Reverse Metabolic Engineering of Bacillus Subtilis for Xylose Utilization and Acetoin/ 2,3-Butanediol Production

Xylose is one of the most abundant sugars in lignocellulosic biomass and efficient utilization of it by bacteria is essential for production of fuels and chemicals from lignocellulosic biomass. However, wild type Bacillus subtilis is not able to utilize xylose as the sole carbon source. In this study, Bacillus subtilis 168 was subjected to laboratory adaptive evolution and a mutant E72 capable of utilizing xylose with a specific growth rate of 0.445 h^-1 was obtained. By whole genome sequencing a total of sixteen mutations were identified in strain E72. Through further analysis of these mutations, three of them, which were in the coding regions of genes araR, sinR and comP, were identified as the beneficial mutations. A reconstructed strain 168ARSRCP harboring these mutations was developed and exhibited maximum and average xylose consumption rates of 3.71 g/l/h and 1.21 g/l/h respectively in bioreactor containing minimal medium. Then acoA gene was deleted and xylA, xylB, sthA gene were co-overexpressed for effective accumulation of acetoin and 2,3-butanediol in glucose-xylose mixtures. Under oxygen-limited conditions, the final engineered strain produced 5.8g/l acetoin and 2.4g/l D-（-）-2,3-butanediol from10g/l glucose and 12 g/l xylose in M9 minimal medium. These results suggested that this strain could be used as a potential platform for production of fuels and chemicals from lignocellulosic biomass.