Development and verification of an FLP/FRT system for gene deletion in Candida amazonensis

Abstract:

[Objective] To develop an FLP/FRT system for gene disruption in Candida amazonensis that can repeatedly use a single selectable marker, and to verify the effectiveness of this system by deleting the PDC gene encoding pyruvate decarboxylase.[Methods] Four promoters (SpXYLp, SpMAL6p, SpMAL1p and SpGAL1p) from Spathaspora passalidarum and ScGAL1p promoter from Saccharomyces cerevisiae were amplified and fused to the reporter gene of green fluorescent protein (gfpm) to study the regulation under corresponding inducible conditions. A strictly inducible promoter was selected to control the expression of the C. amazonensis-adapted FLP gene (caFLP), encoding the site-specific recombinase FLP. The promoter-caFLP fusion fragment was used to ligated with the hphm marker gene that conferred resistance to Hygromycin B, and the ligation product was flanked by direct repeats of the FLP recognition target (FRT). Then with the addition of the homologous arms, we constructed the PDC deletion cassette (PRFgHRP). The cassette was transformed into C. amazonensis CBS 12363 and transformants with hphm were derived. When the transformants were incubated into inducible medium, FLP-mediated recombination resulted in the deletion of DNA located between the repeats.[Results] SpMAL1p (induced by maltose) and SpGAL1p (induced by galactose) were identified to be strictly inducible promoters. SpGAL1p was used to regulate the expression of the FLP, and the PDC deletion cassette (PRFgHRP) was constructed and transformed into C. amazonensis successfully. After selection of Hyg-resistant transformant (designated as C. amazonensis PDC01) in which the deletion cassette was inserted into the PDC target gene, FLP expression was induced by growth of the transformant in galactose-containing medium, and Hyg-sensitive transformant in which hphm and caFLP flippers were excised from the genome was obtained, designated as C. amazonensis PDC02.[Conclusion] It is the first time to construct an FLP/FRT system for gene disruption in C. amazonensis, and we obtained a PDC mutant without resistant marker gene successfully through this system. These research results lay a good foundation for further metabolic engineering of C. amazonensis.