Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and -demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic -demethylation, but they are rarely compared to determine an optimal biocatalytic strategy. Here, two pathways for aromatic -demethylation were compared in KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in , and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate demethylase, PP_3494, a global regulator of vanillate catabolism, and , involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms yielding a platform strain for efficient -demethylation of lignin-related aromatic compounds to value-added products.

中文翻译：

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恶臭假单胞菌 KT2440 芳香族 O-去甲基化途径的进化和改造


从生物质中生物转化木质素为燃料和化学品的可持续生产提供了一种有前景的策略。然而，源自木质素的芳香族化合物通常含有甲氧基，这些底物的去甲基化通常是影响分解代谢效率的限速反应。几个酶家族催化芳香族去甲基化，但很少对它们进行比较以确定最佳的生物催化策略。在此，比较了 KT2440 中芳香族去甲基化的两种途径。天然 Rieske 非血红素铁单加氧酶 (VanAB) 和异源四氢叶酸依赖性脱甲基酶 (LigM) 分别在 中组成型表达，并通过适应性实验室进化 (ALE) 以香草酸作为模型底物对菌株进行优化。所有进化菌株都表现出改善的生长表型，具有天然 VanAB 途径的进化菌株的生长速度比具有异源 LigM 途径的菌株快约 1.8 倍。酶动力学和转录组学研究调查了选定突变对增强香草酸利用率的贡献。 VanAB 过表达菌株包含最有影响力的突变，包括 VanB（香草酸脱甲基酶的还原酶）、PP_3494（香草酸分解代谢的全局调节因子）和参与甲醛解毒的突变。这三种突变被组合成一个菌株，在培养的前 8 小时内，其香草酸消耗速度比野生型菌株快约 5 倍。总体而言，本研究阐明了两种不同酶促机制背景下香草酸分解代谢的细节，产生了用于将木质素相关芳香族化合物有效去甲基化为增值产品的平台菌株。