Abstract
During ethanol production, the fermentation cells are always exposed to stresses like high temperature and low nutritional conditions, which affect their growth and productivity. Stress-tolerant strains with high ethanol yield are highly desirable. Therefore, a recombinant Zymomonas mobilis (Z. mobilis) designated as HYM was constructed by integrating three genes (yfdZ, metB, and Pfu-sHSP) into the genome of Z. mobilis CP4 (CP4) via Tn5 transposon in the present study. The yfdZ and metB genes from E. coli were used to decrease the nutritional requirement. The small heat shock protein gene (Pfu-sHSP) from Pyrococcus furiosus (P. furiosus) was used to increase the heat tolerance. The genomic integration of three genes confers on Z. mobilis the ability to grow in simple chemical defined medium without the addition of amino acid. The HYM not only demonstrated the high tolerance to unfavorable lower nutrition stresses but also the capability of converting glucose to ethanol with high yield at higher temperature. What is more, these genetic characteristics were stable up to 100 generations on nonselective medium. The effects of glucose concentration, fermentation temperature, and initial pH on ethanol production of the mutant strain HYM were optimized using a Box–Behnken design (BBD) experiment. The integration of three genes led to a significant increase in ethanol production by 9 % compared with its original Z. mobilis counterpart. The maximum ethanol production of HYM was as high as 105 g/l.
Similar content being viewed by others
References
Burnett ME, Liu J, Conway T (1992) Molecular characterization of the Zymomonas mobilis enolase (eno) gene. J Bacteriol 174:6548–6553
Conway T, Sewell GW, Ingram LO (1987) Glyceraldehyde-3-phosphate dehydrogenase gene from Zymomonas mobilis: cloning, sequencing, and identification of promoter region. J Bacteriol 169:5653–5662
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 3:426–428
Widiastuti H, Kim JY, Selvarasu S, Karimi IA, Kim H, Seo J-S, Lee D-Y (2010) Genome-scale modeling and in silico analysis of ethanologenic bacteria Zymomonas mobilis. Biotechnol Bioeng 108(3):655–665
Yanase H, Sato D, Yamamoto K, Matsuda S, Yamamoto S, Okamoto K (2007) Genetic engineering of Zymobacter palmae for production of ethanol from xylose. Appl Microbiol Biotechnol 02302–06:2592–2599
Seo JS, Chong H, Park HS, Yoon KO, Jung C, Kim JJ, Hong JH, Kim H, Kim JH, Kil JI, Park CJ, Oh HM, Lee JS, Jin SJ, Um HW, Lee HJ, Oh SJ, Kim JY, Kang HL, Lee SY, Lee KJ, Kang HS (2005) The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4. Nat Biotechnol 23:63–68
Laksanalamai P, Jiemjit A, Bu Z, Maeder DL, Robb FT (2003) Multisubunit assembly of the Pyrococcus furiosus small heat shock protein is essential for cellular protection at high temperature. Extremophiles 7(1):79–83
Laksanalamai P, Maeder DL, Robb FT (2001) Regulation and mechanism of action of the small heat shock protein from the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 183:5198–5202
Skotnicki ML, Lee KJ, Tribe DE, Rogers PL (1982) Genetic alteration of Zymomonas mobilis for ethanol production. Basic Life Sci 19:271–290
Skotnicki ML, Warr RG, Goodman AE, Lee KJ, Rogers PL (1982) High-productivity alcohol fermentations using Zymomonas mobilis. Biochem Soc Symp 19:53–86
Talarico LA, Gil MA, Yomano LP, Ingram LO, Maupin-Furlow JA (2005) Construction and expression of an ethanol production in Gram-positive bacteria. Microbiology 151:4023–4031
Jeffries TW (2005) Ethanol fermentation on the move. Nat Biotechnol 23(1):40–41
Marsden WL, Gray PP, Nippard GJ, Quinlan MR (1982) Evaluation of the DNS method for analysing lignocellulosic hydrolysates. J Chem Technol Biotechnol 32(7–12):1016–1022
Vidal JE, Chen J, Li J, McClane BA (2009) Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13. PLoS ONE 4(7):e6232
Acknowledgments
We gratefully acknowledge the financial support from the National Natural Science Foundation of China (NSFC) (Project No. 30870798), Hubei Provincial Innovative Research Team in University (Project No. T200705), and the Scientific Research Key Project of Hubei Provincial Department of Education (Project No. Z200614001). Haoyong Wang also thanks the Scientific Research Foundation for the Returned Overseas Chinese Scholars (Ministry of Personnel, State Education Ministry and Hubei Provincial Department of Personnel).
Conflict of interest
The authors declare that they have no competing interests.
Author information
Authors and Affiliations
Corresponding author
Additional information
Xianghui Jia, Na Wei, and Tianyv Wang contributed equally to this work.
Rights and permissions
About this article
Cite this article
Jia, X., Wei, N., Wang, T. et al. Use of an EZ-Tn5-based random mutagenesis system to create a Zymomonas mobilis with significant tolerance to heat stress and malnutrition. J Ind Microbiol Biotechnol 40, 811–822 (2013). https://doi.org/10.1007/s10295-013-1287-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10295-013-1287-1