The ratio of the major monensin analogs produced by Streptomyces cinnamonensis is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The meaA gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B12-dependent mutases. Plasmid-based expression of meaA from the ermE* promoter in the S. cinnamonensis C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a meaA mutant, S. cinnamonensis WM2 (generated from the C730.1 strain by insertional inactivation of meaA by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an S. cinnamonensis WD2 strain (an icm meaA mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the meaA gene or the Amycolatopsis mediterranei mutAB genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the meaA gene product is significantly involved in methylmalonyl-CoA production in S. cinnamonensis and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.

译文

肉桂链霉菌产生的主要莫能菌素类似物的比例取决于生物合成前体甲基丙二酰辅酶a (CoA) (莫能菌素A和莫能菌素B) 和乙基丙二酰辅酶a (莫能菌素A) 的相对水平。克隆并测序了该生物的meaA基因,并显示出编码与甲基丙二酰辅酶a变位酶 (MCM) (40%) 和异丁酰辅酶a变位酶 (ICM) 大亚基 (36%) 和小亚基 (52%) 具有显着氨基酸序列同一性的推定74 kDa蛋白有机体。MeaA的预测C末端包含在所有辅酶B12-dependent突变酶中高度保守的结构特征。在肉桂S. cinnamonensis C730.1菌株中,来自ermme * 启动子的meaA基于质粒的表达导致莫能菌素a与莫能菌素B的比例从1:1降低到1:3。相反,在meaA突变体,S. cinnamonensis WM2 (通过使用红霉素抗性基因对meaA进行插入失活而由C730.1菌株产生) 中,该比率增加至4:1。在这两个实验中,总体莫能菌素滴度没有受到显着影响。然而,随着90% 的推移,在肉桂S. cinnamonensis WD2菌株 (一种icmmeaa突变体) 中,莫能菌素滴度确实降低。通过meaA基因或Amycolatopsis mediterranei mutAB基因 (编码MCM) 的基于质粒的表达,将WD2菌株中的莫能菌滴度至少恢复到野生型水平。相反,在0.8 M缬氨酸存在下WD2菌株的生长仅导致莫能菌素滴度的部分恢复 (<25%)。这些结果表明,meaA基因产物显着参与了肉桂草中甲基丙二酰辅酶a的产生,并且在测试条件下,MeaA和ICM的存在对于WD2菌株中的莫能菌素的产生至关重要。这些结果还表明,缬氨酸降解与为许多聚酮化合物生物合成过程提供甲基丙二酰辅酶a前体有关,对WD2突变体中的莫能菌素生物合成没有很大程度的影响。

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