McDaniel R, Ebert-Khosla S, Fu H, Hopwood D A, Khosla C
Department of Chemical Engineering, Stanford University, CA 94305-5025.
Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11542-6. doi: 10.1073/pnas.91.24.11542.
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 roseofulvus (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, dehydromutactin 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.
为了确定芳香族聚酮化合物体内生物合成所需的最小聚酮合酶（PKS）组件集，编码三种不同芳香族PKS亚基的基因组合——来自天蓝色链霉菌A3(2)（放线紫红素生产者）的act、来自玫瑰黄链霉菌（弗氏菌素和纳那霉素生产者）的fren以及来自浅灰链霉菌（四环素霉素生产者）的tcm——在最近开发的链霉菌宿主-载体系统中表达。在有和没有功能性聚酮化合物酮还原酶和/或环化酶的情况下产生“最小”组件（酮合酶/推定的酰基转移酶、链长决定因子和酰基载体蛋白），并对这些重组菌株的聚酮化合物产物进行结构表征。除了本文描述的两种先前未鉴定的聚酮化合物脱氢变肌动蛋白和SEK 15b外，还分离出了几种先前鉴定的聚酮化合物。结果证明，先前报道的几种异常环化产物的生物合成不需要act环化酶。它们也与早期结论一致，即最小PKS控制链长以及第一次环化的区域特异性，并且在没有酮还原酶和环化酶的情况下也能做到这一点。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，它自身无法准确控制该反应的进程。在存在下游酶的情况下，通过环化途径一个分支的通量相对于另一个分支增加。我们提出，这些替代特异性可能是由于下游酶与最小PKS结合并选择性抑制环化途径特定分支的能力。
