pH 7, 37 °C, 150 rpm) to ethanol with the metabolically engineered acetate-tolerant Escherichia coli strain MS04 under non-aerated conditions, producing 35 g/l of ethanol in 18 h (1.94 g_EtOH/l/h), i.e., a conversion yield greater than 80 % of the theoretical value based on total sugars was obtained. Hence, using the procedures developed in this study, 288 l of ethanol can be produced per metric ton of dry corncobs. Strain MS04 can ferment sugars in the presence of acetate, and the amount of furans generated during the sequential thermochemical and enzymatic hydrolysis was low; hence, the detoxification step was avoided. The residual salts, acetic acid, and solubilized lignin present in the syrup did not interfere with the production of ethanol by E. coli MS04 and the results show that this strain can metabolize mixtures of glucose and xylose simultaneously.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture

Clostridium acetobutylicum was metabolically engineered to produce a biofuel consisting of an isopropanol/butanol/ethanol mixture. For this purpose, different synthetic isopropanol operons were constructed and introduced on plasmids in a butyrate minus mutant strain (C. acetobutylicum ATCC 824 Δcac15ΔuppΔbuk). The best strain expressing the isopropanol operon from the thl promoter was selected from batch experiments at pH 5. By further optimizing the pH of the culture, a biofuel mixture with almost no by-products was produced at a titer, a yield and productivity never reached before, opening the opportunities to develop an industrial process for alternative biofuels with Clostridial species. Furthermore, by performing in vivo and in vitro flux analysis of the synthetic isopropanol pathway, this flux was identified to be limited by the [acetate]_int and the high Km of CoA-transferase for acetate. Decreasing the Km of this enzyme using a protein engineering approach would be a good target for improving isopropanol production and avoiding acetate accumulation in the culture medium.     

In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production

Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising bulk chemical with wide industrial applications, along with various byproducts. In this study, the in silico gene knockout simulation of K. oxytoca was carried out for 2,3-BD overproduction by inhibiting the formation of byproducts. The knockouts of ldhA and pflB genes were targeted with the criteria of maximization of 2,3-BD production and minimization of byproducts formation. The constructed K. oxytoca ΔldhA ΔpflB strain showed higher 2,3-BD yields and higher final concentrations than those obtained from the wild-type and ΔldhA strains. However, the simultaneous deletion of both genes caused about a 50 % reduction in 2,3-BD productivity compared with K. oxytoca ΔldhA strain. Based on previous studies and in silico investigation that the agitation speed during 2,3-BD fermentation strongly affected cell growth and 2,3-BD synthesis, the effect of agitation speed on 2,3-BD production was investigated from 150 to 450 rpm in 5-L bioreactors containing 3-L culture media. The highest 2,3-BD productivity (2.7 g/L/h) was obtained at 450 rpm in batch fermentation. Considering the inhibition of acetoin for 2,3-BD production, fed-batch fermentations were performed using K. oxytoca ΔldhA ΔpflB strain to enhance 2,3-BD production. Altering the agitation speed from 450 to 350 rpm at nearly 10 g/L of acetoin during the fed-batch fermentation allowed for the production of 113 g/L 2,3-BD, with a yield of 0.45 g/g, and for the production of 2.1 g/L/h of 2,3-BD.     

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T).

Results

Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed.

Conclusions

Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

     

Identification and manipulation of a novel locus to improve cell tolerance to short-chain alcohols in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli KO11 is a popular ethanologenic strain, but is more sensitive to ethanol than other producers. Here, an ethanol-tolerant mutant EM was isolated from ultraviolet mutagenesis library of KO11. Comparative genomic analysis added by piecewise knockout strategy and complementation assay revealed EKO11_3023 (espA) within the 36.6-kb deletion from KO11 was the only locus responsible for ethanol sensitivity. Interestingly, when espA was deleted in strain W (the parent strain of KO11), ethanol tolerance was dramatically elevated to the level of espA-free hosts [e.g., MG1655 and BL21(DE3)]. And overexpression of espA in strains MG1655 and BL21(DE3) led to significantly enhanced ethanol sensitivity. In addition to ethanol, deletion of espA also improved cell tolerance to other short-chain (C2–C4) alcohols, including methanol, isopropanol, n-butanol, isobutanol and 2-butanol. Therefore, espA was responsible for short-chain alcohol sensitivity of W-strains compared to other cells, which provides a potential engineering target for alcohols production.     

