Functional analysis of type II thioesterase of Streptomyces lydicus AS 4.2501.
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.
Yu
Feng-Ming
FM
Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, PO Box 6888, Tianjin University, Tianjin 300072, P.R. China.
Qiao
Bin
B
Zhu
Feng
F
Wu
Jin-Chuan
JC
Yuan
Ying-Jin
YJ
D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000096:biosynthesis; D001483:Base Sequence; D003001:Cloning, Molecular; D064429:Fatty Acid Synthases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism*
D000617:Aminoglycosides; C100307:streptolydigin; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II