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Development of Pichia stipitis Co-fermenting Cellobiose and Xylose Through Adaptive Evolution

적응진화를 활용한 cellobiose와 xylose 동시발효 Pichia stipitis의 개발

  • Kim, Dae-Hwan (Department of Bioenergy Science and Technology, Chonnam National University) ;
  • Lee, Won-Heong (Department of Bioenergy Science and Technology, Chonnam National University)
  • 김대환 (전남대학교 바이오에너지공학과) ;
  • 이원흥 (전남대학교 바이오에너지공학과)
  • Received : 2019.09.10
  • Accepted : 2019.10.10
  • Published : 2019.12.28

Abstract

Production of biofuels and value-added materials from cellulosic biomass requires the development of a microbial strain capable of efficiently fermenting mixed sugars. In this study, the natural xylose fermenting yeast, Pichia stipitis, was evolved to simultaneously ferment cellobiose and xylose. Serial subcultures of wild-type P. stipitis in 20 g/l cellobiose were performed to increase the rate of cellobiose consumption. A total of ten rounds of the serial subculture led to the isolation of an evolved strain fermenting cellobiose significantly faster than the parental strain. The evolved strain displayed enhanced ethanol yield from 0 to 0.4 g ethanol/g cellobiose. The evolved P. stipitis simultaneously fermented cellobiose and xylose in batch fermentation. The genetic information of our evolved P. stipitis would be valuable in the development of a microbial host for the production of biofuels and biomaterials from cellulosic biomass.

섬유소계 바이오매스로부터 바이오 연료 등과 같은 유용한 물질을 생산하기 위해서는 바이오매스로부터 유래하는 혼합당을 효과적으로 대사할 수 있는 균주의 개발이 필수적이다. 본 연구에서는 xylose를 대사가 가능한 효모인 P. stipitis를 적응진화하여 cellobiose 대사효율이 향상되고 cellobiose와 xylose를 동시에 대사할 수 있는 균주를 개발하고자 하였다. 총 10회의 계대배양을 통해 얻어진 진화된 P. stipitis 돌연변이 균주는 모균주에 비해 6배 이상 증가된 cellobiose 대사속도를 나타내었으며 ethanol 생산수율을 0에서 0.4 (g ethanol/g cellobiose)로 향상시켰다. 아울러 본 실험에서 개발한 돌연변이 균주는 cellobiose와 xylose 혼합당 조건에서 모균주에 비해 2배 가까이 향상된 ethanol 생산 및 생산속도를 나타내었다.

Keywords

References

  1. Amoah J, Kahar P, Ogino C, Kondo A. 2019. Bioenergy and Biorefinery: feedstock, biotechnological conversion and products. Biotechnol. J. 14: e1800494.
  2. Gray KA, Zhao L, Emptage M. 2006. Bioethanol. Curr. Opin. Chem. Biol. 10: 141-146. https://doi.org/10.1016/j.cbpa.2006.02.035
  3. Turner TL, Kim H, Kong II, Liu JJ, Zhang GC, Jin YS. 2018. Engineering and evolution of Saccharomyces cerevisiae to produce biofuels and chemicals. Adv. Biochem. Eng. Biotechnol. 162: 175-215.
  4. Gancedo JM. 1998. Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62: 334-361. https://doi.org/10.1128/mmbr.62.2.334-361.1998
  5. Kayikci O, Nielsen J. 2015. Glucose repression in Saccharomyces cerevisiae. FEMS Yeast Res. 15: fov068. https://doi.org/10.1093/femsyr/fov068
  6. Jin YS, Cate JH. 2017. Metabolic engineering of yeast for lignocellulosic biofuel production. Curr. Opin. Chem. Biol. 41: 99-106. https://doi.org/10.1016/j.cbpa.2017.10.025
  7. Park YC, Oh EJ, Jo JH, Jin YS, Seo JH. 2016. Recent advances in biological production of sugar alcohols. Curr. Opin. Biotechnol. 37: 105-113. https://doi.org/10.1016/j.copbio.2015.11.006
  8. Zhang GC, Liu JJ, Kong II, Kwak S, Jin YS. 2015. Combining C6 and C5 sugar metabolism for enhancing microbial bioconversion. Curr. Opin. Chem. Biol. 29: 49-57. https://doi.org/10.1016/j.cbpa.2015.09.008
  9. Kim SR, Park YC, Jin YS, Seo JH. 2013. Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism. Biotechnol. Adv. 31: 851-861. https://doi.org/10.1016/j.biotechadv.2013.03.004
  10. Kim SR, Skerker JM, Kang W, Lesmana A, Wei N, Arkin AP, et al. 2013. Rational and evolutionary engineering approaches uncover a small set of genetic changes efficient for rapid xylose fermentation in Saccharomyces cerevisiae. PLoS One. 8: e57048. https://doi.org/10.1371/journal.pone.0057048
  11. Tsai CS, Kong II, Lesmana A, Million G, Zhang GC, Kim SR, Jin YS. 2015. Rapid and marker‐free refactoring of xylose‐fermenting yeast strains with Cas9/CRISPR. Biotechnol. Bioeng. 112: 2406-2411. https://doi.org/10.1002/bit.25632
  12. Ha SJ, Galazka JM, Kim SR, Choi JH, Yang X, Seo JH, et al. 2011. Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation. Proc. Natl. Acad. Sci. USA 108: 504-509. https://doi.org/10.1073/pnas.1010456108
  13. Ha SJ, Galazka JM, Oh EJ, Kordić V, Kim H, Jin YS, et al. 2013. Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters. Metab. Eng. 15: 134-143. https://doi.org/10.1016/j.ymben.2012.11.005
  14. Kim SR, Ha SJ, Wei N, Oh EJ, Jin YS. 2012. Simultaneous co-fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol. Trends Biotechnol. 30: 274-282. https://doi.org/10.1016/j.tibtech.2012.01.005
  15. Bae YH, Kang KH, Jin YS, Seo JH. 2014. Molecular cloning and expression of fungal cellobiose transporters and ${\beta}$-glucosidases conferring efficient cellobiose fermentation in Saccharomyces cerevisiae. J. Biotechnol. 169: 34-41. https://doi.org/10.1016/j.jbiotec.2013.10.030
  16. Chomvong K, Kordić V, Li X, Bauer S, Gillespie AE, Ha SJ, et al. 2014. Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose. Biotechnol. Biofuels. 7: 85. https://doi.org/10.1186/1754-6834-7-85
  17. Agbogbo FK, Coward-Kelly G. 2008. Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis. Biotechnol. Lett. 30: 1515-1524. https://doi.org/10.1007/s10529-008-9728-z
  18. Jeffries TW, Grigoriev IV, Grimwood J, Laplaza JM, Aerts A, Salamov A, et al. 2007. Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis. Nat. Biotechnol. 25: 319-326. https://doi.org/10.1038/nbt1290
  19. Perego P, Converti A, Palazzi E, Del Borghi M, Ferraiolo G. 1990. Fermentation of hardwood hemicellulose hydrolysate by Pachysolen tannophilus, Candida shehatae and Pichia stipitis. J. Ind. Microbiol. 6: 157-164. https://doi.org/10.1007/BF01577690
  20. Geiger M, Gibbons J, West T, Hughes SR, Gibbons W. 2012. Evaluation of UV-C mutagenized Scheffersomyces stipitis strains for ethanol production. J. Lab. Autom. 17: 417-424. https://doi.org/10.1177/2211068212452873
  21. Lang GI, Desai MM. 2014. The spectrum of adaptive mutations in experimental evolution. Genomics 104: 412-416. https://doi.org/10.1016/j.ygeno.2014.09.011
  22. Sandberg TE, Salazar MJ, Weng LL, Palsson BO, Feist AM. 2019. The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology. Metab. Eng. 8: 1-16. https://doi.org/10.1016/j.ymben.2005.08.003
  23. Winkler JD, Kao KC. 2014. Recent advances in the evolutionary engineering of industrial biocatalysts. Genomics 104: 406-411. https://doi.org/10.1016/j.ygeno.2014.09.006
  24. Jeffries TW, Van Vleet JRH. 2009. Pichia stipitis genomics, transcriptomics, and gene clusters. FEMS Yeast Res. 9: 793-807. https://doi.org/10.1111/j.1567-1364.2009.00525.x
  25. Ha SJ, Kim H, Lin Y, Jang M-U, Galazka JM, Kim TJ, et al. 2013. Single amino acid substitutions in HXT2. 4 from Scheffersomyces stipitis lead to improved cellobiose fermentation by engineered Saccharomyces cerevisiae. Appl. Environ. Microbiol. 79: 1500-1507. https://doi.org/10.1128/AEM.03253-12
  26. Nelson SS, Van Vleet JH, Jeffries TW. 2010. Presented at the 32nd Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, FL.