Enhanced production of bacterial cellulose in Komagataeibacter xylinus via tuning of biosynthesis genes with synthetic RBS.

Bacterial cellulose (BC) has outstanding physical and chemical properties, including high crystallinity, moisture retention, and tensile strength. Currently, the major producer of BC is Komagataeibacter xylinus. However, due to limited tools of expression, this host is difficult to engineer metabolically to improve BC productivity. In this study, a regulated expression system for K. xylinus with synthetic ribosome binding site (RBS) was developed and used to engineer a BC biosynthesis pathway. A synthetic RBS library was constructed using green fluorescent protein (GFP) as a reporter, and three synthetic RBS (R4, R15, and R6) with different strengths were successfully isolated by fluorescence-activated cell sorting (FACS). Using synthetic RBS, the expression of three homologous genes, pgm, galU, and ndp, responsible for BC production were optimized, and BC production was greatly increased under both static and shaking culture conditions. The final titer of BC under static and shaking conditions was 5.28 and 3.67 g/L, respectively. Our findings demonstrate that reinforced metabolic flux towards BC through quantitative gene expression represents a practical strategy for the improvement of BC productivity.