Plant waxes are interesting substitutes of fossil-derived compounds; however, their limited sources and narrow structural diversity prompted the development of microbial platforms to produce esters with novel chemical structures and properties. One successful strategy was the heterologous expression of the mycocerosic polyketide synthase-based biosynthetic pathway (MAS-PKS, PapA5 and FadD28 enzymes) from Mycobacterium tuberculosis in Escherichia coli. This recombinant strain has the ability to produce a broad spectrum of multimethyl-branched long-chain esters (MBE) with novel chemical structures and high oxidation stability. However, one limitation of this microbial platform was the low yields obtained for MBE derived of short-chain alcohols. In an attempt to improve the titers of the short-chain alcohol-derived MBE, we focused on the PapA5 acyltransferase-enzyme that catalyzes the ester formation reaction. Specific amino acid residues located in the two-substrate recognition channels of this enzyme were identified, rationally mutated, and the corresponding mutants characterized both in vivo and in vitro. The phenylalanine located at 331 position in PapA5 (F331) was found to be a key residue that when substituted by other bulky and aromatic or bulky and polar amino acid residues (F331W, F331Y or F331H), gave rise to PapA5 mutants with improved bioconversion efficiency; showing in average, 2.5 higher yields of short-chain alcohol-derived MBE compared with the wild-type enzyme. Furthermore, two alternative pathways for synthetizing ethanol were engineered into the MBE producer microorganism, allowing de novo production of ethanol-derived MBE at levels comparable with those obtained by the external supply of this alcohol. KEY POINTS: • Mutation in channel 2 changes PapA5 acyltransferase bioconversion efficiency. • Improved production of short-chain alcohol derived multimethyl-branched esters. • Establishing ethanologenic pathways for de novo production of ethanol derived MBE. • Characterization of a novel phenylethanol-derived MBE.

中文翻译：

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修饰PapA5酰基转移酶底物选择性，以优化大肠杆菌中短链醇衍生的多甲基支链酯的生产。

植物蜡是化石衍生化合物的有趣替代品。然而，它们的来源有限和狭窄的结构多样性促使微生物平台的发展，以生产具有新颖化学结构和性质的酯。一种成功的策略是在结核分枝杆菌中以结核分枝杆菌聚酮合成酶为基础的生物合成途径（MAS-PKS，PapA5和FadD28酶）异源表达。此重组菌株具有产生具有新颖化学结构和高氧化稳定性的广谱多甲基支链长链酯（MBE）的能力。但是，这种微生物平台的局限性是由短链醇衍生的MBE所获得的收率低。为了提高源自短链醇的MBE的效价，我们专注于催化酯形成反应的PapA5酰基转移酶。鉴定，合理突变位于该酶的两个底物识别通道中的特定氨基酸残基，并在体内和体外表征相应的突变体。发现位于PapA5（F331）中331位的苯丙氨酸是一个关键残基，当被其他大块的芳香族或大块的极性氨基酸残基（F331W，F331Y或F331H）取代时，会产生具有改善的生物转化效率的PapA5突变体; 结果表明，与野生型酶相比，短链醇衍生的MBE平均产量高2.5。此外，MBE生产者微生物还设计了两种合成乙醇的替代途径，允许从头生产乙醇衍生的MBE，其水平可与外部供应该醇的水平相当。要点：•通道2中的突变改变了PapA5酰基转移酶的生物转化效率。•改进了短链醇衍生的多甲基支化酯的生产。•建立从头生产乙醇衍生的MBE的产乙醇途径。•表征新型苯乙醇衍生的MBE。