Doubling the catabolic reducing power (NADH) output of <i>Escherichia coli</i> fermentation for production of reduced products

Zhou, Shengde; Iverson, Andrew; Grayburn, W. Scott

doi:10.1002/btpr.302

Cited by 21 publications

(20 citation statements)

References 29 publications

(38 reference statements)

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“…Chromosomal gene deletions were constructed using previously described procedures [5,31,44,45]. The chromosomal deletion was veriWed by analysis of PCR product size.…”

Section: Genetic Methodsmentioning

confidence: 99%

“…In previous studies, a non-transgenic homoethanol strain E. coli SZ420 was engineered from E. coli B by chromosomal gene deletions of competing fermentation pathway genes (ldhA, pXB, ackA, frdBC) and anaerobic expression of the pyruvate dehydrogenase operon (pXBp6-aceEF-lpd) [44,45]. The cell growth and ethanol production rates of SZ420 were low in glucose and even lower in xylose fermentation [44].…”

Section: Deletion Of Cra and The Hsr2 Region Of Rngmentioning

confidence: 99%

“…Interestingly, the RNase G HSR2 deletion also increased mRNA expression of many other genes related to glycolysis and the homoethanol pathway, such as glucokinase (158-fold), phosphoglucose isomerase (88-fold), triose phosphate isomerase (78-fold), glyceraldehyde 3-phosphate (47-fold), enolase (164-fold), pyruvate dehydrogenase (aceEF-lpd) (64-fold), and transcriptional regulator Fnr (fnr) (88-fold) [25]. Enhanced expression of the aceEF-lpd operon was probably derived from the improved fnr expression, which enhances anaerobic expression of pXB, because aceEF-lpd was previously transcriptionally fused with a pXB promoter and Fnr binding box [44,45].…”

Section: G L ¡1 Arabinose Fermentationmentioning

confidence: 99%

“…In our previous studies, a non-transgenic Escherichia coli strain was engineered by establishing a native homoethanol pathway ( [20,41,44,45]. The resulting non-transgenic strain, SZ420, fermented xylose to ethanol to greater than 90% of the theoretic yield (1.2 molecule of xylose converted to 2 molecules of ethanol).…”

Section: Introductionmentioning

confidence: 99%

“…The majority of previous eVorts have focused on transgenic approaches, either by cloning homoethanol pathway genes into C5-fermenting hosts [7,36,42], or by inserting the C5 sugar catabolic pathway genes into homoethanolfermenting hosts [9,10,12,21,43]. Few studies have been devoted to development of a non-transgenic strain for homoethanol fermentation from C5 sugars [3,19,40,44,45]. Non-transgenic strains may have fewer concerns than the transgenic ones, at least in certain regions of the world, regarding public perceptions and/or governmental regulations of the genetically modiWed organisms (GMO) when applied to large-scale fermentation.…”

Section: Introductionmentioning

confidence: 99%

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Partial deletion ofrng(RNase G)-enhanced homoethanol fermentation of xylose by the non-transgenicEscherichia coliRM10

Manow

Wang

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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Previously, a native homoethanol pathway was engineered in Escherichia coli B by deletions of competing pathway genes and anaerobic expression of pyruvate dehydrogenase (PDH encoded by aceEF-lpd). The resulting ethanol pathway involves glycolysis, PDH, and alcohol dehydrogenase (AdhE). The E. coli B-derived ethanologenic strain SZ420 was then further improved for ethanol tolerance (up to 40 g l−1 ethanol) through adaptive evolution. However, the resulting ethanol tolerant mutant, SZ470, was still unable to complete fermentation of 75 g l−1 xylose, even though the theoretical maximum ethanol titer would have been less than 40 g l−1 should the fermentation have reached completion. In this study, the cra (encoding for a catabolite repressor activator) and the HSR2 region of rng (encoding for RNase G) were deleted from SZ470 in order to improve xylose fermentation. Deletion of the HSR2 domain resulted in significantly increased mRNA levels (47-fold to 409-fold) of multiple glycolytic genes (pgi, tpiA, gapA, eno), as well as the engineered ethanol pathway genes (aceEF-lpd, adhE) and the transcriptional regulator Fnr (fnr). The higher adhE mRNA level resulted in increased AdhE activity (>twofold). Although not measured, the increase of other mRNAs might also enhance expressions of their encoding proteins. The increased enzymes would then enable the resulting strain, RM10, to achieve increased cell growth and complete fermentation of 75 g l−1 xylose with an 84% improved ethanol titer (35 g l−1), compared to that (19 g l−1) obtained by the parent, SZ470. However, deletion of cra resulted in a negative impact on cell growth and xylose fermentation, suggesting that Cra is important for long-term fermentative cell growth.

