Enhanced hydrogen production from glucose by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

To utilize fermentative bacteria for producing the alternative fuel hydrogen, we performed successive rounds of P1 transduction from the Keio Escherichia coli K-12 library to introduce multiple, stable mutations into a single bacterium to direct the metabolic flux toward hydrogen production. E. coli cells convert glucose to various organic acids (such as succinate, pyruvate, lactate, formate, and acetate) to synthesize energy and hydrogen from formate by the formate hydrogen-lyase (FHL) system that consists of hydrogenase 3 and formate dehydrogenase-H. We altered the regulation of FHL by inactivating the repressor encoded by hycA and by overexpressing the activator encoded by fhlA, removed hydrogen uptake activity by deleting hyaB (hydrogenase 1) and hybC (hydrogenase 2), redirected glucose metabolism to formate by using the fdnG, fdoG, narG, focA, focB, poxB, and aceE mutations, and inactivated the succinate and lactate synthesis pathways by deleting frdC and ldhA, respectively. The best of the metabolically engineered strains, BW25113 hyaB hybC hycA fdoG frdC ldhA aceE, increased hydrogen production 4.6-fold from glucose and increased the hydrogen yield twofold from 0.65 to 1.3 mol H₂/mol glucose (maximum, 2 mol H₂/mol glucose).     

Characterization of hydrogen production by engineered Escherichia coli strains using rich defined media

Fermentation conditions (e.g., pressure and medium) are well-documented to impact the yield of microbial products in bioreactors. In this study we used carefully controlled batch fermentations to characterize hydrogen production from engineered strains of Escherichia coli and developed a rapid method of inducing hydrogen production in previously aerobically grown cells by using a rich defined medium. Our results indicated that rich defined media activated hydrogen production from aerobic pre-cultures with no lag time and yielded more hydrogen and biomass than the commonly used minimal media. Under these conditions, deletion of both uptake hydrogenase 1 (ΔhyaAB) and hydrogenase 2 (ΔhybABC) was shown to increase hydrogen yield from glucose by 10% over the wildtype strain BW25113. However, the deletion of the repressor for the formate-hydrogen-lyase (FHL-1) complex (ΔhycA) did not further increase hydrogen production. Additional deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdBC) of the mixed-acid fermentation pathway increased hydrogen yield by 22 and 23%, respectively. Interestingly, combined elimination of ldhA and frdBC in the uptake and hycA null strain increased hydrogen yield from 1.37 to 1.82 mol/mol glucose, obtaining 91% of the theoretical maximum hydrogen yield. Our results indicated the advantage of using rich defined media for inducing hydrogen production. This study represents the first report of characterizing metabolically engineered E. coli strains in batch hydrogen fermentation using rich defined media under tightly controlled conditions.     

Enhanced production of ethanol from glycerol by engineered Hansenula polymorpha expressing pyruvate decarboxylase and aldehyde dehydrogenase genes from Zymomonas mobilis

To improve production of ethanol from glycerol, the methylotrophic yeast Hansenula polymorpha was engineered to express the pdc and adhB genes encoding pyruvate decarboxylase and aldehyde dehydrogenase II from Zymomonas mobilis, respectively, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The ethanol yield was 3.3-fold higher (2.74 g l^?1) in the engineered yeast compared with the parent strain (0.83 g l^?1). Further engineering to stimulate glycerol utilization in the recombinant strain via expression of dhaD and dhaKLM genes from Klebsiella pneumoniae encoding glycerol dehydrogenase and dehydroxyacetone kinase, respectively, resulted in a 3.7-fold increase (3.1 g l^?1) in ethanol yield.     

