Cloning and Characterization of a Pyruvate Carboxy

Authors: GeYi Fu Yi Lu Zhe Chi GuangLei Liu ShouFeng Zhao Hong Jiang ZhenMing Chi

Publish Date: 2015/10/15

Volume: 18, Issue: 1, Pages: 1-14

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Abstract

In this study a pyruvate carboxylase gene PYC1 from a marine fungus Penicillium rubens I607 was cloned and characterized ORF of the gene accession number KM3973491 had 3534 bp encoding 1177 amino acids with a molecular weight of 127531 kDa and a PI of 620 The promoter of the gene was located at −1200 bp and contained a TATAA box several CAAT boxes and a sequence 5′SYGGRG3′ The PYC1 deduced from the gene had no signal peptide was a homotetramer α4 and had the four functional domains After expression of the PYC1 gene from the marine fungus in the marinederived yeast Yarrowia lipolytica SWJ1b the transformant PR32 obtained had much higher specific pyruvate carboxylase activity 053 U/mg than Y lipolytica SWJ1b 007 U/mg and the PYC1 gene expression 1338  and citric acid production 702 g/l by the transformant PR32 were also greatly enhanced compared to those 100  and 273 g/l by Y lipolytica SWJ1b When glucose concentration in the medium was 600 g/l citric acid CA concentration formed by the transformant PR32 was 361 g/l leading to conversion of 621  of glucose into CA During a 10l fedbatch fermentation the final concentration of CA was 1111 ± 13 g/l the yield was 093 g/g the productivity was 046 g/l/h and only 172 g/l reducing sugar was left in the fermented medium within 240 h HPLC analysis showed that most of the fermentation products were CA However minor malic acid and other unknown products also existed in the culture

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