Efficient 2-O-α-D-glucopyranosyl-sn-glycerol production by single whole-cell biotransformation through combined engineering and expression regulation with novel sucrose phosphorylase from Leuconostoc mesenteroides ATCC 8293

Abstract

2-O-α-D-glucopyranosyl-sn-glycerol (2-αGG) is a high value product with wide applications. Here, an efficient, safe and sustainable bioprocesses for 2-αGG production was designed. A novel sucrose phosphorylase (SPase) was firstly identified from Leuconostoc mesenteroides ATCC 8293. Subsequently, SPase mutations were processed with computer-aided engineering, of which the activity of SPaseK138C was 160% higher than that of the wild-type. Structural analysis revealed that K138C was a key functional residue moderating substrate binding pocket and thus influences catalytic activity. Furthermore, Corynebacterium glutamicum was employed to construct microbial cell factories along with ribosome binding site (RBS) fine-tuning and a two-stage substrate feeding control strategy. The maximum production of 2-αGG by these combined strategies reached 351.8 g·L^-1 with 98% conversion rate from 1.4 M sucrose and 3.5 M glycerol in a 5-L bioreactor. This was one of the best performance reported in single-cell biosynthesis of 2-αGG, which paved effective ways for industrial-scale preparation of 2-αGG.

Keywords: 2-αGG; Biosynthesis; Corynebacterium glutamicum; Novel SPase; Protein engineering.