Escherichia coli genome-scale metabolic gene knockout of lactate dehydrogenase (ldhA), increases succinate production from glycerol

Genome-scale metabolic model (GEM) of Escherichia coli has been published with applications in predicting metabolic engineering capabilities on different carbon sources and directing biological discovery. The use of glycerol as an alternative carbon source is economically viable in biorefinery. The use of GEM for predicting metabolic gene deletion of lactate dehydrogenase (ldhA) for increasing succinate production in Escherichia coli from glycerol carbon source remained largely unexplored. Here, I hypothesized that metabolic gene knockout of ldhA in E. coli from glycerol could increase succinate production. A proof-of-principle strain was constructed and designated as E. coli BMS5 (ΔldhA), by predicting increased succinate production in E. coli GEM and confirmed the predicted outcomes using wet cell experiments. The mutant GEM (ΔldhA) predicted 11% increase in succinate production from glycerol compared to its wild-type model (iAF1260), and the E. coli BMS5 (ΔldhA) showed 1.05 g/l and its corresponding wild-type produced .05 g/l (23-fold increase). The proof-of-principle strain constructed in this study confirmed the aforementioned hypothesis and further elucidated the fact that E. coli GEM can prospectively and effectively predict metabolic engineering interventions using glycerol as substrate and could serve as platform for new strain design strategies and biological discovery.     

Engineering glycolysis branch pathways of Escherichia coli to improve heterologous protein expression

Escherichia coli expression systems are still preferred to other bacterial expression systems. However, by-product formation via glycolytic pathways inhibits protein production efficiency. In this paper, by-product-forming pathways were engineered to evaluate their effect on foreign protein production. Elimination of d-lactate dehydrogenase (encoded by ldhA) resulted in enhanced cell performance and 17.8% increase in recombinant β-mannanase production. Single deletions of pflB (encoding pyruvate formate lyase), pps (encoding phoenolpyruvate synthase) or poxB (encoding pyruvate oxidase) also had an affirmative impact on recombinant protein production. Furthermore, simultaneous deletions of ldhA, pflB, pps and poxB increased cell mass by 29% and β-mannanase production by 56% under shake-flasks cultivation. Meanwhile, overall acetate concentration showed a decrease of 33%. This quadruple mutant showed the best performance under bioreactor process, in which volume and specific activity of β-mannanase increased by 1.9 and 1.46 fold compared to the control strain respectively. The approach shown here indicated that rational engineering of glycolytic pathways can efficiently improve foreign protein production in E. coli.     

Erratum to: Production of isobutanol from crude glycerol by a genetically-engineered Klebsiella pneumoniae strain

Klebsiella pneumoniae was engineered to produce isobutanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and α-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, ?ldhA/pBR-iBO (ilvIH–ilvC–ilvD–kivd–adhA), produced isobutanol (160 mg l^?1) from crude glycerol. To increase the yield of isobutanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating α-acetolactate decarboxylase (adc). This further engineering step improved the yield of isobutanol from 160 to 320 mg l^?1. This represents the first successful attempt to produce isobutanol from crude glycerol.     

Production of 2-butanol from crude glycerol by a genetically-engineered Klebsiella pneumoniae strain

Klebsiella pneumoniae was engineered to produce 2-butanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and α-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, ?ldhA/pBR-iBO (ilvIH–ilvC–ilvD–kivd–adhA), produced 2-butanol (160 mg l^?1) from crude glycerol. To increase the yield of 2-butanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating α-acetolactate decarboxylase (adc). This further engineering step improved the yield of 2-butanol from 160 to 320 mg l^?1. This represents the first successful attempt to produce 2-butanol from crude glycerol.     

Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase

Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83 g/l (0.06 g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84 g/l (0.41 g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60 g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain.     

