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Comprehensive Characterization of Mutant Pichia stipitis Co-Fermenting Cellobiose and Xylose through Genomic and Transcriptomic Analyses

  • Dae-Hwan Kim (Department of Bioenergy Science and Technology, Chonnam National University) ;
  • Hyo-Jin Choi (Department of Bioenergy Science and Technology, Chonnam National University) ;
  • Yu Rim Lee (Interdisciplinary Program of Agriculture and Life Science, Chonnam National University) ;
  • Soo-Jung Kim (Department of Integrative Food, Bioscience and Biotechnology, Chonnam National University) ;
  • Sangmin Lee (Gwangju Bio/Energy R&D Center, Korea Institute of Energy Research) ;
  • Won-Heong Lee (Department of Bioenergy Science and Technology, Chonnam National University)
  • Received : 2022.09.05
  • Accepted : 2022.10.06
  • Published : 2022.11.28

Abstract

The development of a yeast strain capable of fermenting mixed sugars efficiently is crucial for producing biofuels and value-added materials from cellulosic biomass. Previously, a mutant Pichia stipitis YN14 strain capable of co-fermenting xylose and cellobiose was developed through evolutionary engineering of the wild-type P. stipitis CBS6054 strain, which was incapable of co-fermenting xylose and cellobiose. In this study, through genomic and transcriptomic analyses, we sought to investigate the reasons for the improved sugar metabolic performance of the mutant YN14 strain in comparison with the parental CBS6054 strain. Unfortunately, comparative whole-genome sequencing (WGS) showed no mutation in any of the genes involved in the cellobiose metabolism between the two strains. However, comparative RNA sequencing (RNA-seq) revealed that the YN14 strain had 101.2 times and 5.9 times higher expression levels of HXT2.3 and BGL2 genes involved in cellobiose metabolism, and 6.9 times and 75.9 times lower expression levels of COX17 and SOD2.2 genes involved in respiration, respectively, compared with the CBS6054 strain. This may explain how the YN14 strain enhanced cellobiose metabolic performance and shifted the direction of cellobiose metabolic flux from respiration to fermentation in the presence of cellobiose compared with the CBS6054 strain.

Keywords

Acknowledgement

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (No. NRF-2022R1F1A1074296). This study was also supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) and the Korea Smart Farm R&D Foundation (KosFarm) through the Smart Farm Innovation Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) and the Ministry of Science and ICT (MSIT), Rural Development Administration (RDA) (No. 421045-03). This study was also supported by Regional Innovation Strategy (RIS) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021RIS-002).

