Construction of Saccharomyces cerevisiae haploid mutant deficient in lanosterol synthase gene.
- Author:
Li-Li GAO
;
Qing-Hua WANG
;
Hui-Chao LIANG
;
Ting GONG
;
Jin-Ling YANG
;
Ping ZHU
- Publication Type:Journal Article
- MeSH:
Chromatography, High Pressure Liquid;
DNA Primers;
Down-Regulation;
Ergosterol;
metabolism;
Haploidy;
Intramolecular Transferases;
genetics;
Polymerase Chain Reaction;
Saccharomyces cerevisiae;
genetics;
Squalene;
analogs & derivatives;
metabolism
- From:
Acta Pharmaceutica Sinica
2014;49(5):742-746
- CountryChina
- Language:Chinese
-
Abstract:
Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.
