Nothing Special   »   [go: up one dir, main page]
Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Engineering a xylose fermenting yeast for lignocellulosic ethanol production

Nature Chemical Biology (2024)Cite this article

Subjects

Abstract

Lignocellulosic ethanol is produced by yeast fermentation of lignocellulosic hydrolysates generated by chemical pretreatment and enzymatic hydrolysis of plant cell walls. The conversion of xylose into ethanol in hydrolysates containing microbial inhibitors is a major bottleneck in biofuel production. We identified sodium salts as the primary yeast inhibitors, and evolved a Saccharomyces cerevisiae strain overexpressing xylose catabolism genes in xylose or glucose-mixed medium containing sodium salts. The fully evolved yeast strain can efficiently convert xylose in the hydrolysates to ethanol on an industrial scale. We elucidated that the amplification of xylA, XKS1 and pentose phosphate pathway-related genes TAL1, RPE1, TKL1, RKI1, along with mutations in NFS1, TRK1, SSK1, PUF2 and IRA1, are responsible and sufficient for the effective xylose utilization in corn stover hydrolysates containing high sodium salts. Our evolved or reverse-engineered yeast strains enable industrial-scale production of lignocellulosic ethanol and the genetic foundation we uncovered can also facilitate transfer of the phenotype to yeast cell factories producing chemicals beyond ethanol.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Component analysis and fermentations confirm that sodium salts are key inhibitors.
Fig. 2: Industrial bioethanol yeast eGXI-1.0 was generated by metabolic rewiring and adaptive evolution.
Fig. 3: Reverse engineering to reproduce the fermentation phenotype of eGXI-1.0.
Fig. 4: Mechanism analysis of TRK1 and SSK1 mutations.
Fig. 5: Mechanism analysis of PUF2R243C by combining genetic verification and omics.

Similar content being viewed by others

Data availability

The raw data of whole-genome sequencing and RNA sequencing in this work have been deposited in the NCBI under the project accession number PRJNA827654. Haploid genome assembly and annotation have been deposited under the NCBI project accession number PRJNA556784. The proteomics sequencing datasets have been submitted to the iProX database with the project ID IPX0004391000. Source data are provided with this paper.

References

  1. Robertson, G. P. et al. Cellulosic biofuel contributions to a sustainable energy future: choices and outcomes. Science 356, eaal2324 (2017).

    Article  PubMed  Google Scholar 

  2. Lynd, L. R. The grand challenge of cellulosic biofuels. Nat. Biotechnol. 35, 912–915 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Wang, L., Bilal, M., Tan, C., Jiang, X. & Li, F. Industrialization progress of lignocellulosic ethanol. Syst. Microbiol. Biomanufacturing 2, 246–258 (2021).

    Article  Google Scholar 

  4. Service, R. F. Renewable energy. Cellulosic ethanol at last? Science 345, 1111 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Huang, H. et al. Identification of crucial yeast inhibitors in bio-ethanol and improvement of fermentation at high pH and high total solids. Bioresour. Technol. 102, 7486–7493 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Lam, F. H. et al. Engineered yeast tolerance enables efficient production from toxified lignocellulosic feedstocks. Sci. Adv. 7, eabf7613 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Humbird, D. et al. Dilute-Acid Pretreatment and Enzymatic Hydrolysis of Corn Stover (National Renewable Energy Laboratory, 2011).

  8. Cunha, J. T., Romani, A., Costa, C. E., Sa-Correia, I. & Domingues, L. Molecular and physiological basis of Saccharomyces cerevisiae tolerance to adverse lignocellulose-based process conditions. Appl. Microbiol. Biotechnol. 103, 159–175 (2019).

    Article  CAS  PubMed  Google Scholar 

  9. Vanmarcke, G., Demeke, M. M., Foulquie-Moreno, M. R. & Thevelein, J. M. Identification of the major fermentation inhibitors of recombinant 2G yeasts in diverse lignocellulose hydrolysates. Biotechnol. Biofuels 14, 92 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kim, S. R., Park, Y. C., Jin, Y. S. & Seo, J. H. Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism. Biotechnol. Adv. 31, 851–861 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Diao, L. et al. Construction of fast xylose-fermenting yeast based on industrial ethanol-producing diploid Saccharomyces cerevisiae by rational design and adaptive evolution. BMC Biotechnol. 13, 110 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Runquist, D., Hahn-Hagerdal, B. & Radstrom, P. Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae. Biotechnol. Biofuels 3, 5 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Hou, J. et al. Characterization and evolution of xylose isomerase screened from the bovine rumen metagenome in Saccharomyces cerevisiae. J. Biosci. Bioeng. 121, 160–165 (2016).

