New molecular genetic tools for the non-conventional yeasts Candida sonorensis and Candida methanosorbosa

New molecular genetic tools were developed for Candida sonorensis and Candida methanosorbosa enabling their genetic engineering for the first time. Transformation was based on antibiotic markers and, alternatively, a dominant non-antibiotic marker, the MEL5 gene from Saccharomyces cerevisiae, enabling growth on melibiose as sole carbon source. Endogenous promoters were cloned from C. sonorensis and used to control the expression of the heterologous genes in both hosts. Introduction of the lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus into C. sonorensis and C. methanosorbosa resulted in moderate (~10 g/l, ~20% yield) production of lactic acid with co-production of ethanol. Targeted gene deletions were accomplished in C. sonorensis. Two genes encoding pyruvate decarboxylase (PDC) were isolated. Strains deleted of PDC1, PDC2, or both PDC genes were constructed generating C. sonorensis strains producing reduced amounts or no ethanol. The PDC2 encoded enzyme was identified as the major activity contributing to ethanol production. Integration of the PDC1 and PDC2 replacement constructs into both homologous and non-homologous sites occurred in C. sonorensis. This work clearly demonstrates the feasibility of genetically engineering novel, non-conventional yeast species, and illustrates the usefulness of the molecular tools for analysing gene function.