Furfural-tolerant Zymomonas mobilis derived from error-prone PCR-based whole genome shuffling and their tolerant mechanism
Furfural-tolerant strain is essential for the fermentative production of biofuels or chemicals from lignocellulosic biomass. In this study, Zymomonas mobilis CP4 was for the first time subjected to error-prone PCR-based whole genome shuffling, and the resulting mutants F211 and F27 that could tolerate 3 g/L furfural were obtained. The mutant F211 under various furfural stress conditions could rapidly grow when the furfural concentration reduced to 1 g/L. Meanwhile, the two mutants also showed higher tolerance to high concentration of glucose than the control strain CP4. Genome resequencing revealed that the F211 and F27 had 12 and 13 single-nucleotide polymorphisms. The activity assay demonstrated that the activity of NADH-dependent furfural reductase in mutant F211 and CP4 was all increased under furfural stress, and the activity peaked earlier in mutant than in control. Also, furfural level in the culture of F211 was also more rapidly decreased. These indicate that the increase in furfural tolerance of the mutants may be resulted from the enhanced NADH-dependent furfural reductase activity during early log phase, which could lead to an accelerated furfural detoxification process in mutants. In all, we obtained Z. mobilis mutants with enhanced furfural and high concentration of glucose tolerance, and provided valuable clues for the mechanism of furfural tolerance and strain development.
KeywordsFurfural tolerance Zymomonas mobilis Furfural reductase Error-prone PCR-based whole genome shuffling Genome resequencing
We thank Dr. Jie Bao for providing plasmid pHW20a.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Carey VC, Ingram LO (1983) Lipid composition of Zymomonas mobilis: effects of ethanol and glucose. J Bacteriol 1291–1300Google Scholar
- Kádár Z, Maltha SF, Szengyel Z, Réczey K, De Laat W (2007) Ethanol fermentation of various pretreated and hydrolyzed substrates at low initial pH. Appl Biochem Biotech 137(1–12):847–858Google Scholar
- Liu ZL, Moon J (2009) A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. Gene 446(1):1–10. https://doi.org/10.1016/j.gene.2009.06.018 CrossRefPubMedGoogle Scholar
- Liu ZL, Moon J, Andersh BJ, Slininger PJ, Weber S (2008) Multiple gene-mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 81(4):743–753. https://doi.org/10.1007/s00253-008-1702-0 CrossRefPubMedGoogle Scholar
- Shui ZX, Qin H, Wu B, Ruan ZY, Wang LS, Tan FR, Wang JL, Tang XY, Dai LC, Hu GQ (2015) Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors. Appl Microbiol Biotechnol 99(13):5739–5748. https://doi.org/10.1007/s00253-015-6616-z CrossRefPubMedGoogle Scholar
- Wang X, Miller EN, Yomano LP, Zhang X, Shanmugam KT, Ingram LO (2011) Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate. Appl Environ Microbiol 77(15):5132–5140. https://doi.org/10.1128/AEM.05008-11 CrossRefPubMedPubMedCentralGoogle Scholar
- Wang X, Ma M, Liu ZL, Xiang Q, Li X, Liu N, Zhang X (2016a) GRE2 from Scheffersomyces stipitis as an aldehyde reductase contributes tolerance to aldehyde inhibitors derived from lignocellulosic biomass. Appl Microbiol Biotechnol 100(15):6671–6682. https://doi.org/10.1007/s00253-016-7445-4 CrossRefPubMedGoogle Scholar
- Yi X, Gu H, Gao Q, Liu ZL, Bao J (2015) Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment. Biotechnol Biofuels 8(1):153. https://doi.org/10.1186/s13068-015-0333-9 CrossRefPubMedPubMedCentralGoogle Scholar