Concomitant cell‐free biosynthesis of optically pure D‐(−)‐acetoin and xylitol via a novel NAD⁺ regeneration in two‐enzyme cascade

Abstract

BACKGROUND: Acetoin and xylitol are widely used as high-value platform chemicals with numerous potential applications. The chiral enantiomers L-(+)-and D-(−)-acetoin are used as pharmaceutical intermediates. Cell-free biosynthesis has many applications in chemicals production, but efficient methods for production of optically pure acetoin were rarely reported. RESULTS:A novel two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase was constructed to co-produce acetoin and xylitol with NAD + regeneration. Four BDHs from four candidates, as well as xylose reductase from Candida tenuis were first purified and characterized. The best BDH was then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g L −1 D-(−)-acetoin with an optical purity of 95.2% was produced in 6 h. The yield and productivity of acetoin was 0.97 g g −1 and 4.75 g L −1 h −1 . The titer of co-product xylitol was 40.29 g L −1 , and the yield and productivity of xylitol reached 0.98 g g −1 and 6.72 g L −1 h −1 . CONCLUSIONS: To our best knowledge, this is the first report on the production of optically pure D-(−)-acetoin using xylose reductase to regenerate NAD + , as well as the highest acetoin titer among enzymatic synthesis methods. This work therefore provides an economical and green alternative for the in vitro production of D-(−)-acetoin.

In vitro biosynthesis of optically pure d‐(−)‐acetoin from meso‐2,3‐butanediol using 2,3‐butanediol dehydrogenase and NADH oxidase

“…CG‐BDH was well‐characterized in our previous work . Its optimal temperature and pH were 52 °C and 8.5, and its K m values for meso ‐2,3‐BD and NAD + were 2.6 and 0.2 mmol L −1 , respectively.…”

“…One‐pot enzymatic reactions were conducted in 400 μL of 100 mmol L −1 sodium phosphate buffer (pH 6.5) containing 50 mmol L −1 meso ‐2,3‐BD, 5 mmol L −1 NAD + , 0.5 U mL −1 CG‐BDH, 0.5 U mL −1 NOX and the relevant FAD, as described previously . The reactions were performed at 35 °C and 220 rpm.…”

“…To realize an economical and environmentally friendly process for the biosynthesis of chiral AC in vitro , chiral meso ‐2,3‐butanediol ( meso ‐2,3‐BD) has been favored as a promising substrate, because it is innocuous, and cheaper than AC, especially chiral acetoin . Moreover, there is no carbon loss from 2,3‐butanediol to AC . Kochius and coworkers achieved an AC titer of 4.23 g L −1 from meso ‐2,3‐BD with almost the maximum theoretical yield through an electroenzymatic process, but its productivity was only 0.176 g L −1 h −1 , which may be due to the inefficiency of electrochemical nicotinamide adenine dinucleotide ( NAD) + regeneration.…”

Enhanced production of optical (S)-acetoin by a recombinant Escherichia coli whole-cell biocatalyst with NADH regeneration

“…12,13 Native microorganisms generally produce acetoin as a mixture of two stereoisomers, in which (R)-acetoin is predominant and (S)-acetoin exists as a minor by-product, leading to very low yield of (S)-acetoin and high cost of chiral separation. [14][15][16] Liu et al developed an engineered Lactococcus lactis to produce (S)-acetoin from glucose. 17 However, the nonenzymatic oxidative decarboxylation of a-acetolactate to diacetyl occurred spontaneously and inefficiently, the engineered strain could only produce 5.8 g L À1 of (S)-acetoin with a productivity of 0.19 g (L h) À1 .…”

Engineering central pathways for industrial-level (3R)-acetoin biosynthesis in Corynebacterium glutamicum

“…The biotechnological production of safe and natural acetoin could be more ecological and sustainable than their chemical counterparts [8,9]. These methods, including microbial fermentation [8,10,11], whole-cell biocatalysis [12,13] and enzymatic biocatalysis [14,15], have consequently gained great attention over the past decades. Saccharomyces cerevisiae JHY617-SDN was able to efficiently accumulate 100.2 g/L acetoin from glucose during fed-batch fermentation [16].…”