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KMID : 0545119970070040229
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Journal of Microbiology and Biotechnology
1997 Volume.7 No. 4 p.229 ~ p.236
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Cloning, Nucleotide Sequence and Expression of Gene Coding for Poly-3-hydroxybutyric Acid(PHB) Synthase of Rhodobacter sphaeroides 2.4.1
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Kim Ji-Hoe
Lee Jeong-Kug
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Abstract
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A gene, phbC_2.4.1 encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring phbA,B_AC in pRK415, which code for β-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of phbC_2.4.1 in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [H^+]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of phbC_2.4.1 with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SalI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring phbA,B_AC. The promoter (s) of the phbC_2.4.1 were localized within a 340-bp DNA region upstream of the phbC_2.4.1 start codon according to heterologous expression analysis.
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