[Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].
Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.
Lu
Jie
J
Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China. nic3602@scu.edu.cn
Yao
Yufeng
Y
Jiang
Weihong
W
Jiao
Ruishen
R
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000235:genetics*; D000192:Actinomycetales / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D054889:Fatty Acid Synthase, Type II; D008969:Molecular Sequence Data; D014158:Transcription, Genetic
D002352:Carrier Proteins; D001710:Biotin; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein