Replication data for : Raw HPLC-MS data of S. pristinaespiralis wild type, the S. pristinaespiralis pathway inactivation mutant and S. virginiae and SAXS data of VirD, VirE, holoACP5b-VirC, holoACP5b-VirD, holoACP5b-VirE.

During biosynthesis by multi-modular trans-AT polyketide synthases (PKSs), polyketide structural space can be expanded by conversion of initially-formed electrophilic beta-ketones into beta-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase (HMGS) cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT PKS. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for beta-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation, identifies a metabolite bearing a second beta-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin beta-branching programming. Furthermore, imperfections in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives.

在多模块反酰基转移酶聚酮合酶（trans-AT polyketide synthases, PKSs）催化的生物合成过程中，初始生成的亲电β-酮基可通过转化为β-烷基来拓展聚酮的结构空间。此类多步转化反应由3-羟基-3-甲基戊二酰合酶（3-hydroxy-3-methylgluratryl synthase, HMGS）酶盒催化完成。尽管这类反应的机制细节已得到阐明，但目前关于此类酶盒如何靶向选择特定聚酮中间体的相关信息仍较为匮乏。本研究借助整合结构生物学（integrative structural biology）技术，解析了维吉尼亚霉素M反酰基转移酶聚酮合酶（virginiamycin M trans-AT PKS）的模块5选择底物的分子基础。此外，体外实验证实，至少模块7可作为β-甲基化的潜在额外作用位点。通过高效液相色谱-质谱联用（high performance liquid chromatography-mass spectrometry, HPLC-MS）结合同位素标记与通路失活分析，我们确实鉴定到了一种在预期位置带有第二个β-甲基的代谢产物。综上，本研究结果表明，多种协同作用的调控机制共同支撑了β支化编程过程。进一步而言，该调控过程中存在的疏漏——无论是天然存在还是人为引入——均为聚酮结构的多样化改造提供了可行途径，以制备高价值衍生物。