References

  1. Amoah J, Kahar P, Ogino C, Kondo A. 2019. Bioenergy and Biorefinery: feedstock, biotechnological conversion and products. Biotechnol. J. 14: 1800494.
  2. Rosales-Calderon O, Arantes V. 2019. A review on commercial-scale high-value products that can be produced alongside cellulosic ethanol. Biotechnol. Biofuels 12: 240.
  3. 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
  4. 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
  5. 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
  6. Hou J, Qiu C, Shen Y, Li H, Bao X. 2017. Engineering of Saccharomyces cerevisiae for the efficient co-utilization of glucose and xylose. FEMS. Yeast Res. 17. doi: 10.1093/femsyr/fox034.
  7. Parisutham V, Chandran SP, Mukhopadhyay A, Lee SK, Keasling JD. 2017. Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries. Bioresour. Technol. 239: 496-506. https://doi.org/10.1016/j.biortech.2017.05.001
  8. Ha SJ, Kim SR, Kim H, Du J, Cate JH, Jin YS. 2013. Continuous co-fermentation of cellobiose and xylose by engineered Saccharomyces cerevisiae. Bioresour. Technol. 149: 525-531. https://doi.org/10.1016/j.biortech.2013.09.082
  9. 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
  10. Lee WH, Jin YS. 2021. Observation of cellodextrin accumulation resulted from non-conventional secretion of intracellular β-glucosidase by engineered Saccharomyces cerevisiae fermenting cellobiose. J. Microbiol. Biotechnol. 31: 1035-1043. https://doi.org/10.4014/jmb.2105.05018
  11. Chomvong K, Kordi? V, Li X, Bauer S, Gillespie AE, Ha SJ, Oh EJ, et al. 2014. Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose. Biotechnol. Biofuels. 7: 85.
  12. 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
  13. 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
  14. Jeffries TW, Van Vleet JR. 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
  15. Kim DH, Lee WH. 2019. Development of Pichia stipitis co-fermenting cellobiose and xylose through adaptive evolution. Microbiol. Biotechnol. Lett. 47: 565-573. https://doi.org/10.4014/mbl.1909.09005
  16. 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
  17. Lambrechts MG, Bauer FF, Marmur J, Pretorius IS. 1996. Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast. Proc. Natl. Acad. Sci. USA 93: 8419-8424. https://doi.org/10.1073/pnas.93.16.8419
  18. Liu T, Zou W, Liu L, Chen J. 2012. A constraint-based model of Scheffersomyces stipitis for improved ethanol production. Biotechnol. Biofuels 5: 72.
  19. Nelson SS, Van Vleet JH, Jeffries TW. 2010. Presented at the 32nd Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach, FL.
  20. Graves JA, Henry SA. 2000. Regulation of the yeast INO1 gene: The products of the INO2, INO4 and OPI1 regulatory genes are not required for repression in response to inositol. Genetics 154: 1485-1495. https://doi.org/10.1093/genetics/154.4.1485
  21. Ding J, Huang X, Zhang L, Zhao N, Yang D, Zhang K. 2009. Tolerance and stress response to ethanol in the yeast Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 85: 253-263. https://doi.org/10.1007/s00253-009-2223-1
  22. Weierstall T, Hollenberg CP, Boles E. 1999. Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipitis. Mol. Microbiol. 31: 871-883. https://doi.org/10.1046/j.1365-2958.1999.01224.x
  23. Young E, Poucher A, Comer A, Bailey A, Alper H. 2011. Functional survey for heterologous sugar transport proteins, using Saccharomyces cerevisiae as a host. Appl. Environ. Microbiol. 77: 3311-3319. https://doi.org/10.1128/AEM.02651-10
  24. Watanabe S, Utsumi Y, Sawayama S, Watanabe Y. 2016. Identification and characterization of D-arabinose reductase and D-arabinose transporters from Pichia stipitis. Biosci. Biotechnol. Biochem. 80: 2151-2158. https://doi.org/10.1080/09168451.2016.1204221
  25. Moon J, Liu ZL, Ma M, Slininger PJ. 2013. New genotypes of industrial yeast Saccharomyces cerevisiae engineered with YXI and heterologous xylose transporters improve xylose utilization and ethanol production. Biocatal. Agric. Biotechnol. 2: 247-254. https://doi.org/10.1016/j.bcab.2013.03.005
  26. Glerum DM, Shtanko A, Tzagoloff A. 1996. Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase. J. Biol. Chem. 271: 14504-14509. https://doi.org/10.1074/jbc.271.24.14504
  27. Gamberi T, Puglia M, Bianchi L, Gimigliano A, Landi C, Magherini F, et al. 2012. Evaluation of SCO1 deletion on Saccharomyces cerevisiae metabolism through a proteomic approach. Proteomics 12: 1767-1780. https://doi.org/10.1002/pmic.201100285
  28. Wang Z, Wang Y, Hegg EL. 2009. Regulation of the heme A biosynthetic pathway: differential regulation of heme A synthase and heme O synthase in Saccharomyces cerevisiae. J. Biol. Chem. 284: 839-847. https://doi.org/10.1074/jbc.M804167200
  29. Barrientos A, Gouget K, Horn D, Soto IC, Fontanesi F. 2009. Suppression mechanisms of COX assembly defects in yeast and human: insights into the COX assembly process. Biochim. Biophys. Acta 1793: 97-107. https://doi.org/10.1016/j.bbamcr.2008.05.003
  30. Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schagger H. 1999. ATP synthase of yeast mitochondria: Isolation of subunit j and disruption of the ATP18 gene. J. Biol. Chem. 274: 36-40. https://doi.org/10.1074/jbc.274.1.36
  31. Salsaa M, Pereira B, Liu J, Yu W, Jadhav S, Huttemann M, et al. 2020. Valproate inhibits mitochondrial bioenergetics and increases glycolysis in Saccharomyces cerevisiae. Sci. Rep. 10: 11785.
  32. Pan Y. 2011. Mitochondria, reactive oxygen species, and chronological aging: a message from yeast. Exp. Gerontol. 46: 847-852. https://doi.org/10.1016/j.exger.2011.08.007
  33. Perrone GG, Tan S-X, Dawes IW. 2008. Reactive oxygen species and yeast apoptosis. Biochim. Biophys. Acta 1783: 1354-1368. https://doi.org/10.1016/j.bbamcr.2008.01.023
  34. Ramos-Alonso L, Romero AM, Martinez-Pastor MT, Puig S. 2020. Iron regulatory mechanisms in Saccharomyces cerevisiae. Front. Microbiol. 11: 582830.
  35. De Freitas JM, Liba A, Meneghini R, Valentine JS, Gralla EB. 2000. Yeast lacking Cu-Zn superoxide dismutase show altered iron homeostasis: role of oxidative stress in iron metabolism. J. Biol. Chem. 275: 11645-11649. https://doi.org/10.1074/jbc.275.16.11645
  36. De Freitas J, Wintz H, Hyoun Kim J, Poynton H, Fox T, Vulpe C. 2003. Yeast, a model organism for iron and copper metabolism studies. Biometals 16: 185-197. https://doi.org/10.1023/A:1020771000746
  37. Stearman R, Yuan DS, Yamaguchi-Iwai Y, Klausner RD, Dancis A. 1996. A permease-oxidase complex involved in high-affinity iron uptake in yeast. Science 271: 1552-1557. https://doi.org/10.1126/science.271.5255.1552
  38. Zara G, Nardi T. 2021. Yeast metabolism and its exploitation in emerging winemaking trends: from sulfite tolerance to sulfite reduction. Fermentation 7: 57.
  39. Sa-Correia I, dos Santos SC, Teixeira MC, Cabrito TR, Mira NP. 2009. Drug: H+  antiporters in chemical stress response in yeast. Trends Microbiol. 17: 22-31. https://doi.org/10.1016/j.tim.2008.09.007
  40. Felder T, Bogengruber E, Tenreiro S, Ellinger A, Sa-Correia I, Briza P. 2002. Dtr1p, a multidrug resistance transporter of the major facilitator superfamily, plays an essential role in spore wall maturation in Saccharomyces cerevisiae. Eukaryot. Cell 1: 799-810. https://doi.org/10.1128/EC.1.5.799-810.2002
  41. Jungwirth H, Kuchler K. 2006. Yeast ABC transporters - a tale of sex, stress, drugs and aging. FEBS Lett. 580: 1131-1138. https://doi.org/10.1016/j.febslet.2005.12.050