BACKGROUNDOptically pure acetoin is an important potential pharmaceutical intermediate. It has also been widely used to synthesize novel optically active -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. RESULTSA synthetic pathway involved the budRAB genes from Serratia marcescens and NADH oxidase gene from Lactobacillus 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%. CONCLUSIONAn 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. (c) 2013 Society of Chemical Industry