ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - DISSECTION OF THE CATALYTIC SPECIFICITY OF THE ACT KETOREDUCTASE
ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - DISSECTION OF THE CATALYTIC SPECIFICITY OF THE ACT KETOREDUCTASE
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DOI：
10.1021/ja00089a003
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发表时间：
1994-05-18
影响因子：
15
通讯作者：
KHOSLA, C
中科院分区：
文献类型：
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作者：
FU, H;EBERTKHOSLA, S;KHOSLA, C
天蓝色链霉菌A3(2)和灰链霉菌四环素(2)的放线菌素(1)的聚酮合成酶(PKS)亚单位的基因组合(分别为ACI和TCM)在最近开发的链霉菌宿主载体系统中进行了功能表达。缺乏与PKS相关的任何酮还原酶活性的重组菌株产生了新的多酮，利用核磁共振和高分辨质谱学结合同位素标记实验对其结构进行了表征。正如预期的那样，ACT和TCM PKS产生了完全未还原的聚酮，其骨架分别来自8个和10个醋酸酯单元(9和10)。然而，这两个分子中环化的区域特异性不同于它们的还原对应分子，并且以前没有在这类细菌聚酮中观察到。结合早期的发现，我们的结果为通过ACTE酮还原酶与不同链长特性的PKS(包括那些与自然界中任何酮还原酶无关的PKS)的组合表达新型多酮的生物合成提供了另一种自由度的证据。通过比较本文报道的新型聚酮与先前报道的新型聚酮的结构和推导的骨架，支持一种生物合成模型，其中第一次环化的区域特异性部分由链延长酶本身控制，而不受酮还原酶的影响；然而，负责控制第二次环化的区域特异性的环酶可以区分不同长度和不同还原程度的聚酮骨架。
Combinations of genes (aci and tcm, respectively) encoding subunits of polyketide synthases (PKSs) for actinorhodin (1) from Streptomyces coelicolor A3(2) and tetracenomycin (2) from Streptomyces glaucescens were functionally expressed in a recently developed Streptomyces host-vector system. Recombinant strains lacking any ketoreductase activity associated with the PKS produced novel polyketides, which were structurally characterized using NMR and high-resolution mass spectroscopy in combination with isotopic labeling experiments. As expected, the act and tcm PKSs produced completely unreduced polyketides with backbones derived from 8 and 10 acetate units, respectively (9 and 10). However, the regiospecificities of cyclizations in these two molecules differed from their reduced counterparts, and have not been previously observed in this class of bacterial polyketides. Taken together with earlier findings, our results provide evidence for yet another degree of freedom for the biosynthesis of novel polyketides through combinatorial expression of the act ketoreductase with PKSs of varying chain length specificities (including those that are not associated with any ketoreductase in nature). By comparing the structures and deduced backbones of the novel polyketides reported here with those of novel polyketides described earlier, a biosynthetic model is favored in which the regiospecificity of the first cyclization is controlled in part by the chain elongation enzymes themselves and is not influenced by the ketoreductase; however, the cyclase responsible for controlling the regiospecificity of the second cyclization can discriminate between polyketide backbones of different lengths as well as different degrees of reduction.