    Article  CAS  PubMed  Google Scholar 

  14. Kuyper, M. et al. Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. FEMS Yeast Res. 5, 925–934 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Sato, T. K. et al. Directed evolution reveals unexpected epistatic interactions that alter metabolic regulation and enable anaerobic xylose use by Saccharomyces cerevisiae. PLoS Genet. 12, e1006372 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Zhou, H., Cheng, J. S., Wang, B. L., Fink, G. R. & Stephanopoulos, G. Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae. Metab. Eng. 14, 611–622 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. Frazzon, J. & Dean, D. R. Formation of iron-sulfur clusters in bacteria: an emerging field in bioinorganic chemistry. Curr. Opin. Chem. Biol. 7, 166–173 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Garland, S. A., Hoff, K., Vickery, L. E. & Culotta, V. C. Saccharomyces cerevisiae ISU1 and ISU2: members of a well-conserved gene family for iron-sulfur cluster assembly. J. Mol. Biol. 294, 897–907 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Rouault, T. A. & Tong, W. H. Iron-sulphur cluster biogenesis and mitochondrial iron homeostasis. Nat. Rev. Mol. Cell Biol. 6, 345–351 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Dos Santos, L. V. et al. Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains. Sci. Rep. 6, 38676 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Palermo, G. C. L., Coutoune, N., Bueno, J. G. R., Maciel, L. F. & Dos Santos, L. V. Exploring metal ion metabolisms to improve xylose fermentation in Saccharomyces cerevisiae. Microb. Biotechnol. 14, 2101–2115 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Li, L., Chen, O. S., McVey Ward, D. & Kaplan, J. CCC1 is a transporter that mediates vacuolar iron storage in yeast. J. Biol. Chem. 276, 29515–29519 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Haro, R. & Rodriguez-Navarro, A. Molecular analysis of the mechanism of potassium uptake through the TRK1 transporter of Saccharomyces cerevisiae. Biochim. Biophys. Acta 1564, 114–122 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Haro, R. & Rodrı́guez-Navarro, A. Functional analysis of the M2D helix of the TRK1 potassium transporter of Saccharomyces cerevisiae. Biochim. Biophys. Acta Biomembranes 1613, 1–6 (2003).

    Article  CAS  Google Scholar 

  25. Xu, X., Williams, T. C., Divne, C., Pretorius, I. S. & Paulsen, I. T. Evolutionary engineering in Saccharomyces cerevisiae reveals a TRK1-dependent potassium influx mechanism for propionic acid tolerance. Biotechnol. Biofuels 12, 97 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Hohmann, S. Control of high osmolarity signalling in the yeast Saccharomyces cerevisiae. FEBS Lett. 583, 4025–4029 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Posas, F. & Saito, H. Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator. EMBO J. 17, 1385–1394 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Horie, T., Tatebayashi, K., Yamada, R. & Saito, H. Phosphorylated Ssk1 prevents unphosphorylated Ssk1 from activating the Ssk2 mitogen-activated protein kinase kinase kinase in the yeast high-osmolarity glycerol osmoregulatory pathway. Mol. Cell. Biol. 28, 5172–5183 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hsiao, W. Y., Wang, Y. T. & Wang, S. W. Fission yeast Puf2, a pumilio and FBF family RNA-binding protein, links stress granules to processing bodies. Mol. Cell. Biol. 40, e00589-19 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Porter, D. F., Koh, Y. Y., VanVeller, B., Raines, R. T. & Wickens, M. Target selection by natural and redesigned PUF proteins. Proc. Natl Acad. Sci. USA 112, 15868–15873 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gerber, A. P., Herschlag, D. & Brown, P. O. Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS Biol. 2, E79 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Montllor-Albalate, C. et al. Extra-mitochondrial Cu/Zn superoxide dismutase (Sod1) is dispensable for protection against oxidative stress but mediates peroxide signaling in Saccharomyces cerevisiae. Redox Biol. 21, 101064 (2019).

    Article  CAS  PubMed  Google Scholar 

  33. Kanehisa, M. & Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27–30 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yenush, L. Potassium and sodium transport in yeast. Adv. Exp. Med Biol. 892, 187–228 (2016).

    Article  CAS  PubMed  Google Scholar 

  35. Wang, X., Yang, J., Yang, S. & Jiang, Y. Unraveling the genetic basis of fast l-arabinose consumption on top of recombinant xylose-fermenting Saccharomyces cerevisiae. Biotechnol. Bioeng. 116, 283–293 (2019).

