Metabolic engineering ofEscherichia colifor efficient production of (3R)-acetoin

Abstract

BACKGROUND: Optically pure acetoin is an important potential pharmaceutical intermediate. It has also been widely used to synthesize novel optically active α-hydroxyketone derivatives and liquid crystal composites. Recombinant Escherichia coli was developed for efficient (3R)-acetoin production. Culture medium optimization and process control were carried out to improve (3R)-acetoin yield by the engineered strain. RESULTS: A synthetic pathway involved the budRAB genes from Serratia marcescens and NADH oxidase gene fromLactobacillus brevis in E. coli was developed for efficient (3R)-acetoin production. Batch culture showed that 23.4 g L −1 of (3R)-acetoin could be obtained from 60 g L −1 glucose by the engineered strain. Chiral-column GC analysis indicated that the stereoisomeric purity of (3R)-acetoin produced was 97.3%. Further, the medium composition was optimized in shake flasks by an orthogonal design method. Under optimal conditions, (3R)-acetoin concentration reached 38.3 g L −1 in flask fermentation. Fed-batch fermentation based on a suitable agitation speed was carried out in a 5 L bioreactor, and maximum (3R)-acetoin concentration of 60.3 g L −1 was achieved with a productivity of 1.55 g L −1 h −1 and yield 86.3%. CONCLUSION: An engineering E. coli for efficient (3R)-acetoin production was constructed. The optimization of fermentation variables and fed-batch culture resulted in a maximum (3R)-acetoin concentration of 60.3 g L −1 . DISCUSSIONChiral AC has recently been paid increasing attention due to its potential industrial and pharmaceutical applications. 7 However, natural microorganisms usually produce a mixture of (3R)-AC and (3S)-AC, which is difficult to accumulate due to a large J Chem Technol Biotechnol 2015; 90: 93-100

High acetoin production by a newly isolated marine Bacillus subtilis strain with low requirement of oxygen supply

“…The sea sediment samples were obtained from Pusamiao (1), Zhangzi Island (2), Beijingzi (3), Daludao (4), Dalianwan (5) and Xiaobaodao (6) of Dalian, China. The isolation of strains was carried out in two steps.…”

“…Direct production of acetoin from 2,3-butanediol was also an efficient way and the acetoin concentration reached 89.2 g/l by Gluconobacter oxydans DSM 2003 [4]. The study of single isomer production was focused on metabolically engineered strains [5,6], and 60.3 g/l D-acetoin was obtained by recombinant E. coli [6]. However, until now, very few reports have been published on the fermentation of one single isomer of acetoin by a native strain.…”

Production of 2,3‐butanediol from diverse saccharides via fermentation

“…Glucose is enzymatically fermented and transformed into pyruvic acid (PA) by glycolysis, and galactose is transformed by the Leloir pathway into glucose‐6‐phosphate, which is transformed into PA by glycolysis . Then, 3 successive enzymatic reactions are performed: i) from PA to α‐acetolactate by the enzyme α‐acetolactate synthase (ALS); ii) from α‐acetolactate to A by the enzyme α‐acetolactate decarboxylase (ALDC); and iii) from A to 2,3‐BD by the enzyme 2,3‐butanediol dehydrogenase (BDH) . Under aerobic conditions, which may inhibit the ALS enzyme, α‐acetolactate may be transformed into diacetyl, and then into A.…”

“…Metabolic pathway to produce 2,3‐BD from lactose fermentation in the presence of Escherichia coli JFR12 . Relevant reactions to transform the pyruvic acid into 2,3‐BD are represented by the names of the corresponding enzymes from Enterobacter cloacae : α‐acetolactate synthase (ALS); α‐acetolactate decarboxylase (ALDC); and 2,3‐butanediol dehydrogenase (BDH); and the reduced (NADH) and oxidized (NAD + ) form of the coenzyme nicotinamide adenine dinucleotide (NAD).…”

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

“…Acetoin and its isomers are widely applied in foods, cosmetics, chemical synthesis, especially in asymmetric synthesis of optically active pharmaceuticals and liquid crystals. 10,11 Its derivate, 4-chloro-4,5-dimethyl-1,3dioxolan-2-one, has been extensively utilized in modifying antibiotics to synthesize optically active drugs, which could enhance drug targeting properties and reduce side effects. 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.…”