Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Cloning and Overexpression of Phytoene Desaturase (CrtI) from Paracoccus haeundaensis

  • Choi, Seong-Seok (Department of Microbiology, College of Natural Sciences, Pukyong National University) ;
  • Seo, Yong Bae (Department of Microbiology, College of Natural Sciences, Pukyong National University) ;
  • Lim, Han Kyu (Department of Marine and Fisheries Resources, College of Natural Sciences, Mokpo National University) ;
  • Nam, Soo-Wan (Division of Applied Bioengineering, Biomedical Engineering and Biotechnology Major, College of Engineering, Dong-Eui University) ;
  • Kim, Gun-Do (Department of Microbiology, College of Natural Sciences, Pukyong National University)
  • Received : 2018.02.21
  • Accepted : 2018.05.17
  • Published : 2018.06.28
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Abstract

Among the carotenoid biosynthesis genes, crtI gene encodes the phytoene desaturase (CrtI) enzyme, and phytoene desaturase convert phytoene to lycopene. Phytoene desaturase is involved in the dehydrogenation reaction, in which four single bonds in the phytoene are introduced into a double bond, eliminating eight hydrogen atoms in the process. Phytoene desaturase is one of the key regulating enzyme in carotenoid biosynthetic pathway of various carotenoid biosynthetic organisms. The crtI gene in genomic DNA of Paracoccus haeundaensis was amplified and cloned into a T-vector to analyze the nucleotide sequence. As a result, the crtI gene coding for phytoene desaturase from P. haeundaensis consists of 1,503 base pairs encoding 501 amino acids residues. An expression plasmid containing the crtI gene was constructed, and Escherichia coli cells containing this plasmid produced the recombinant protein of approximately 55 kDa, equivalent to the molecular weight of phytoene desaturase. The expressed protein in cell lysate showed enzymatic activity similar to phytoene desaturase. Phytoene and lycopene were analyzed by HPLC and measured at wavelength of 280 nm and 470 nm, respectively. The $K_m$ values for phytoene and NADPH were $11.1{\mu}M$ and $129.3{\mu}M$, respectively.

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References

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