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Reprogramming methanol utilization pathways to convert Saccharomyces cerevisiae to a synthetic methylotroph

Nature Catalysis volume 6, pages 435–450 (2023)Cite this article

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Abstract

Methanol, an organic one-carbon (C1) compound, represents an attractive alternative carbon source for microbial fermentation. Despite considerable advancements in methanol utilization by prokaryotes such as Escherichia coli, engineering eukaryotic model organisms such as Saccharomyces cerevisiae into synthetic methylotrophs remains challenging. Here, an engineered module circuit strategy combined with adaptive laboratory evolution was applied to engineer S. cerevisiae to use methanol as the sole carbon source. We revealed that the evolved glyoxylate-based serine pathway plays an important role in methanol-dependent growth by promoting formaldehyde assimilation. Further, we determined that the isoprenoid biosynthetic pathway was upregulated, resulting in an increased concentration of squalene and ergosterol in our evolved strain. These changes could potentially alleviate cell membrane damage in the presence of methanol. This work sets the stage for expanding the potential of exploiting S. cerevisiae as a potential organic one-carbon platform for biochemical or biofuel production.

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Fig. 1: Evaluation of combinatorial methanol metabolic pathways in S. cerevisiae.
Fig. 2: Peroxisomal compartmentalization of the Aox-XuMP pathway.
Fig. 3: Modularization-strategy-enhanced methanol utilization in methylotrophic S. cerevisiae.
Fig. 4: Adaptive laboratory evolution strategy to promote methanol and/or xylose utilization in engineering yeast.
Fig. 5: Evaluation of phenotypes and gene targets in the parental and evolved strains.
Fig. 6: gSerine pathway improved formaldehyde assimilation in methanol-containing medium.
Fig. 7: The Shm2-dependent gSerine pathway promoted methanol assimilation.
Fig. 8: Production of flaviolin by introducing type-III PKS RppA.

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Data availability

The RNA-seq raw data can be downloaded from the Genome Expression Omnibus website (https://www.ncbi.nlm.nih.gov/geo/) with series no. GSE173802. The accession no. for the genome sequence data of evolved strains reported in this paper is [NCBI.SRA]:[PRJNA728351]. All other data that support the findings in this study are available on request. All plasmids and strains used in this study can be obtained from Z.B. under a material transfer agreement. Source Data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant no. 21908077 to C.Z.), the National Key R&D Program of China (grant no. 2021YFC2100203 to Y.Y.), the national first-class discipline program of Light Industry Technology and Engineering (grant no. LITE2018-24), the 111 Project (111-2-06) and the DOE Joint BioEnergy Institute (https://www.jbei.org), and funded by the US Department of Energy, Office of Science, Office of Biological, and Environmental Research through contract no DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.

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Author notes

  1. These authors contributed equally: Chunjun Zhan, Xiaowei Li.

Authors and Affiliations

  1. National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi, China

    Chunjun Zhan, Yankun Yang, Yang Sun, Shijie Wang & Zhonghu Bai

  2. Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

    Chunjun Zhan, Xiaowei Li, Yanyan Wang, Guokun Wang, Jens Nielsen & Yun Chen

  3. Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA

    Chunjun Zhan, Guangxu Lan, Edward E. K. Baidoo, Yuzhong Liu & Jay D. Keasling

  4. Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, China

    Chunjun Zhan, Yankun Yang, Yang Sun, Shijie Wang & Zhonghu Bai

  5. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China

    Chunjun Zhan, Yankun Yang, Yang Sun, Shijie Wang & Zhonghu Bai

  6. Joint BioEnergy Institute, Emeryville, CA, USA

    Chunjun Zhan, Guangxu Lan, Edward E. K. Baidoo, Yuzhong Liu & Jay D. Keasling

  7. Institute of Microbial Engineering, Laboratory of Bioresource and Applied Microbiology, School of Life Sciences, Henan University, Kaifeng, China

    Yang Sun

  8. BioInnovation Institute, Copenhagen, Denmark

    Jens Nielsen

  9. Departments of Chemical and Biomolecular Engineering and of Bioengineering, University of California, Berkeley, Berkeley, CA, USA

    Jay D. Keasling

  10. Center for Biosustainability, Danish Technical University, Lyngby, Denmark

    Jay D. Keasling

  11. Center for Synthetic Biochemistry, Shenzhen Institutes for Advanced Technologies, Shenzhen, China

    Jay D. Keasling

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  1. Chunjun Zhan
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Contributions

C.Z. conceived the study. C.Z. and X.L. designed the study, performed the experiments and data processing, and analysed the data. Z.B., J.D.K., J.N. and Y.C. supervised the project. E.E.K.B. performed the 13C-labelled experiments and analysed the data. S.W. conducted scanning electron microscopy experiment. Y.W. was involved in constructing some of the plasmids needed for the study. C.Z. and X.L. wrote the manuscript. J.D.K., G.L. and Y.L. revised the manuscript. Y.Y., Y.S. and G.W. contributed to the review, editing and final approval of the manuscript. They provided valuable insights and suggestions to improve the quality of the research paper.

Corresponding authors

Correspondence to Jens Nielsen, Jay D. Keasling, Yun Chen or Zhonghu Bai.

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Competing interests

J.D.K. has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. None of these companies work in the field explored in this study. All other authors declare no competing interests.

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Nature Catalysis thanks Elke Nevoigt and the other, anonymous, reviewer for their contribution to the peer review of this work.

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Zhan, C., Li, X., Lan, G. et al. Reprogramming methanol utilization pathways to convert Saccharomyces cerevisiae to a synthetic methylotroph. Nat Catal 6, 435–450 (2023). https://doi.org/10.1038/s41929-023-00957-w

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