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

Abstract

BACKGROUND: Acetoin, especially the chiral L-(+)-or D-(−)-enantiomer, is a high-value industrial product that can be used as a pharmaceutical intermediate. However, the production of optically pure acetoin via conventional chemical synthesis is expensive. Classical microbial fermentations satisfy the criteria of green chemistry, but have numerous disadvantages, such as complicated process engineering, unsatisfactory titers or productivities, and difficult product separation due to the inevitable generation of by-products and overly complex media. By contrast, enzymatic biocatalysis in cell-free biosystems has numerous advantages in production of chemicals with high purity, whereas efficient enzymatic methods for the production of optically pure acetoin have rarely been reported. RESULTS: An efficient coupled two-enzyme system composed of 2,3-butanediol dehydrogenase and H 2 O-forming NADH oxidase was constructed to produce optically pure acetoin without introducing any substrate or by-product. A 2,3-butanediol dehydrogenase and three NADH oxidases were purified, characterized and assembled into different two-enzyme systems. The best system, composed of LL-NOX and CG-BDH, was then selected according to the acetoin titer. After optimization of the reaction conditions, 94.0% of meso-2,3-butanediol was converted to D-(−)-acetoin. Finally, 399.7 mmol L −1 D-(−)-acetoin with an enantiomeric excess of 91.2% was produced in 6 h without any detected by-products. CONCLUSIONS: To our best knowledge, this is the first report on the production of optically pure D-(−)-acetoin from meso-2,3-butanediol in vitro using NADH oxidase to regenerate NAD + , as well as the best substrate conversion and acetoin titer among enzymatic biosynthesis. This work therefore provides an economical and green alternative for the in vitro production of D-(−)-acetoin. Industry well characterized. 46 As shown in Fig. 2, BS-NOX, CA-NOX and LL-NOX were overexpressed in E. coli BL21 (DE3) and all the NOXs and CG-BDH were successfully produced. The molecular masses of CG-BDH and the three NOX proteins were approximately 29.5, J Chem Technol Biotechnol 2019; 94: 2547-2554

Preparation of a whole cell catalyst overexpressing acetohydroxyacid synthase of Thermotoga maritima and its application in the syntheses of α-hydroxyketones

“…Stereoselectivity analyses of the TmcAHAS catalyzed reactions were performed also on the Aglient 7820A GC system equipped with an FID and an Agilent CP-Chirasil capillary column (20 m × 250 μm × 0.25 μm). For AC and HP analyses, the temperatures of the injector and detector were both set at 260°; The GC program was set as follows: 60 °C (3 min), ramp to 80 °C (7 min) at 10 °C min −1 , then a ramp of 20 °C min −1 to 125 °C (10 min) 1 . For R -PAC analysis, the temperatures of the injector and detector were both set at 220 °C; The GC program was set as follows: 100 °C (3 min), ramp to 120 °C (30 min) at 5 °C min −1 .…”

“…Moreover, AC can also be used as a flavor enhancer of butter, cheese, coffee and nut containing food because of its pleasant odor. Up to now, quite many biotransformation methods have been established to produce AC, mainly through oxidation of 2,3-butanediol by 2,3-butanediol dehydrogenase of different microorganisms or reduction of diacetyl by carbonyl reductase 1 – 4 . A cell-free cascade for preparation of AC using thermostable acetolactate synthase (ALS) from Caldicellulosiruptor owensensis and α-acetolactate decarboxylase from Bacillus subtilis has also been reported 5 .…”

Non-natural Aldol Reactions Enable the Design and Construction of Novel One-Carbon Assimilation Pathways in vitro

“…The cell pellets were lysed using a high-pressure homogenizer at 4°C, and subsequently centrifuged at 6,000 × g and 4°C for 40 min to remove cell debris. The clear lysate was onto a Ni-NTA His-binding column and concentrated as described previously (Cui et al, 2018(Cui et al, , 2019. The purity of the enzymes was analyzed by 12% SDS-PAGE (Supplementary Figure 1) and quantified using a bicinchoninic acid (BCA) Kit (CWBiotech, Beijing, China).…”

Efficient 1-Hydroxy-2-Butanone Production from 1,2-Butanediol by Whole Cells of Engineered E. coli

“…Fortunately, various diol dehydrogenases have shown the abilities in the oxidation of diol to the corresponding ketone alcohol [6,7]. Further, 2,3-Butanediol dehydrogenase (BDH), one of diol dehydrogenases, can catalyze the dehydrogenation of 2,3-butanediol (2,3-BD) into acetoin (AC, a stereoisomer of HB) [8][9][10][11][12]. Furthermore, the BDH enzymes from Serratia marcescens H30 and Paenibacillus polymyxa ATCC 12321 also showed the activities for 1,2-pentadiol and 1,2-propanediol as substrates [13,14].…”