Efficient Whole-Cell Biocatalyst for Acetoin Production with NAD+ Regeneration System through Homologous Co-Expression of 2,3-Butanediol Dehydrogenase and NADH Oxidase in Engineered Bacillus subtilis

An auto-inducing expression system was developed that could express target genes in S. marcescens MG1. Using this system, MG1 was constructed as a whole-cell biocatalyst to produce 2,3-butanediol/acetoin. Formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were expressed together to build an NADH regeneration system to transform diacetyl to 2,3-butanediol. After fermentation, the extract of recombinant S. marcescens MG1ABC (pETDuet-bdhA-fdh) showed 2,3-BDH activity of 57.8 U/mg and FDH activity of 0.5 U/mg. And 27.95 g/L of 2,3-BD was achieved with a productivity of 4.66 g/Lh using engineered S. marcescens MG1(Pswnb+pETDuet-bdhA-fdh) after 6 h incubation. Next, to produce 2,3-butanediol from acetoin, NADH oxidase and 2,3-butanediol dehydrogenase from Bacillus subtilis were co-expressed to obtain a NAD+ regeneration system. After fermentation, the recombinant strain S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC) showed AR activity of 212.4 U/mg and NOX activity of 150.1 U/mg. We obtained 44.9 g/L of acetoin with a productivity of 3.74 g/Lh using S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC). This work confirmed that S. marcescens could be designed as a whole-cell biocatalyst for 2,3-butanediol and acetoin production.

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Efficient (3R)-Acetoin Production from meso-2,3-Butanediol Using a New Whole-Cell Biocatalyst with Co-Expression of meso-2,3-Butanediol Dehydrogenase, NADH Oxidase, and Vitreoscilla Hemoglobin

Journal of Microbiology and Biotechnology ◽

10.4014/jmb.1608.08063 ◽

2017 ◽

Vol 27 (1) ◽

pp. 92-100 ◽

Cited By ~ 15

Author(s):

Zewang Guo ◽

Xihua Zhao ◽

Yuanzhi He ◽

Tianxing Yang ◽

Huifang Gao ◽

...

Keyword(s):

Nadh Oxidase ◽

Vitreoscilla Hemoglobin ◽

Whole Cell ◽

Whole Cell Biocatalyst ◽

Acetoin Production ◽

Butanediol Dehydrogenase

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Efficient L-Xylulose Production Using Whole-Cell Biocatalyst with NAD+ Regeneration System through Co-Expression of Xylitol Dehydrogenase and NADH Oxidase in Escherichia coli

Biochemical Engineering Journal ◽

10.1016/j.bej.2021.108137 ◽

2021 ◽

pp. 108137

Author(s):

Mesfin Angaw Tesfay ◽

Xin Win ◽

Huibin Lin ◽

Yujie Liu ◽

Can Li ◽

...

Keyword(s):

Escherichia Coli ◽

Nadh Oxidase ◽

Xylitol Dehydrogenase ◽

Regeneration System ◽

Whole Cell ◽

Whole Cell Biocatalyst

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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.

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