Cloning and Characterization of a Pyruvate Carboxylase Gene from Penicillium rubens and Overexpression of the Genein the Yeast Yarrowia lipolytica for Enhanced Citric Acid Production

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• 作者：Ge-Yi Fu ; Yi Lu ; Zhe Chi ; Guang-Lei Liu ; Shou-Feng Zhao ; Hong Jiang…
• 关键词：Pyruvate carboxylase ; Citric acid ; Yarrowia lipolytica ; Gene expression ; Fed fermentation ; Marine fungi
• 刊名：Marine Biotechnology
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：18
• 期：1
• 页码：1-14
• 全文大小：2,216 KB
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• 作者单位：Ge-Yi Fu (1)
  Yi Lu (1)
  Zhe Chi (1)
  Guang-Lei Liu (1)
  Shou-Feng Zhao (1)
  Hong Jiang (1)
  Zhen-Ming Chi (1)
  
  1. College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China
  
• 刊物类别：Earth and Environmental Science
• 刊物主题：Earth sciences
  Oceanography
  
• 出版者：Springer New York
• ISSN：1436-2236

文摘

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: KM397349.1) had 3534 bp encoding 1177 amino acids with a molecular weight of 127.531 kDa and a PI of 6.20. The promoter of the gene was located at −1200 bp and contained a TATAA box, several CAAT boxes and a sequence 5′-SYGGRG-3′. 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 marine-derived yeast Yarrowia lipolytica SWJ-1b, the transformant PR32 obtained had much higher specific pyruvate carboxylase activity (0.53 U/mg) than Y. lipolytica SWJ-1b (0.07 U/mg), and the PYC1 gene expression (133.8 %) and citric acid production (70.2 g/l) by the transformant PR32 were also greatly enhanced compared to those (100 % and 27.3 g/l) by Y. lipolytica SWJ-1b. When glucose concentration in the medium was 60.0 g/l, citric acid (CA) concentration formed by the transformant PR32 was 36.1 g/l, leading to conversion of 62.1 % of glucose into CA. During a 10-l fed-batch fermentation, the final concentration of CA was 111.1 ± 1.3 g/l, the yield was 0.93 g/g, the productivity was 0.46 g/l/h, and only 1.72 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. Keywords Pyruvate carboxylase Citric acid Yarrowia lipolytica Gene expression Fed fermentation Marine fungi