Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Alissa C. Bleem , Eugene Kuatsjah , Josefin Johnsen , Elsayed T. Mohamed , William G. Alexander , Zoe A. Kellermyer , Austin L. Carroll , Riccardo Rossi , Ian B. Schlander , George L. Peabody V , Adam M. Guss , Adam M. Feist , Gregg T. Beckham
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引用次数: 0

Abstract

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 O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, 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 O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, 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 O-demethylation of lignin-related aromatic compounds to value-added products.

普氏假单胞菌 KT2440 中芳香族 O-脱甲基途径的进化和工程化。
生物转化生物质中的木质素为燃料和化学品的可持续生产提供了一种前景广阔的策略。然而,从木质素中提取的芳香族化合物通常含有甲氧基,这些底物的 O-去甲基化通常是影响分解效率的限速反应。有几个酶家族可以催化芳香族 O-脱甲基反应,但它们很少在体内进行比较,以确定最佳的生物催化策略。本文比较了假单胞菌 KT2440 中芳香族 O-去甲基化的两种途径。在假单胞菌(P. putida)中组成型表达了原生的 Rieske 非血红素铁单加氧酶(VanAB),并分别表达了异源的四氢叶酸依赖性脱甲基酶(LigM),以香草酸盐为模型底物,通过适应性实验室进化(ALE)对菌株进行了优化。所有进化菌株的生长表型都有所改善,其中携带原生 VanAB 通路的进化菌株的生长速度比携带异源 LigM 通路的进化菌株快 1.8 倍。酶动力学和转录组学研究调查了所选突变对提高香草酸利用率的贡献。VanAB过表达菌株含有影响最大的突变,包括香草酸O-脱甲基酶的还原酶VanB、香草酸分解的全局调控因子PP_3494和参与甲醛解毒的fghA。将这三种突变结合到一个菌株中,在培养的前 8 小时内,该菌株的香草酸消耗速度比野生型菌株快约 5 倍。总之,这项研究通过两种不同的酶学机制阐明了香草酸分解代谢的细节,为木质素相关芳香化合物的高效 O-去甲基化转化为高附加值产品提供了一个平台菌株。
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来源期刊
Metabolic engineering
Metabolic engineering 工程技术-生物工程与应用微生物
CiteScore
15.60
自引率
6.00%
发文量
140
审稿时长
44 days
期刊介绍: Metabolic Engineering (MBE) is a journal that focuses on publishing original research papers on the directed modulation of metabolic pathways for metabolite overproduction or the enhancement of cellular properties. It welcomes papers that describe the engineering of native pathways and the synthesis of heterologous pathways to convert microorganisms into microbial cell factories. The journal covers experimental, computational, and modeling approaches for understanding metabolic pathways and manipulating them through genetic, media, or environmental means. Effective exploration of metabolic pathways necessitates the use of molecular biology and biochemistry methods, as well as engineering techniques for modeling and data analysis. MBE serves as a platform for interdisciplinary research in fields such as biochemistry, molecular biology, applied microbiology, cellular physiology, cellular nutrition in health and disease, and biochemical engineering. The journal publishes various types of papers, including original research papers and review papers. It is indexed and abstracted in databases such as Scopus, Embase, EMBiology, Current Contents - Life Sciences and Clinical Medicine, Science Citation Index, PubMed/Medline, CAS and Biotechnology Citation Index.
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