Cloning and expression in Saccharomyces cerevisiae of delta5-fatty acid desaturase gene from Phaeodactylum tricornutum.
- Author:
Zhe YANG
1
;
Dongsheng WEI
;
Laijun XING
;
Mingchun LI
Author Information
1. Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, Nankai University, Tianjin 300071, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Diatoms;
enzymology;
genetics;
Fatty Acid Desaturases;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
Saccharomyces cerevisiae;
genetics;
metabolism
- From:
Chinese Journal of Biotechnology
2009;25(2):195-199
- CountryChina
- Language:Chinese
-
Abstract:
Delta5-fatty acid desaturase is the key enzyme in synthesis of arachidonic acid. Two specific fragment was cloned from genomic DNA and total cDNA of Phaeodactylum tricornutum through PCR with primer designed according to the reported sequences, respectively 1520 bp and 1410 bp. Comparison of the genomic and cDNA sequences revealed that the delta5-fatty Acid Desaturase gene from genomic DNA had an 110 bp intron. The 1.4 kb was subcloned into the yeast-E. coli shuttle vector pYES2.0, then an expression recombinant plasmid pYPTD5 containerizing target gene was constructed. The plasmid pYPTD5 was transformed into defective mutant INCSc 1 of Saccharomyces cerevisiae for expression by electrotransformation method. Dihomo-gamma-linolenic acid was provided as an exogenous substrate to the yeast cultures, with galactose as inducer. By GC detecting, the recombinant S. cerecisiae had arachidonic acid. The results indicated that high level expression of delta5-fatty acid desaturase, and the substrate conversion reached 45.9%.
