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
{"title":"Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440","authors":"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","doi":"10.1016/j.ymben.2024.06.009","DOIUrl":null,"url":null,"abstract":"<div><p>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 <em>O</em>-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic <em>O</em>-demethylation, but they are rarely compared <em>in vivo</em> to determine an optimal biocatalytic strategy. Here, two pathways for aromatic <em>O</em>-demethylation were compared in <em>Pseudomonas putida</em> KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in <em>P. putida</em>, 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 <em>O-</em>demethylase, PP_3494, a global regulator of vanillate catabolism, and <em>fghA</em>, 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<em>,</em> yielding a platform strain for efficient <em>O</em>-demethylation of lignin-related aromatic compounds to value-added products.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":6.8000,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic engineering","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S109671762400082X","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
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.
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.