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

Abstract

Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD+ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD+ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD+ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD+ regeneration may also be a useful way for improving the productivity of NAD+-dependent chemistry-based products.

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

“…Expression of KSDD was also detected by western blotting with a mouse monoclonal anti-His6 antibody. The samples were treated as described by Bao et al [26], and the bands were detected by chemiluminescence with luminol and peroxide with the help of a Bio-Rad Chemidoc XRS. Bradford method was employed to determine the protein concentration by using BSA as standard protein [27].…”

Enhanced intracellular soluble production of 3‐ketosteroid‐Δ1‐dehydrogenase from Mycobacterium neoaurum in Escherichia coli and its application in the androst‐1,4‐diene‐3,17‐dione production

“…The AD conversion rate decreased when the pH shifted away from the optimal pH, which indicated that the KSDD enzyme could only retain limited activity in these pH conditions. Temperature is also an important factor in whole‐cell catalytic processes, and affects enzyme activity and cellular maintenance . Reactions were carried out at temperatures ranging from 20 to 50°C and used to determine the optimal temperature.…”

“…Among all these approaches, due to its mild and simple reaction conditions, high catalytic efficiency and selectivity, environmental compatibility, and easy purification process, enzymatic synthesis using KSDD to catalyze AD dehydrogenation to ADD is a favorable way . With the constant improvement in environmental protection consciousness and the further development of green technology, non‐pollution and non‐toxic biological technology has inevitably become the main direction of industrial development . So far, several microorganisms including Mycobacterium , Rhodococcus , Nocardia , and Arthrobacter were found capable of transforming AD to ADD .…”

Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ1-dehydrogenase and catalase inBacillus subtilis

“…To achieve a higher product yield, fed‐batch strategies could efficiently improve the production of the aimed products (Bao et al . ; Shao et al . ).…”

“…With the constant improvement in environmental protection consciousness and further development of green technology, nonpollution and nontoxic biological technology has inevitably become the main direction of industrial development (Bao et al . ; Shao et al . ).…”