聚酮
酰基转移酶
聚酮合酶
化学
蛋白质工程
生物合成
立体化学
酰基载体蛋白
酰基转移酶
基质(水族馆)
计算生物学
生物化学
酶
生物
生态学
作者
Elias Englund,Matthias Schmidt,Alberto A. Nava,Anna Lechner,Kai Deng,Renee Jocic,Yingxin Lin,Jacob B. Roberts,Veronica T. Benites,Ramu Kakumanu,Jennifer Gin,Yan Chen,Yuzhong Liu,Young-Mo Kim,Edward E. K. Baidoo,Trent Northen,Paul D. Adams,Leonard Katz,Satoshi Yuzawa,Jay D. Keasling
摘要
Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.
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