Improving the productivity of Candida glycerinogenes in the fermentation of ethanol from non-detoxified sugarcane bagasse hydrolysate by a hexose transporter mutant.

Abstract

In this study, we attempted to increase the productivity of Candida glycerinogenes yeast for ethanol production from non-detoxified sugarcane bagasse hydrolysates (NDSBH) by identifying the hexose transporter in this yeast that makes a high contribution to glucose consumption, and by adding additional copies of this transporter and enhancing its membrane localisation stability (MLS). Based on the knockout and overexpression of key hexose transporter genes and the characterisation of their promoter properties, we found that Cghxt4 and Cghxt6 play major roles in the early and late stages of fermentation, respectively, with Cghxt4 contributing most to glucose consumption. Next, subcellular localisation analysis revealed that a common mutation of two ubiquitination sites (K9 and K538) in Cghxt4 improved its MLS. Finally, we overexpressed this Cghxt4 mutant (Cghxt4.2A) using a strong promoter, PCgGAP , which resulted in a significant increase in the ethanol productivity of C. glycerinogenes in the NDSBH medium. Specifically, the recombinant strain showed 18 and 25% higher ethanol productivity than the control in two kinds of YP-NDSBH medium (YP-NDSBH1G160 and YP-NDSBH2G160 ), respectively. The hexose transporter mutant Cghxt4.2A (Cghxt4K9A,K538A ) with multiple copies and high MLS was able to significantly increase the ethanol productivity of C. glycerinogenes in NDSBH. Our results provide a promising strategy for constructing efficient strains for ethanol production.