Metabolic Engineering of <i>Escherichia coli</i> KO11 with the NADH Regeneration System for Enhancing Ethanol Production

Interests remain in searching for cofactor regeneration system with higher efficiency at lower substrate cost. Glucose dehydrogenase (GDH) system has been dominant in NADH regeneration, but it only has a theoretical yield of one NADH per glucose molecule. This work sought to explore the utility of a two-step ethanol utilization pathway (EUP) in pathway-based NADH regeneration. The pathway runs from ethanol to acetaldehyde and to acetyl-CoA with each step generating one NADH, that together results in a higher theoretical yield of two NADH per ethanol molecule. In this project, anaerobic biotransformation of ketone (acetophenone or butanone) to alcohol by cpsADH from Candida parapsilosis was used as readout for evaluating relative efficacy and operating modes for EUP cofactor regeneration in Escherichia coli BL21 (DE3). Experiment tests validated that EUP was more efficient than GDH in NADH regeneration. Further, growing cell delivered higher biotransformation efficiency compared to resting cell due to the driving force generated by cell growth. Finally, preculture or cultivation in M9 + 10 g/L ethanol medium delivered higher biotransformation efficiency compared to LB medium. Overall, EUP could help regenerate NADH in support of a biocatalytic reaction, and is more efficient in cofactor regeneration than GDH.

Download Full-text

Flux through Citrate Synthase Limits the Growth of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.68.3.1071-1081.2002 ◽

2002 ◽

Vol 68 (3) ◽

pp. 1071-1081 ◽

Cited By ~ 63

Author(s):

S. A. Underwood ◽

M. L. Buszko ◽

K. T. Shanmugam ◽

L. O. Ingram

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Ethanol Production ◽

Citrate Synthase ◽

Corn Steep Liquor ◽

Pyruvate Decarboxylase ◽

Luria Broth ◽

Mineral Salts ◽

E Coli ◽

Steep Liquor

ABSTRACT Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD+. Expression of low-Km Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.

Download Full-text

Allitol bioproduction by recombinant Escherichia coli with NADH regeneration system co-expressing Ribitol Dehydrogenase (RDH) and Formate Dehydrogenase (FDH) in individual or in fusion

Electronic Journal of Biotechnology ◽

10.1016/j.ejbt.2021.11.007 ◽

2021 ◽

Author(s):

Xin Wen ◽

Huibin Lin ◽

Yilin Ren ◽

Can Li ◽

Chengjia Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Formate Dehydrogenase ◽

Recombinant Escherichia Coli ◽

Regeneration System ◽

Nadh Regeneration ◽

Ribitol Dehydrogenase

Download Full-text

Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for ε-caprolactone production

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00370-w ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Jinghui Xiong ◽

Hefeng Chen ◽

Ran Liu ◽

Hao Yu ◽

Min Zhuo ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Regeneration System ◽

Nadph Regeneration ◽

Whole Cell ◽

Cyclohexanone Monooxygenase ◽

Ribosome Binding ◽

Ribosome Binding Sites ◽

Whole Cell Biocatalyst ◽

Substrate Tolerance

Abstractε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

Download Full-text