title: Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans.
abstract: Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance.
authors.affiliation: Genoscope-CEA, CNRS-UMR8030, Université d'Évry, Évry, France atolonen@genoscope.cns.fr.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Tolonen
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Trevor R
authors.initials: TR
authors.lastname: Zuroff
authors.affiliation: Department of Genetic Engineering, SRM University, Kattankulathur, India.
authors.forename: Mohandass
authors.initials: M
authors.lastname: Ramya
authors.affiliation: Genoscope-CEA, CNRS-UMR8030, Université d'Évry, Évry, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Boutard
authors.affiliation: Genoscope-CEA, CNRS-UMR8030, Université d'Évry, Évry, France.
authors.forename: Tristan
authors.initials: T
authors.lastname: Cerisy
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Curtis
mesh_terms: D000220:Adaptation, Biological; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics* / Q000378:metabolism*; D004361:Drug Tolerance; D000431:Ethanol / Q000378:metabolism* / Q000633:toxicity; D015870:Gene Expression; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D011767:Pyruvate Decarboxylase / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017355:Zymomonas / Q000201:enzymology / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D000431:Ethanol; D002482:Cellulose; D000426:Alcohol Dehydrogenase; D011767:Pyruvate Decarboxylase