Establishment of a transient CRISPR-Cas9 genome editing system in Candida glycerinogenes for co-production of ethanol and xylonic acid

Abstract

Candida glycerinogenes, an industrial yeast with excellent multi-stress tolerance, has been applied to glycerol production for decades. However, its genetic manipulation was limited by the absence of meiosis, the diploid genome, and the lack of molecular tools. We described here the implementation of a transient CRISPR-Cas9 system for efficient genome editing in C.glycerinogenes. By targeting the counterselectable marker genes (TRP1, URA3), single and double gene knock-outs were achieved and the auxotroph obtained can be used as a background for targeting other gene (HOG1) at a mutation efficiency of 80%. Further, a xylonic acid producing C.glycerinogenes strain was constructed by knock-in of the xylose dehydrogenase gene, which produced up to 28g/L ethanol and 9g/L xylonic acid simultaneously from simulated lignocellulosic hydrolysate (contained 70g/L glucose and 24g/L xylose). These results indicated that the CRSIPR-Cas9 system developed here can facilitate the study of gene functions and metabolic pathways in C.glycerinogenes.