Characterization of Phosphoglycerate Kinase Promoter from Candida tropicalis

Abstract:

Candida tropicalis is an important industrial strain and can overproduce long chain dicarboxylic acid. However，genetic manipulations were still difficult because of lack of effective expression elements such as promoter and plasmid. Therefore，screening novel promoter is of great significance for metabolic engineering of C. tropicalis. In this study，phosphoglycerate kinase gene (PGK1) promoter was cloned from C. tropicalis ATCC 20336 and its function was investigated. The 846 bp PGK1 promoter was fused into a yeGFP3 reporter gene and transformed into C. tropicalis XZX host. The results showed that promoter could effectively regulate yeGFP3 expression. Then，a series of truncated promoters (including 300，550，750 bp) were cloned and used for expression of yeGFP3 reporter gene in C. tropicalis. Transformants harboring various integrative yeGFP3 reporter gene cassettes were obtained and confirmed. The relative fluorescence intensities of 300，550，750 bp promoters were 4.98%，9.84% and 48.4%，respectively，when using 846 bp PGK1 promoter as a control. Furthermore，the transcriptional levels of yeGFP3 in different transformants were detected by RT-qPCR and the results agreed to the fluorescence intensity evaluations. The results indicated that two upstream activation sequences presumably existed in the PGK1 promoter. In summary，PGK1 promoter would provide a fundamental expression element for engineering C. tropicalis in future.