title: Cloning and transcription analysis of the Candida rugosa propionyl-CoA dehydrogenase gene and its expression in Pichia pastoris.
abstract: The propionyl-CoA dehydrogenase (PACD) gene was cloned from Candida rugosa by the cDNA RACE technique. The full cDNA of the PACD gene has a length of 1408 bp, which contains a complete open reading frame (ORF) of 1329 bp, coding for 442 amino acids. The cDNA of PACD was cloned into the expression plasmid pPIC9K and transformed into Pichia pastoris GS115. The recombinant protein was purified by Ni-NTA affinity chromatography, and its size was observed to be approximately 49 kDa as estimated by SDS-PAGE. Anti-His antibodies were used to characterise the recombinant PACD by western-blot analysis. The recombinant protein retained the activity of catalysing propionyl-CoA to acryloyl-CoA. The results of dot-blotting hybridisation using a PACD cDNA probe indicated that the PACD mRNA level was modified at different stages: mRNA levels were low for the first 36 h, then increased through 48 h and eventually reached a stable level. These results indicate that propionate induction could significantly activate PACD mRNA expression. Information from this study will be helpful in elucidating the metabolic pathway for 3-hydroxypropionic acid production in C. rugosa.
authors.affiliation: Key Laboratory of Biofuel, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101, China. zhoufl@qibebt.ac.cn
authors.forename: Feng-Li
authors.initials: FL
authors.lastname: Zhou
authors.forename: Ru-Bing
authors.initials: RB
authors.lastname: Zhang
authors.forename: Yong-Guang
authors.initials: YG
authors.lastname: Zhang
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Jian-Hang
authors.initials: JH
authors.lastname: Zhu
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002175:Candida / Q000201:enzymology* / Q000235:genetics*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D000214:Acyl Coenzyme A; D011994:Recombinant Proteins; C009061:propionyl-coenzyme A; C047011:acryloyl-coenzyme A; D000429:Alcohol Oxidoreductases