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Title: Improving olefin tolerance and production in E. coli using native and evolved AcrB

Microorganisms can be engineered for the production of chemicals utilized in the polymer industry. However many such target compounds inhibit microbial growth and might correspondingly limit production levels. Here, we focus on compounds that are precursors to bioplastics, specifically styrene and representative alpha-olefins; 1-hexene, 1-octene, and 1-nonene. We evaluated the role of the Escherichia coli efflux pump, AcrAB-TolC, in enhancing tolerance towards these olefin compounds. AcrAB-TolC is involved in the tolerance towards all four compounds in E. coli. Both styrene and 1-hexene are highly toxic to E. coli. Styrene is a model plastics precursor with an established route for production in E. coli (McKenna and Nielsen, 2011). Though our data indicates that AcrAB-TolC is important for its optimal production, we observed a strong negative selection against the production of styrene in E. coli. Thus we used 1-hexene as a model compound to implement a directed evolution strategy to further improve the tolerance phenotype towards this alpha-olefin. We focused on optimization of AcrB, the inner membrane domain known to be responsible for substrate binding, and found several mutations (A279T, Q584R, F617L, L822P, F927S, and F1033Y) that resulted in improved tolerance. Several of these mutations could also be combined in a synergisticmore » manner. Our study shows efflux pumps to be an important mechanism in host engineering for olefins, and one that can be further improved using strategies such as directed evolution, to increase tolerance and potentially production.« less
Authors:
 [1] ;  [1] ;  [2] ;  [2] ;  [3] ;  [1] ;  [2]
  1. Total New Energies, Inc., Emeryville, CA (United States)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
OSTI Identifier:
1213444
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Biotechnology and Bioengineering
Additional Journal Information:
Journal Volume: 112; Journal Issue: 5; Journal ID: ISSN 0006-3592
Research Org:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES directed evolution; host engineering; olefin production; solvent tolerance