Enhancement of xylitol production in glycerol kinase disrupted Candida tropicalis by co-expression of three genes involved in glycerol metabolic pathway

Glycerol can be used as a primary carbon source by yeasts, little is known regarding glycerol metabolism in Candida tropicalis. In this study, glycerol kinase gene (gk) was disrupted from xylitol dehydrogenase gene (XYL2) knockout C. tropicalis strain BSXDH-3. The resultant gk knockout C. tropicalis strain was incapable to grow on glycerol. The cells growth on glycerol was resumed by co-expressing Scheffersomyces stipitis gcy1, 2 and 3 genes, which respectively encode NADP⁺-dependent glycerol dehydrogenase 1, 2 and 3, under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed “GK”, than in BSXDH-3. In fermentation experiments using glycerol as co-substrate with xylose, strain GK produced xylitol 0.85 and 1.28 g l^?1 h^?1 at the time periods of 16 and 24 h, respectively, which is 30 and 18 % higher at same time intervals in BSXDH-3. This is the first report of gk gene disruption and co-expression of gcy1, 2 and 3 genes for NADPH regeneration and enhanced xylitol production in C. tropicalis.     

Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering

With microbial production becoming the primary choice for natural product synthesis, increasing precursor and cofactor availability has become a chief hurdle for the generation of efficient production platforms. As such, we employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability in Escherichia coli. Specifically, two different model objectives were used to identify possible genotypes that exhibited either improved overall NADPH production or an improved flux through an artificial reaction coupling NADPH yield to biomass. The top single, double and triple gene deletion candidates were constructed and as a case study evaluated for their ability to produce two polyphenols, leucocyanidin and (+)-catechin. Each is derived from their common precursor dihydroquercetin using two recombinant NADPH-dependent enzymes: dihydroflavonol 4-reductase and leucoanthocyanidin reductase. The best engineered strain carrying Δpgi, Δppc and ΔpldA deletions accumulated up to 817 mg/L of leucocyanidin and 39 mg/L (+)-catechin in batch culture with 10 g/L glucose in modified M9 medium, a 4-fold and 2-fold increase, respectively, compared to the wild-type control.     

Engineering of Corynebacterium glutamicum for High-Yield l-Valine Production under Oxygen Deprivation Conditions

We previously demonstrated efficient l-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the l-valine yield. Eliminating these by-products therefore was deemed key to improving the l-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and l-valine production dropped considerably due to the severely elevated intracellular NADH/NAD⁺ ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher l-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM l-valine at a yield of 88% mol mol of glucose⁻¹ after 24 h under oxygen deprivation, a vastly improved yield over our previous best.     

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.     

Metabolic engineering to enhance bacterial hydrogen production

Hydrogen fuel is renewable, efficient and clean, and fermentative bacteria hold great promise for its generation. Here we use the isogenic Escherichia coli K‐12 KEIO library to rapidly construct multiple, precise deletions in the E. coli genome to direct the metabolic flux towards hydrogen production. Escherichia coli has three active hydrogenases, and the genes involved in the regulation of the formate hydrogen lyase (FHL) system for synthesizing hydrogen from formate via hydrogenase 3 were also manipulated to enhance hydrogen production. Specifically, we altered regulation of FHL by controlling the regulators HycA and FhlA, removed hydrogen consumption by hydrogenases 1 and 2 via the hyaB and hybC mutations, and re‐directed formate metabolism using the fdnG, fdoG, narG, focA, fnr and focB mutations. The result was a 141‐fold increase in hydrogen production from formate to create a bacterium (BW25113 hyaB hybC hycA fdoG/pCA24N‐FhlA) that produces the largest amount of hydrogen to date and one that achieves the theoretical yield for hydrogen from formate. In addition, the hydrogen yield from glucose was increased by 50%, and there was threefold higher hydrogen production from glucose with this strain.     

Model-assisted formate dehydrogenase-O (fdoH) gene knockout for enhanced succinate production in Escherichia coli from glucose and glycerol carbon sources

Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H⁺ conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l^?1 (41.2-fold in 7 days) from glycerol and .39 g l^?1 (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.     

Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains

We improved the hydrogen yield from glucose using a genetically modified Escherichia coli. E. coli strain SR15 (ΔldhA, ΔfrdBC), in which glucose metabolism was directed to pyruvate formate lyase (PFL), was constructed. The hydrogen yield of wild-type strain of 1.08 mol/mol glucose, was enhanced to 1.82 mol/mol glucose in strain SR15. This figure is greater than 90 % of the theoretical hydrogen yield of facultative anaerobes (2.0 mol/mol glucose). Moreover, the specific hydrogen production rate of strain SR15 (13.4 mmol h⁻¹ g⁻¹ dry cell) was 1.4-fold higher than that of wild-type strain. In addition, the volumetric hydrogen production rate increased using the process where cells behaved as an effective catalyst. At 94.3 g dry cell/l, a productivity of 793 mmol h⁻¹ l⁻¹ (20.2 l h⁻¹ l⁻¹ at 37 °C) was achieved using SR15. The reported productivity substantially surpasses that of conventional biological hydrogen production processes and can be a trigger for practical applications.     

Engineering of the glycerol decomposition pathway and cofactor regulation in an industrial yeast improves ethanol production

Glycerol is a major by-product of industrial ethanol production and its formation consumes up to 4 % of the sugar substrate. This study modified the glycerol decomposition pathway of an industrial strain of Saccharomyces cerevisiae to optimize the consumption of substrate and yield of ethanol. This study is the first to couple glycerol degradation with ethanol formation, to the best of our knowledge. The recombinant strain overexpressing GCY1 and DAK1, encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, in glycerol degradation pathway, exhibited a moderate increase in ethanol yield (2.9 %) and decrease in glycerol yield (24.9 %) compared to the wild type with the initial glucose concentration of 15 % under anaerobic conditions. However, when the mhpF gene, encoding acetylating NAD⁺-dependent acetaldehyde dehydrogenase from Escherichia coli, was co-expressed in the aforementioned recombinant strain, a further increase in ethanol yield by 5.5 % and decrease in glycerol yield by 48 % were observed for the resultant recombinant strain GDMS1 when acetic acid was added into the medium prior to inoculation compared to the wild type. The process outlined in this study which enhances glycerol consumption and cofactor regulation in an industrial yeast is a promising metabolic engineering strategy to increase ethanol production by reducing the formation of glycerol.     

Enhancement of ethanol production from glycerol in a Klebsiella pneumoniae mutant strain by the inactivation of lactate dehydrogenase

Previously, using γ-irradiation treatment, we isolated a mutant strain of Klebsiella pneumoniae (named GEM167) that showed high-level ethanol production from glycerol. In the present study, in an effort to enhance ethanol production, we used a deletion of the lactate dehydrogenase gene to engineer a mutant strain incapable of lactate synthesis. In the ΔldhA mutant of GEM167, the production of ethanol was significantly increased from 21.5 g/l to 28.9 g/l and from 0.93 g/(l h) to 1.2 g/(l h). Introduction of the Zymomonas mobilis pdc and adhII genes encoding pyruvate decarboxylase and aldehyde dehydrogenase, respectively, further improved the ethanol production level from glycerol to 31.0 g/l; this is the highest level reported to date.     

Engineering a native homoethanol pathway in Escherichia coli B for ethanol production

A native homoethanol pathway (pyruvate-to-acetyl-CoA-to-acetaldehyde-to-ethanol) was engineered in Escherichia coli B. The competing fermentation pathways were eliminated by chromosomal deletions of the genes encoding for fumarate reductase (frdABCD), lactate dehydrogenase (ldhA), acetate kinase (ackA), and pyruvate formate lyase (pflB). For redox balance and anaerobic cell growth, the pyruvate dehydrogenase complex (aceEF-lpd, a typical aerobically-expressed operon) was highly expressed anaerobically using a native anaerobic inducible promoter. The resulting strain SZ420 (ΔfrdBC ΔldhA ΔackA ΔfocA-pflB ΔpdhR::pflBp6-pflBrbs-aceEF-lpd) contains no foreign genes and/or promoters and efficiently ferments glucose and xylose into ethanol with a yield of 90% under anaerobic conditions.