A polyketide-based biosynthetic platform for diols, amino alcohols and hydroxyacids

Modular polyketide synthases (PKSs) have been proposed as promising megasynthases for retrobiosynthesis because of their fitness for rational carbon skeleton design. However, over the last three decades, all engineered PKSs produce carboxylic acids via terminal thioesterase (TE), which significantly narrows available chemical scope. We proposed the possibility of expanding PKS chemical diversity via terminal thioreductase (TR) engineering, and comprehensively characterised PKS TRs as NADPH-dependent enzymes to terminate polyketides with aldehyde. These aldehyde products can be readily converted to industrially valuable alcohols and amines, demonstrated by an engineered rimocidin PKS-TR producing 1,3-butanediol, 1,3-pentanediol, and 1,3-hexanediol with a total titer of 1008 mg/L in Streptomyces albus, or producing 554 mg/L 1-amino-3-alcohols via post-PKS transamination, both in shake flasks. Furthermore, efficient control of the product profile was achieved by coenzyme A (CoA) substrate regulation, as elevating butyryl-CoA level resulted in 1,3-hexanediol product ratio increased from 11% to 77%. We also demonstrated that PKS-TR can produce biosynthetically challenging branched-chain diols, culminating in production of 166 mg/L branched-chain 2-methyl-1,3-diols via acyltransferase exchange.

模块化聚酮合酶（modular polyketide synthases, PKSs）因适配理性碳骨架设计的特性，被视为逆生物合成（retrobiosynthesis）领域极具潜力的巨型合酶。然而在过去三十年中，所有经工程化改造的聚酮合酶均通过末端硫酯酶（thioesterase, TE）催化生成羧酸，这极大压缩了其可合成的化学空间范围。本研究提出通过改造末端硫还原酶（thioreductase, TR）拓展聚酮合酶化学多样性的可行路径，并对聚酮合酶硫还原酶开展了系统性表征，证实其为以醛基终止聚酮链合成的NADPH依赖型酶。此类醛基产物可便捷转化为具备工业应用价值的醇类与胺类化合物：经工程化改造的里莫霉素（rimocidin）聚酮合酶-硫还原酶在白色链霉菌（Streptomyces albus）中可合成1,3-丁二醇、1,3-戊二醇与1,3-己二醇，总效价达1008 mg/L；或通过聚酮合酶后转氨作用合成554 mg/L的1-氨基-3-醇类化合物，上述实验均在摇瓶体系中完成。此外，本研究通过辅酶A（coenzyme A, CoA）底物调控实现了产物谱的精准调控：提升丁酰辅酶A（butyryl-CoA）水平可使1,3-己二醇的产物占比从11%提升至77%。本研究还证实，聚酮合酶-硫还原酶可合成生物合成难度较高的支链二醇；通过酰基转移酶（acyltransferase）交换策略，最终实现了166 mg/L的支链2-甲基-1,3-二醇合成。