ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - INFLUENCE OF A DOWNSTREAM ENZYME ON THE CATALYTIC SPECIFICITY OF A MINIMAL AROMATIC POLYKETIDE SYNTHASE
ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - INFLUENCE OF A DOWNSTREAM ENZYME ON THE CATALYTIC SPECIFICITY OF A MINIMAL AROMATIC POLYKETIDE SYNTHASE
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DOI：
10.1073/pnas.91.24.11542
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发表时间：
1994-11-22
影响因子：
11.1
通讯作者：
KHOSLA, C
中科院分区：
文献类型：
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作者：
MCDANIEL, R;EBERTKHOSLA, S;KHOSLA, C
为了确定芳香族聚酮在体内生物合成所需的聚酮合成酶（PKS）组分的最小集合，在最近开发的链霉菌宿主载体系统中表达了编码三种不同的芳香族PKS亚基的基因组合——act来自链霉菌coelicolor A3（一种放线菌素产生物），fren来自链霉菌reseofulvus（一种异丙霉素和纳霉素产生物），tcm来自链霉菌glaucescens（一种四环素产生物）。“最小”组分（酮合酶/假定的酰基转移酶、链长决定因子和酰基载体蛋白）在有和没有功能的聚酮酮还原酶和/或环化酶的情况下产生，并对这些重组菌株的聚酮产物进行了结构表征。除了本文描述的两种先前未鉴定的聚酮，脱氢突变素和SEK 15b，还分离出了几种先前鉴定的聚酮。结果证明，行为环化酶在生物合成中不需要一些先前报道的异常环化产物。他们也与早期的结论一致，即最小PKS控制链长度以及第一次环化的区域特异性，并且它可以在没有酮还原酶和环化酶的情况下做到这一点。然而，最小的tcm PKS合成两种不同的单环化中间体的能力表明，它无法准确地控制该反应的过程。在下游酶存在的情况下，通过环化途径的一个分支的通量相对于另一个分支增加。我们提出，这些选择性特异性可能是由于下游酶与最小PKS结合的能力，并选择性地抑制环化途径的特定分支。
To identify the minimum set of polyketide synthase (PKS) components required for in vivo biosynthesis of aromatic polyketides, combinations of genes encoding subunits of three different aromatic PKSs-act from Streptomyces coelicolor A3(2) (an actinorhodin producer), fren from Streptomyces reseofulvus (a frenolicin and nanaomycin producer), and tcm from Streptomyces glaucescens (a tetracenomycin producer)-were expressed in a recently developed Streptomyces host-vector system. The ''minimal'' components (ketosynthase/putative acyltransferase, chain length-determining factor, and acyl carrier protein) were produced with and without a functional polyketide ketoreductase and/or cyclase, and the polyketide products of these recombinant strains were structurally characterized. Several previously identified polyketides were isolated in addition to two previously unidentified polyketides, dehydromutacin and SEK 15b, described here. The results proved that the act cyclase is not required for the biosynthesis of several aberrantly cyclized products that have been previously reported. They are also consistent with earlier conclusions that the minimal PKS controls chain length as well as the regiospecificity of the first cyclization and that it can do so in the absence of both a ketoreductase and a cyclase. However, the ability of the minimal tcm PKS to synthesize two different singly cyclized intermediates suggests that it is unable to accurately control the course of this reaction by itself. In the presence of a downstream enzyme, the flux through one branch of the cyclization pathway increases relative to the other. We Propose that these alternative specificities may be due to the ability of downstream enzymes to associate with the minimal PKS and to selectively inhibit a particular branch of the cyclization pathway.