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首页> 外文期刊>Biochemistry >Metabolic Engineering of a Methymalonyl-CoA Mutase-Epimerase Pathway for Complex Polyketde Biosynthesis in Escherichia coli
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Metabolic Engineering of a Methymalonyl-CoA Mutase-Epimerase Pathway for Complex Polyketde Biosynthesis in Escherichia coli

机译：甲基丙二酰辅酶A突变酶-表异构酶途径的代谢工程，用于大肠杆菌中复杂聚酮的生物合成

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A barrier to heerologous of complex polyketides in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a comon extender substrate for the biosynthesis of complex polyketides by modular polyketide synthases. One biosynthetic route to (2S)-methylmalonyl-CoA involves the sequential actions of two enzymes, methylmalonyl-CoA mutase andmethylmalonyl-CoA epimerase, which convert succinyl-CoA to (2R)-and then to (2S)-methylmalonyl-CoA. As reported [McKie, N., et al. (1990) Biochem. J. 269,293-298; Haller, T., et al. (2000) Biochemistry 39, 4622-4629], when inactive ncoding coenzyme B_(12)-dependent methlmalonyl-CoA mutases were expressed in E. coli, the inactive apo-enzyme was produced. However, when cells harboring the mutase genes from Propionibacterium shermanii or E. coli were treated with the B(12) precursor hydroxocobalamin, active holo-enyzme was isolated, and (2R)-methylmalonyl-CoA represented approx10% of the intracelualr CoA pool. When the E. coli BAP1 cell line [Pfeifer, B.A., et al. (2001) Science 291,1790-1792] harboring plasmids that expressed P. shermnii methymalonyl-Coa mutase, Streptomyces coelicolor methylmaloyl-CoA epimerase, and the polyketide synthase DEBS (6-deoxyerythronolide B synthease) was fed propionate and hydroxocobalamin, the polyketide 6-deoxyerthronolide B (6-dEB) was produced. Isotopic labeling studies using [~(13)C]propionate showed that the starter unit for polyketide synthesis was derived exclusively from exogenous propionate, while the extender units stemmed from methymalonyl-CoA via the mutase-epimerase pathway. Thus, the introduction of an engineered mutase-epimerase pathway in E. coli enabled the uncoupling of carbon sources used to produce starter and extender units of polyketides.
机译：大肠杆菌中复杂聚酮化合物异源性的一个障碍是缺少（2S）-甲基丙二酰-CoA，后者是通过模块化聚酮化合物合酶生物合成复杂聚酮化合物的常用补充底物。 （2S）-甲基丙二酰-CoA的一种生物合成途径涉及两种酶的顺序作用，即甲基丙二酰-CoA突变酶和甲基丙二酰-CoA表异构酶，它们将琥珀酰-CoA转化为（2R）-，然后再转化为（2S）-甲基丙二酰-CoA。如报道的[McKie，N。，等人。 （1990）生物化学。 J.269,293-298; Haller，T。等。 （2000）Biochemistry 39，4622-4629]，当在大肠杆菌中表达失活的编码辅酶B_（12）-依赖的甲基丙二酰辅酶A突变酶时，产生了失活的脱辅基酶。但是，当用B（12）前体羟考巴兰素处理保留了来自谢尔曼丙酸杆菌或大肠杆菌的突变酶基因的细胞时，活性全酶被分离出来，并且（2R）-甲基丙二酰-CoA代表了细胞内CoA池的约10％。当大肠杆菌BAP1细胞系[Pfeifer，B.A.，et al。 （2001）Science 291,1790-1792]携带表达丙酸疟原虫甲基丙二酰-Coa突变酶，链霉菌coelicolor甲基丙二酰-CoA差向异构酶和聚酮化合物合酶DEBS（6-脱氧赤藓醇B合成酶）的质粒进料丙酸酯和羟考巴宁，聚酮化合物6。产生了-脱氧乙氧乙内酯B（6-dEB）。使用[〜（13）C]丙酸酯的同位素标记研究表明，聚酮化合物合成的起始单元仅衍生自外源丙酸酯，而增量剂单元则来自甲基丙二酰辅酶A，通过突变酶-表观异构酶途径。因此，在大肠杆菌中引入工程化的突变酶-表异构酶途径使得能够解开用于生产聚酮化合物的起始和延伸单元的碳源。

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