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“…Chromosomal gene deletions were constructed using previously described procedures [5,31,44,45]. The chromosomal deletion was veriWed by analysis of PCR product size.…”

Section: Genetic Methodsmentioning

confidence: 99%

Section: Deletion Of Cra and The Hsr2 Region Of Rngmentioning

confidence: 99%

Section: G L ¡1 Arabinose Fermentationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Partial deletion ofrng(RNase G)-enhanced homoethanol fermentation of xylose by the non-transgenicEscherichia coliRM10

Manow

Wang

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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Butyrate production enhancement by Clostridium tyrobutyricum using electron mediators and a cathodic electron donor

Choi

Sang

2012

Biotech & Bioengineering

135

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Electron mediators and electron supply through a cathode were examined to enhance the reducing power for butyrate production by an acidogenic clostridium strain, Clostridium tyrobutyricum BAS 7. Among the tested electron mediators, methyl viologen (MV)-amended cultures showed an increase of butyrate productivity (1.3 times), final concentration (1.4 times), and yield (1.3 times). The electron flow altered by MV addition from the ferredoxin pool to the NADH pool was shown by one electron model, implying that more available NADH increased butyrate production. In the cathode compartment poised at -400 mV versus the Ag/AgCl electrode, the neutral red (NR)-amended cultures of Clostridium tyrobutyricum BAS 7 increased butyrate concentration (from 5 to 8.8 g/L) and yield (from 0.33 up to 0.44 g/g) with no acetate production at all. Given that electrically reduced NR (NR(red) , yellow) by the cathode was re-oxidized (NR(ox) , red) in the cells on the basis of color change, electron flow from NR(red) to NAD(+) (i.e., NADH generation) induced an increase in butyrate production. This is the first report to show the increase of butyric acid production by electrically driven acidogenesis. These results show that the electron flow altered NADH formation by electron mediators and by the cathodic electron donor, increasing the yield and selectivity of reduced end-products like butyrate.

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Adaptive evolution of nontransgenic Escherichia coli KC01 for improved ethanol tolerance and homoethanol fermentation from xylose

Wang

Manow

Finan

et al. 2010

J Ind Microbiol Biotechnol

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Due to its excellent capability to ferment five-carbon sugars, Escherichia coli has been considered one of the platform organisms to be engineered for production of cellulosic ethanol. Nevertheless, genetically engineered ethanologenic E. coli lacks the essential trait of alcohol tolerance. Development of ethanol tolerance is required for cost-effective ethanol fermentation. In this study, we improved alcohol tolerance of a nontransgenic E. coli KC01 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) through adaptive evolution. During ~350 generations of adaptive evolution, a gradually increased concentration of ethanol was used as a selection pressure to enrich ethanol-tolerant mutants. The evolved mutant, E. coli SZ470, was able to grow anaerobically at 40 g l(-1) ethanol, a twofold improvement over parent KC01. When compared with KC01 for small-scale (500 ml) xylose (50 g l(-1)) fermentation, SZ470 achieved 67% higher cell mass, 48% faster volumetric ethanol productivity, and 50% shorter time to complete fermentation with ethanol titer of 23.5 g l(-1) and yield of 94%. These results demonstrate that an industry-oriented nontransgenic E. coli strain could be developed through incremental improvements of desired traits by a combination of molecular biology and traditional microbiology techniques.

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Doubling the catabolic reducing power (NADH) output of Escherichia coli fermentation for production of reduced products

Cited by 21 publications

References 29 publications

Partial deletion ofrng(RNase G)-enhanced homoethanol fermentation of xylose by the non-transgenicEscherichia coliRM10

Partial deletion ofrng(RNase G)-enhanced homoethanol fermentation of xylose by the non-transgenicEscherichia coliRM10

Butyrate production enhancement by Clostridium tyrobutyricum using electron mediators and a cathodic electron donor

Adaptive evolution of nontransgenic Escherichia coli KC01 for improved ethanol tolerance and homoethanol fermentation from xylose

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