Improving Olefin Tolerance and Production in E. coli Using Native and Evolved AcrB

被引:42
作者
Mingardon, Florence [1 ]
Clement, Camille [1 ]
Hirano, Kathleen [2 ,3 ]
Nhan, Melissa [2 ,3 ]
Luning, Eric G. [3 ]
Chanal, Angelique [1 ]
Mukhopadhyay, Aindrila [2 ,3 ]
机构
[1] Total New Energies Inc, Emeryville, CA 94608 USA
[2] Joint BioEnergy Inst, Emeryville, CA 94608 USA
[3] Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Phys Biosci Div, Berkeley, CA 94720 USA
关键词
directed evolution; host engineering; olefin production; solvent tolerance; MULTIDRUG EFFLUX PUMP; TRANSPORTER ACRB; SUBSTRATE PATH; MECHANISMS; EXPORTER; SUPPORT; TRIMER;
D O I
10.1002/bit.25511
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 synergistic 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. Biotechnol. Bioeng. 2015;112: 879-888. (c) 2015 Wiley Periodicals, Inc.
引用
收藏
页码:879 / 888
页数:10
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