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Title: Comprehensive molecular characterization of Methylobacterium extorquens AM1 adapted for 1-butanol tolerance

In this study, the toxicity of alcohols is one of the major roadblocks of biological fermentation for biofuels production. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, has been engineered to generate 1-butanol from cheap carbon feedstocks through a synthetic metabolic pathway. However, M. extorquens AM1 is vulnerable to solvent stress, which impedes further development for 1-butanol production. Only a few studies have reported the general stress response of M. extorquens AM1 to solvent stress. Therefore, it is highly desirable to obtain a strain with ameliorated 1-butanol tolerance and elucidate the molecular mechanism of 1-butnaol tolerance in M. extorquens AM1 for future strain improvement. In this work, adaptive laboratory evolution was used as a tool to isolate mutants with 1-butanol tolerance up to 0.5 %. The evolved strains, BHBT3 and BHBT5, demonstrated increased growth rates and higher survival rates with the existence of 1-butanol. Whole genome sequencing revealed a SNP mutation at kefB in BHBT5, which was confirmed to be responsible for increasing 1-butanol tolerance through an allelic exchange experiment. Global metabolomic analysis further discovered that the pools of multiple key metabolites, including fatty acids, amino acids, and disaccharides, were increased in BHBT5 in response to 1-butanol stress. Additionally, the carotenoidmore » synthesis pathway was significantly down-regulated in BHBT5. In conclusion, we successfully screened mutants resistant to 1-butanol and provided insights into the molecular mechanism of 1-butanol tolerance in M. extorquens AM1. This research will be useful for uncovering the mechanism of cellular response of M. extorquens AM1 to solvent stress, and will provide the genetic blueprint for the rational design of a strain of M. extorquens AM1 with increased 1-butanol tolerance in the future.« less
Authors:
 [1] ;  [2] ;  [3] ;  [2] ;  [2] ;  [2] ;  [3] ;  [4]
  1. Univ. of Washington, Seattle, WA (United States); Intrexon Corp., South San Francisco, CA (United States)
  2. Qingdao Agricultural Univ., Qingdao (China)
  3. Univ. of Washington, Seattle, WA (United States)
  4. Qingdao Agricultural Univ., Qingdao (China); Tianjin Univ., Tianjin (China)
Publication Date:
OSTI Identifier:
1253902
Grant/Contract Number:
SC0006871
Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Research Org:
Univ. of Washington, Seattle, WA (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 09 BIOMASS FUELS Methylobacterium extorquens AM1; 1-butanol tolerance; adaptive evolution; whole genome sequencing; global metabolome analysis; carotenoid