Construction of an
            <i>Escherichia coli</i>
            K-12 Mutant for Homoethanologenic Fermentation of Glucose or Xylose without Foreign Genes

Kim, Youngnyun; Ingram, L. O.; Shanmugam, K. T.

doi:10.1128/aem.02456-06

Cited by 128 publications

(98 citation statements)

References 31 publications

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“…In T. saccharolyticum ALK2, one of the two moles of NAD(P)H required to make a mole of ethanol from acetyl-CoA originates from glycolysis, and the second originates from the action of FNOR. A similar redirection of electron flow was recently reported for a pyruvate decarboxylase-minus strain of E. coli-metabolizing pyruvate via NADH-forming pyruvate dehydrogenase (19). The results reported here support the feasibility of engineering the large group of bacteria that metabolize pyruvate by POR to achieve high ethanol yields, including C. thermocellum which exhibits one of the highest rates of cellulose utilization known (13).…”

Section: Discussionsupporting

confidence: 68%

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

Shaw

Podkaminer²,

Desai³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

330

242

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We report engineering Thermoanaerobacterium saccharolyticum, a thermophilic anaerobic bacterium that ferments xylan and biomass-derived sugars, to produce ethanol at high yield. Knockout of genes involved in organic acid formation (acetate kinase, phosphate acetyltransferase, and L-lactate dehydrogenase) resulted in a strain able to produce ethanol as the only detectable organic product and substantial changes in electron flow relative to the wild type. Ethanol formation in the engineered strain (ALK2) utilizes pyruvate:ferredoxin oxidoreductase with electrons transferred from ferredoxin to NAD(P), a pathway different from that in previously described microbes with a homoethanol fermentation. The homoethanologenic phenotype was stable for >150 generations in continuous culture. The growth rate of strain ALK2 was similar to the wild-type strain, with a reduction in cell yield proportional to the decreased ATP availability resulting from acetate kinase inactivation. Glucose and xylose are co-utilized and utilization of mannose and arabinose commences before glucose and xylose are exhausted. Using strain ALK2 in simultaneous hydrolysis and fermentation experiments at 50°C allows a 2.5-fold reduction in cellulase loading compared with using Saccharomyces cerevisiae at 37°C. The maximum ethanol titer produced by strain ALK2, 37 g/liter, is the highest reported thus far for a thermophilic anaerobe, although further improvements are desired and likely possible. Our results extend the frontier of metabolic engineering in thermophilic hosts, have the potential to significantly lower the cost of cellulosic ethanol production, and support the feasibility of further cost reductions through engineering a diversity of host organisms.bioenergy ͉ cellulosic ethanol ͉ thermophile

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Section: Discussionsupporting

confidence: 68%

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

Shaw

Podkaminer²,

Desai³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

330

242

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“…Formate is an indicator of PFL activity because these strains lack formate hydrogen-lyase activity. The small amount of ethanol and acetate (<5% of glucose carbon) produced by strain QZ19 is probably derived from acetyl-CoA produced by pyruvate dehydrogenase complex (25).…”

Section: Resultsmentioning

confidence: 99%

“…To confirm that the gldA101 allele in strain QZ19 encodes D-LDH activity, the gene was cloned from strain QZ19 (plasmid pQZ115) and introduced into E. coli strain AH242. E. coli strain AH242 is anaerobic minus due to mutations in ldhA and pflB that abolished the ability to oxidize NADH produced during glycolysis (25). Anaerobic growth of strain AH242 was restored by plasmid pQZ115 with D(−)-lactate as the fermentation product.…”

Section: Identification Of Glycerol Dehydrogenase As the Source Of D(mentioning

confidence: 96%

Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose

Wang

Ingram

Shanmugam

2011

Proc. Natl. Acad. Sci. U.S.A.

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Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40°C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50°C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(−)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L −1 of optically pure D(−)-lactic acid from glucose in <48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min −1 ðmg proteinÞ −1 . By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50°C and pH 5.0), the cellulase usage could be reduced to 1∕3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(−) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.

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“…Industrial production of ethanol is dominated by the conversion of sugars from starch or sucrose to ethanol by the yeast Saccharomyces cerevisiae [55]. Significant research efforts have focused on engineering other microorganisms to produce maximal ethanol yields and titers from lignocellulosic derived sugars [56][57][58][59][60]. Despite the success of these engineering efforts, the oxidation state of sugars such as glucose dictates that about half of their weight is lost as CO 2 during the fermentation process (Figure 3), reducing the product yield.…”

Section: Propionic Acidmentioning

confidence: 99%

Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals

Clomburg

González

2013

Trends in Biotechnology

271

184

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Construction of an Escherichia coli K-12 Mutant for Homoethanologenic Fermentation of Glucose or Xylose without Foreign Genes

Cited by 128 publications

References 31 publications

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose

Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals

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