Gene Editing of Candida glycerinogenes by Designed Toxin-Antitoxin Cassette

Candida glycerinogenes is an industrial yeast with excellent multistress resistance. However, due to the diploid genome and the lack of meiosis and screening markers, its molecular genetic operation is limited. Here, a gene editing system using the toxin-antitoxin pair relBE from the type II toxin-antitoxin system in Escherichia coli as a screening marker was constructed. The RelBE complex can specifically and effectively regulate cell growth and arrest through a conditionally controlled toxin RelE switch, thereby achieving the selection of positive recombinants. The constructed editing system achieved precise gene deletion, replacement, insertion, and gene episomal expression in C. glycerinogenes. Compared with the traditional amino acid deficiency complementation editing system, this editing system produced higher biomass and the gene deletion efficiency was increased by 3.5 times. Using this system, the production of 2-phenylethanol by C. glycerinogenes was increased by 11.5-13.5% through metabolic engineering and tolerance engineering strategies. These results suggest that the stable gene editing system based on toxin-antitoxin pairs can be used for gene editing of C. glycerinogenes to modify metabolic pathways and promote industrial applications. Therefore, the constructed gene editing system is expected to provide a promising strategy for polyploid industrial microorganisms lacking gene manipulation methods.