ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - MANIPULATION AND ANALYSIS OF AN AROMATIC POLYKETIDE SYNTHASE WITH UNPROVED CATALYTIC SPECIFICITIES

被引:82
作者
MCDANIEL, R
EBERTKHOSLA, S
HOPWOOD, DA
KHOSLA, C
机构
[1] STANFORD UNIV, DEPT CHEM ENGN, STANFORD, CA 94305 USA
[2] JOHN INNES INST, JOHN INNES CTR, DEPT GENET, NORWICH NR4 7UH, NORFOLK, ENGLAND
关键词
D O I
10.1021/ja00078a002
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Genes for a putative aromatic polyketide synthase (PKS) of unproven catalytic specificity from Streptomyces roseofulvus, which produces both frenolicin B (1) and nanaomycin A (2), were functionally expressed in Streptomyces coelicolor A3(2) using a heterologous expression system in combination with components of the actinorhodin (3) PKS gene cluster. Spectroscopic characterization and isotopic labeling experiments showed that the primary product of the recombinant strain was a novel tricyclic molecule (6), derived from a nonaketide backbone that undergoes a single ketoreduction. In addition, two octaketides (4 and 7) were produced by this PKS, suggesting that the chain length determining factor (CLF) of this PKS has a relaxed specificity and providing strong support for the supposition that the cloned genes encoded the PKS for frenolicin and nanaomycin biosynthesis. The regiospecificity of ketoreduction of the putative backbone of one of the octaketides (7) argues in favor of a biosynthetic model in which ketoreduction occurs only after the complete polyketide chain has been synthesized. Construction and analysis of hybrid PKSs containing different subunits from the actinorhodin and the above PKS confirmed earlier conclusions regarding the protein determinants of chain length, ketoreduction, and cyclization specificities.
引用
收藏
页码:11671 / 11675
页数:5
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