1. Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics
- Author
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Volker Döring, Magali Boutard, Marcel Salanoubat, Ivan Dubois, Tiffany Souterre, Wahiba Berrabah, Andrew C. Tolonen, Ismael Torres-Romero, Julien Patrouix, Tristan Cerisy, Karine Labadie, Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Génomique métabolique (UMR 8030), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), This work was funded by the Genoscope-CEA, a CNRS Chaire d'Excellence (to A.C.T.), and the Agence Nationale de la Recherche Grant ANR-16-CE05-0020 (to A.C.T.)., We thank P. Marlière for the GM3, E. Darii for mass spectrometry, and the Genoscope-CEA sequencing platform for DNA and RNA sequencing., ANR-16-CE05-0020,Phytocell,Développement des bactéries cellulolytiques Clostridium phytofermentans et Clostridium cellulolyticum comme biocatalyseurs pour la conversion de la biomasse végétale en alcools supérieurs(2016), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,Lignocellulosic biomass ,MESH: Biological Evolution ,MESH: Bacterial Proteins/genetics ,MESH: Clostridium/genetics ,Lignin ,7. Clean energy ,Applied Microbiology and Biotechnology ,MESH: Biodegradation, Environmental ,MESH: Fermentation ,Clostridia ,Ferulic acid ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,MESH: Biomass ,Botany ,evolution ,Environmental Microbiology ,genomics ,Biomass ,Food science ,MESH: Cellulose/metabolism ,Cellulose ,Clostridium ,2. Zero hunger ,[SDV.GEN]Life Sciences [q-bio]/Genetics ,Phenol ,Ecology ,biology ,food and beverages ,Plants ,Clostridium phytofermentans ,biology.organism_classification ,Biological Evolution ,Biodegradation, Environmental ,030104 developmental biology ,chemistry ,Fermentation ,clostridia ,MESH: Bacterial Proteins/metabolism ,Bacteria ,Food Science ,Biotechnology - Abstract
Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans ), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, long-term growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors. IMPORTANCE Fermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacterium Clostridium phytofermentans resists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors.
- Published
- 2017