Functional analysis of type II thioesterase of Streptomyces lydicus AS 4.2501 |
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Authors: | Feng-Ming Yu Bin Qiao Feng Zhu Jin-Chuan Wu Ying-Jin Yuan |
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Institution: | (1) Department of Pharmaceutical Engineering School of Chemical Engineering and Technology, Tianjin University, PO Box 6888, 300072 Tianjin, P.R. China |
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Abstract: | Constructing a mutant strain of single gene disruption is the basis for the study of gene function and metabolomics. Systematic
and complete genome sequencing is the basis of genetic manipulation. In the case of a little knowledge about the Streptomyces lydicus genome and the speculation that polyketide synthases (type I) might be responsible for the polyketide side chain biosynthesis
of streptolydigin, a 588-bp fragment was amplified by polymerase chain reaction (PCR) according to the homology existing in
the same functional genes among Streptomyces. A mutant strain of this gene was constructed by single crossover homologous recombination. The results of sequence analysis
as well as the metabolite analysis of the mutant and the original strain by liquid chromatography/mass spectroscopy indicated
that this fragment was part of type II thioesterase (TE) gene, which was required for streptolydigin biosynthesis like other
type II TEs function in related antibiotics biosynthesis. Furthermore, targeted gene manipulation based on PCR was a powerful
tool for studying gene function and metabolomics, especially when little was known about the genomic sequence of streptomyces. |
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Keywords: | Type II thioesterase polymerase chain reaction mutant strain construction functional analysis single crossover homologous recombination Streptomyces lydicus AS 4 2501 |
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