Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors
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Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84 % theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89 %. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the “omics” level, which will provide some useful information for inverse metabolic engineering.
KeywordsAdaptive laboratory evolution (ALE) Lignocellulosic hydrolysates Furfural Acetic acid Bioethanol Zymomonas mobilis
This work was supported by Open Funds of State Key Laboratory of Microbial Technology (Shandong University, M2013-07), Open Funds of Key Laboratory of Microbial Resources Collection and Preservation (Ministry of Agriculture, MOA, 2013). Partially supported by Open Funds of Xinjiang Production and Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin (Tarim University, BRZD1403). Partially supported by Youth Science and Technology Foundation of Sichuan Province (2015JQO047).
Conflict of interests
The authors declare that they have no competing interests.
- Demeke MM, Dietz H, Li Y, Foulquié-Moreno MR, Mutturi S, Deprez S, Den Abt T, Bonini BM, Liden G, Dumortier F (2013) Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering. Biotechnol Biofuels 6(1):89PubMedCentralPubMedCrossRefGoogle Scholar
- Gutierrez-Padilla MGD, Karim MN (2005) Influence of furfural on the recombinant Zymomonas mobilis strain CP4 (pZB5) for ethanol production. J Am Sci 1(1):24–27Google Scholar
- Viitanen PV, Tao L, Knoke K, Zhang Y, Caimi PG, Zhang M, Chou Y, Franden MA (2009) Process for the production of ethanol from a medium comprising xylose, employing a recombinant Zymomonas strain having a reduced himA expression. Patent WO/2009/058938Google Scholar
- Viitanen PV, Tao L, Knoke K, Zhang Y, Caimi PG, Zhang M, Chou Y-c, Franden MA (2012) Zymomonas with improved ethanol production in medium containing concentrated sugars and acetate. EP Patent 2,209,899Google Scholar
- Wang Y (2008) Development of acetic-acid tolerant Zymomonas mobilis strains through adaptation. Master Thesis: Georgia Institute of TechnologyGoogle Scholar
- Yamada T, Fatigati M, Zhang M (2002) Performance of Immobilized Zymomonas mobilis 31821 (pZB5) on Actual Hydrolysates Produced by Arkenol Technology. In: Finkelstein M, McMillan J, Davison B (eds) Biotechnology for fuels and chemicals. Applied biochemistry and biotechnology. Humana Press, Totowa, pp 899–907CrossRefGoogle Scholar
- Yang S, Land ML, Klingeman DM, Pelletier DA, Lu TY, Martin SL, Guo HB, Smith JC, Brown SD (2010a) Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 107(23):10395–10400PubMedCentralPubMedCrossRefGoogle Scholar