Premium
Cloning and characterization of a novel NAD + ‐dependent glyceraldehyde‐3‐phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter
Author(s) -
Zhang Cheng,
Zhuge Bin,
Zhan Xiaobei,
Fang Huiying,
Zong Hong,
Zhuge Jian
Publication year - 2013
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.2946
Subject(s) - biology , microbiology and biotechnology , open reading frame , nad+ kinase , gene , saccharomyces cerevisiae , dehydrogenase , biochemistry , peptide sequence , enzyme
Abstract A 3950 bp genomic fragment from Candida glycerinogenes , WL2002‐5, containing the Cg GAP gene encoding a glyceraldehyde‐3‐phosphate dehydrogenase homologous to GAP genes in other yeasts using degenerate primers, was cloned and characterized with inverse PCR. Sequence analysis revealed a 1164 bp open reading frame encoding a putative peptide of 387 deduced amino acids, with a molecular mass of 36 kDa. The Cg GAP protein consisted of an N‐terminal NAD + ‐binding domain and a central catalytic domain. Six stress‐response elements were found in the upstream region of the Cg GAP gene. The influence of Cg GAP on glycolysis was investigated. Functional analysis revealed that Saccharomyces cerevisiae transformed with Cg GAP was restored to the wild‐type phenotype when cultured in high‐osmolarity medium, suggesting that it is a functional GAP protein. Promoter studies in S . cerevisiae using the green fluorescent protein ( gfp ) gene as a reporter showed that the GAP promoter (P Cg GAP ) is constitutively expressed in S . cerevisiae cells grown on glucose. Copyright © 2013 John Wiley & Sons, Ltd.