Generation of a platform strain for ionic liquid tolerance using adaptive laboratory evolution

Microbial Cell Factories

Research

Blake A. Simmons¹  Steven W. Singer¹  Shizeng Wang²  Markus J. Herrgård³  Elsayed T. Mohamed³  Rebecca M. Lennen³  Adam M. Feist⁴ 

[1] Joint Bioenergy Institute, Emeryville, CA, USA;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA;Joint Bioenergy Institute, Emeryville, CA, USA;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA;State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, 100029, Beijing, People’s Republic of China;Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, 2800, Kgs. Lyngby, Denmark;Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, 2800, Kgs. Lyngby, Denmark;Department of Bioengineering, University of California, 9500 Gilman Drive La Jolla, 92093, San Diego, CA, USA;

关键词: Escherichia coli;    Renewable feedstocks;    Ionic liquids;    Adaptive laboratory evolution;   

DOI  :  10.1186/s12934-017-0819-1

 received in 2017-08-25, accepted in 2017-11-09,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundThere is a need to replace petroleum-derived with sustainable feedstocks for chemical production. Certain biomass feedstocks can meet this need as abundant, diverse, and renewable resources. Specific ionic liquids (ILs) can play a role in this process as promising candidates for chemical pretreatment and deconstruction of plant-based biomass feedstocks as they efficiently release carbohydrates which can be fermented. However, the most efficient pretreatment ILs are highly toxic to biological systems, such as microbial fermentations, and hinder subsequent bioprocessing of fermentative sugars obtained from IL-treated biomass.MethodsTo generate strains capable of tolerating residual ILs present in treated feedstocks, a tolerance adaptive laboratory evolution (TALE) approach was developed and utilized to improve growth of two different Escherichia coli strains, DH1 and K-12 MG1655, in the presence of two different ionic liquids, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3-methylimidazolium chloride ([C4C1Im]Cl). For multiple parallel replicate populations of E. coli, cells were repeatedly passed to select for improved fitness over the course of approximately 40 days. Clonal isolates were screened and the best performing isolates were subjected to whole genome sequencing.ResultsThe most prevalent mutations in tolerant clones occurred in transport processes related to the functions of mdtJI, a multidrug efflux pump, and yhdP, an uncharacterized transporter. Additional mutations were enriched in processes such as transcriptional regulation and nucleotide biosynthesis. Finally, the best-performing strains were compared to previously characterized tolerant strains and showed superior performance in tolerance of different IL and media combinations (i.e., cross tolerance) with robust growth at 8.5% (w/v) and detectable growth up to 11.9% (w/v) [C2C1Im][OAc].ConclusionThe generated strains thus represent the best performing platform strains available for bioproduction utilizing IL-treated renewable substrates, and the TALE method was highly successful in overcoming the general issue of substrate toxicity and has great promise for use in tolerance engineering.

【 授权许可】

CC BY   
© The Author(s) 2017