Vanillin Resistance Induced by BssS Overexpression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Overexpression of the BssS gene, a biofilm formation regulator, in planktonic Escherichia coli cells has been shown to confer the vanillin-resistant phenotype Van^r to the bacteria. The MG1655P_L-tac-bssS strain started growing in liquid aerated LB medium with 2 g/L vanillin after a lag phase of 17 ± 2 h, whereas the original MG1655 strain did not grow under these conditions. The role of aldehyde reductase YqhD, a vanillin- degrading enzyme, in Van^r phenotype formation has been assessed. However, the Van^r trait in the MG1655P_L-tac-bssS strain primarily depended on autoinducer-2 (AI-2), which formed in E. coli cells with an intact luxS gene. We supposed that BssS acts together with autoinducer-2 (which presumably accumulated during the prolonged lag phase) to induce vanillin resistance determined by changes in the expression of a range of genes.     

Synthesis of methyl ketones by metabolically engineered Escherichia coli

Methyl ketones are a group of highly reduced platform chemicals with widespread applications in the fragrance, flavor and pharmacological industries. Current methods for the industrial production of methyl ketones include oxidation of hydrocarbons, but recent advances in the characterization of methyl ketone synthases from wild tomato have sparked interest towards the development of microbial platforms for the industrial production of methyl ketones. A functional methyl ketone biosynthetic pathway was constructed in Escherichia coli by over-expressing two genes from Solanum habrochaites: shmks2, encoding a 3-ketoacyl-ACP thioesterase, and shmks1, encoding a beta-decarboxylase. These enzymes enabled methyl ketone synthesis from 3-ketoacyl-ACP, an intermediate in the fatty acid biosynthetic cycle. The production of 2-nonanone, 2-undecanone, and 2-tridecanone by MG1655 pTH-shmks2-shmks1 was initially detected by nuclear magnetic resonance and gas chromatography–mass spectrometry analyses at levels close to 6?mg/L. The deletion of major fermentative pathways leading to ethanol (adhE), lactate (ldhA), and acetate (pta, poxB) production allowed for the carbon flux to be redirected towards methyl ketone production, doubling total methyl ketone concentration. Variations in methyl ketone production observed under different working volumes in flask experiments led to a more detailed analysis of the effects of oxygen availability on methyl ketone concentration in order to determine optimal levels of oxygen. The methyl ketone concentration achieved with MG1655 ?adhE ?ldhA ?poxB ?pta pTrcHis2A-shmks2-shmks1, the best performer strain in this study, was approximately 500?mg/L, the highest reported for an engineered microorganism. Through the establishment of optimal operating conditions and by executing rational metabolic engineering strategies, we were able to increase methyl ketone concentrations by almost 75-fold from the initial confirmatory levels.     

Improved 2-methyl-1-propanol production in an engineered Bacillus subtilis by constructing inducible pathways

High-level constitutive gene expression can result in cellular metabolic imbalance and limit production. To circumvent these problems, a P _alsSD -controlled auto-inducible 2-ketoisovalerate biosynthetic pathway and a P _spac -controlled IPTG-inducible Ehrlich pathway were constructed in Bacillus subtilis to modulate gene expression. Based on the precise gene expression characteristics of the two inducible pathways, the optimal IPTG induction time point and dose for 2-methyl-1-propanol biosynthesis were determined as 9.5?h and 300?μM, respectively. Under the optimized conditions, strain BSUΔL-03 with inducible pathways produced up to 3.83?±?0.46?g 2-methyl-1-propanol/l, which was about 60?% higher than BSUL04 with constitutive pathways.     

Engineering a homobutanol fermentation pathway in Escherichia coli EG03

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition.