Thorsten Lechtenberg, Benedikt Wynands, Moritz-Fabian Müller, Tino Polen, Stephan Noack, Nick Wierckx
{"title":"通过实验室自动适应性进化(ALE)提高台湾假单胞菌 VLB120 对 5-(羟甲基)糠醛(HMF)的耐受性","authors":"Thorsten Lechtenberg, Benedikt Wynands, Moritz-Fabian Müller, Tino Polen, Stephan Noack, Nick Wierckx","doi":"10.1016/j.mec.2024.e00235","DOIUrl":null,"url":null,"abstract":"<div><p>The aldehyde 5-(hydroxymethyl)furfural (HMF) is of great importance for a circular bioeconomy. It is a renewable platform chemical that can be converted into a range of useful compounds to replace petroleum-based products such as the green plastic monomer 2,5-furandicarboxylic acid (FDCA). However, it also exhibits microbial toxicity for example hindering the efficient biotechnological valorization of lignocellulosic hydrolysates. Thus, there is an urgent need for tolerance-improved organisms applicable to whole-cell biocatalysis. Here, we engineer an oxidation-deficient derivative of the naturally robust and emerging biotechnological workhorse <em>P. taiwanensis</em> VLB120 by robotics-assisted adaptive laboratory evolution (ALE). The deletion of HMF-oxidizing enzymes enabled for the first time evolution under constant selection pressure by the aldehyde, yielding strains with consistently improved growth characteristics in presence of the toxicant. Genome sequencing of evolved clones revealed loss-of function mutations in the LysR-type transcriptional regulator-encoding <em>mexT</em> preventing expression of the associated efflux pump <em>mexEF</em>-<em>oprN</em>. This knowledge allowed reverse engineering of strains with enhanced aldehyde tolerance, even in a background of active or overexpressed HMF oxidation machinery, demonstrating a synergistic effect of two distinct tolerance mechanisms.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":"18 ","pages":"Article e00235"},"PeriodicalIF":3.7000,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S221403012400004X/pdfft?md5=d7f841437723f702451889b3caf5d32c&pid=1-s2.0-S221403012400004X-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Improving 5-(hydroxymethyl)furfural (HMF) tolerance of Pseudomonas taiwanensis VLB120 by automated adaptive laboratory evolution (ALE)\",\"authors\":\"Thorsten Lechtenberg, Benedikt Wynands, Moritz-Fabian Müller, Tino Polen, Stephan Noack, Nick Wierckx\",\"doi\":\"10.1016/j.mec.2024.e00235\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>The aldehyde 5-(hydroxymethyl)furfural (HMF) is of great importance for a circular bioeconomy. It is a renewable platform chemical that can be converted into a range of useful compounds to replace petroleum-based products such as the green plastic monomer 2,5-furandicarboxylic acid (FDCA). However, it also exhibits microbial toxicity for example hindering the efficient biotechnological valorization of lignocellulosic hydrolysates. Thus, there is an urgent need for tolerance-improved organisms applicable to whole-cell biocatalysis. Here, we engineer an oxidation-deficient derivative of the naturally robust and emerging biotechnological workhorse <em>P. taiwanensis</em> VLB120 by robotics-assisted adaptive laboratory evolution (ALE). The deletion of HMF-oxidizing enzymes enabled for the first time evolution under constant selection pressure by the aldehyde, yielding strains with consistently improved growth characteristics in presence of the toxicant. Genome sequencing of evolved clones revealed loss-of function mutations in the LysR-type transcriptional regulator-encoding <em>mexT</em> preventing expression of the associated efflux pump <em>mexEF</em>-<em>oprN</em>. This knowledge allowed reverse engineering of strains with enhanced aldehyde tolerance, even in a background of active or overexpressed HMF oxidation machinery, demonstrating a synergistic effect of two distinct tolerance mechanisms.</p></div>\",\"PeriodicalId\":18695,\"journal\":{\"name\":\"Metabolic Engineering Communications\",\"volume\":\"18 \",\"pages\":\"Article e00235\"},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2024-05-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S221403012400004X/pdfft?md5=d7f841437723f702451889b3caf5d32c&pid=1-s2.0-S221403012400004X-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Metabolic Engineering Communications\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S221403012400004X\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic Engineering Communications","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S221403012400004X","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Improving 5-(hydroxymethyl)furfural (HMF) tolerance of Pseudomonas taiwanensis VLB120 by automated adaptive laboratory evolution (ALE)
The aldehyde 5-(hydroxymethyl)furfural (HMF) is of great importance for a circular bioeconomy. It is a renewable platform chemical that can be converted into a range of useful compounds to replace petroleum-based products such as the green plastic monomer 2,5-furandicarboxylic acid (FDCA). However, it also exhibits microbial toxicity for example hindering the efficient biotechnological valorization of lignocellulosic hydrolysates. Thus, there is an urgent need for tolerance-improved organisms applicable to whole-cell biocatalysis. Here, we engineer an oxidation-deficient derivative of the naturally robust and emerging biotechnological workhorse P. taiwanensis VLB120 by robotics-assisted adaptive laboratory evolution (ALE). The deletion of HMF-oxidizing enzymes enabled for the first time evolution under constant selection pressure by the aldehyde, yielding strains with consistently improved growth characteristics in presence of the toxicant. Genome sequencing of evolved clones revealed loss-of function mutations in the LysR-type transcriptional regulator-encoding mexT preventing expression of the associated efflux pump mexEF-oprN. This knowledge allowed reverse engineering of strains with enhanced aldehyde tolerance, even in a background of active or overexpressed HMF oxidation machinery, demonstrating a synergistic effect of two distinct tolerance mechanisms.
期刊介绍:
Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.