    Article  CAS  PubMed  Google Scholar 

  36. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25, 402–408 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Lippert, C. et al. FaST linear mixed models for genome-wide association studies. Nat. Methods 8, 833–835 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Panja, C., Setty, R. K., Vaidyanathan, G. & Ghosh, S. Label-Free proteomic analysis of Flavohemoglobin deleted strain of Saccharomyces cerevisiae. Int. J. Proteom. 2016, 8302423 (2016).

    Article  Google Scholar 

  39. Boonekamp, F. J. et al. Design and experimental evaluation of a minimal, innocuous watermarking strategy to distinguish near-identical DNA and RNA sequences. ACS Synth. Biol. 9, 1361–1375 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Mi, H., Muruganujan, A., Ebert, D., Huang, X. & Thomas, P. D. PANTHER version 14: more genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools. Nucleic Acids Res. 47, D419–D426 (2019).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank H.-Y. Ren, W. Li and H. Yang from Novozymes for corn stover hydrolysates preparation, phenotype confirmation and research suggestions; J. Chen, Y.-M. Liu, L.-Y. Diao, X. Wang, J.-L. Liu, Y.-Y. Kong, J. Zhu, L.-Y. Wang and S. Xu from S.Y.’s laboratory for assistance with the experiments; X. Li and X.-T. Li from the Chinese Academy of Science for GWAS analyses; and Z.-J. Li from Angel Yeast, Z.-B. Shen from Tianguan and F.-Y. Bai and F.-L. Li from the Chinese Academy of Science for helpful suggestions on yeast chassis selection. This work was supported by National Natural Science Foundation of China (grant nos. 21825804 and 31921006), National Key R&D Program of China (grant nos. 2021YFC2103703, 2023YFF1000202) to S.Y.

Author information

Author notes

  1. These authors contributed equally: Yi-Wen Zhang, Jun-Jie Yang.

Authors and Affiliations

  1. Key Laboratory of Synthetic Biology, Center for Excellence of Molecular Plant Science, Chinese Academy of Sciences, Shanghai, China

    Yi-Wen Zhang, Jun-Jie Yang & Sheng Yang

  2. College of Life Science, University of Chinese Academy of Sciences, Beijing, China

    Yi-Wen Zhang

  3. Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China

    Feng-Hui Qian, Yu Jiang & Sheng Yang

  4. Novozymes North America Inc., Durham, NC, USA

    Kate Brandon Sutton

  5. Novozymes A/S, Bagsvaerd, Denmark

    Carsten Hjort

  6. Novozymes China Investment Co. Ltd, Beijing, China

    Wen-Ping Wu

Authors
  1. Yi-Wen Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jun-Jie Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Feng-Hui Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kate Brandon Sutton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Carsten Hjort
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wen-Ping Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yu Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sheng Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.-P.W. and S.Y. conceived the project. S.Y., W.-P.W., C.H., J.-J.Y. and Y.-W.Z. designed the experiments. F.-H.Q. and Y.-W.Z. performed most of the experiments. S.Y., Y.J., J.-J.Y. and Y.-W.Z. analyzed the data. C.H. and K.B.S. helped with experiments. J.-J.Y. performed the bioinformatic analyses in this study. S.Y., Y.-W.Z., J.-J.Y. and Y.J. discussed the data and wrote the paper.

Corresponding author

Correspondence to Sheng Yang.

Ethics declarations

Competing interests

This work was partially funded by Novozymes. Patent applications have been filed by the Novozymes, Shanghai Research and Development Center of Industrial Biotechnology (CIBT) and Center for Excellence of Molecular Plant Science (CEMPS) based on this work. The pending patent application (application number WO2016045569) covers the method for producing lignocellulosic ethanol using evolved yeasts in this study. The patent applicant is Novozymes and K.B.S., Y.J., S.Y. and F.-H.Q. are listed as inventors. S.Y., J.-J.Y. and Y.J. are inventors of a granted patent related to the NFS1 variant for improving xylose use in S. cerevisiae (patent number CN202010475833.9). The patent applicant is CEMPS. The pending patent application (application number CN2023074360) covers the other gene targets for improving S. cerevisiae’s performance in lignocellulosic hydrolysates in this study. The patent applicant is CEMPS and CIBT and S.Y., Y.-W.Z., J.-J.Y., F.-H.Q. and Y.J. are listed as inventors. S.Y. has contributed to the scientific advisory board at Angel Yeast. Some authors receive royalty payment from xylose yeast customers. The other authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks Jens Nielsen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–13.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–7.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, YW., Yang, JJ., Qian, FH. et al. Engineering a xylose fermenting yeast for lignocellulosic ethanol production. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01771-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41589-024-01771-6

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research