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Biosynthesis of catharanthine in engineered Pichia pastoris

Nature Synthesis volume 2, pages 231–242 (2023)Cite this article

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Abstract

Catharanthine can be coupled with vindoline to synthesize vinblastine and vincristine, which have been used clinically as potent anticancer drugs. However, the structural complexity and low abundance in nature hamper bulk chemical synthesis and plant extraction, leading to a limited supply and a high cost of this plant natural product. Here, we engineer the methylotrophic yeast Pichia pastoris for complete biosynthesis of catharanthine from simple carbon sources. Through the selection of stable integration sites, screening of biosynthetic pathway enzymes with higher activity and/or specificity, amplification of flux-limiting enzyme encoding genes, rewiring of cellular metabolism and process optimization, we achieve de novo biosynthesis of catharanthine with a titre as high as 2.57 mg l−1, which also represents the most complicated molecule heterologously synthesized in a non-model microorganism. Our study establishes P. pastoris as a cell factory for producing plant natural products with complex biosynthetic pathways.

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Fig. 1: Pathway diagram of catharanthine biosynthesis.
Fig. 2: Genome-wide profiling and characterization of stable integration sites in P. pastoris.
Fig. 3: Reconstitution and optimization of CAN module.
Fig. 4: Reconstitution and optimization of the STR module.
Fig. 5: Reconstitution and optimization of NPT module for de novo biosynthesis of catharanthine.
Fig. 6: Optimization and fed-fed fermentation of catharanthine-producing strains.

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Data availability

The data involved in the research are included in the manuscript, supplementary materials and supplementary data. NGS data have been deposited into NCBI with an accession number of PRJNA859235.

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Acknowledgements

This work was supported by National Key Research and Development Program of China (grant nos. 2018YFA0901800 and 2021YFC2103200 to J.L.), Natural Science Foundation of Zhejiang Province (grant no. LR20B060003 to J.L.), Natural Science Foundation of China (grant no. 22278361 to J.L.) and Fundamental Research Funds for the Central Universities (grant nos. 226-2022-00214 and 226-2022-00055 to J.L.). We thank S. E. O’Connor from Max Planck Institute of Chemical Ecology for kindly sharing Strain4 and we thank Y. Yuan and W. Xiao from Tianjin University for generously providing pJGZ200. We also would like to thank iBioFoundry and Core Facility at the Institute for Intelligent Bio/Chem Manufacturing, ZJU-Hangzhou Global Scientific and Technological Innovation Center for analytical support.

Author information

Authors and Affiliations

  1. Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China

    Jucan Gao, Yimeng Zuo, Junhao Xu, Cuifang Ye, Linjuan Feng, Leijie Jiang, Tengfei Liu, Di Gao, Lei Huang, Zhinan Xu & Jiazhang Lian

  2. ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, China

    Jucan Gao, Yimeng Zuo, Feng Xiao, Yiling Wang, Dongfang Li, Junhao Xu, Cuifang Ye, Linjuan Feng, Bin Ma, Lei Huang & Jiazhang Lian

  3. Zhejiang Key Laboratory of Smart Biomaterials, Zhejiang University, Hangzhou, China

    Jucan Gao, Yimeng Zuo & Jiazhang Lian

  4. Institute of Soil and Water Resources and Environmental Science, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, China

    Yiling Wang & Bin Ma

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  1. Jucan Gao
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Contributions

J.L. conceptualized and supervised the study. J.G. performed experiments, analysed data and drafted the manuscript. J.G., Y.Z., J.X., C.Y., L.F. and L.J. selected and characterized the integration sites. J.G., T.L. and D.G. designed and constructed the catharanthine biosynthetic pathway. J.G. and F.X. performed the bioreactor fermentation. J.G. and D.L. performed the LC–MS analysis. J.G. and Y.W. performed the whole-genome sequencing and analysis. J.L., B.M., L.H. and Z.X. revised the manuscript. All authors approved the manuscript.

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Correspondence to Jiazhang Lian.

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Extended data

Extended Data Fig. 1 Evaluation of the stability of heterologous genes integrated into the selected intergenic regions.

Evaluation of the stability of heterologous genes integrated into the selected intergenic regions. A single yeast colony containing the mCherry expression cassette was cultured in YPD medium for ~30 generations, and then streak out into YPD agar plates. The stability of evaluated by the percentage of mCherry fluorescent (pink) colonies. IntE4, IntE5, IntE12, IntE15, IntE16, IntE20, IntE24, IntE30, and IntE31 were shown as representative examples.

Extended Data Fig. 2 Multiplex genome integration efficiency.

Multiplex genome integration efficiency. mVenus, mCherry, and HygB were chosen as the reporter genes to test the efficiency of editing several loci simultaneously. For two-loci integration, mCherry (TEF1p-mCherry-PRX5t) and mVenus (GAPp-mVenus-AOX1t) were used as reporters and the genome integration efficiency was calculated as the percentage of cells showing both mCherry and mVenus fluorescence. For three-loci integration, mCherry (TEF1p-mCherry-PRX5t), mVenus (GAPp-mVenus-AOX1t), and HygB (TEF1p-HygB-PRX5t) were used as reporters and the genome integration efficiency was calculated as the percentage of colonies showing mCherry and mVenus fluorescence as well as hygromycin resistance. The results represent the mean ± s.d. of biological triplicates (n = 3).

Source data

Extended Data Fig. 3 LC-MS analysis of the production of catharanthine.

LC-MS analysis of the production of catharanthine. MRM chromatograms and spectra (m/z = 337.1 > 144.0 and m/z = 337.1 > 93.1) for CAN4A sample (a) and catharanthine standard (b). (c) Proposed ion structures of the two MS/MS fragments of catharanthine with an m/z of 144.0 and 93.1, respectively.

Extended Data Fig. 4 Confocal microscopy analysis of the localization of PAS variants expressed in P. pastoris.

Confocal microscopy analysis of the localization of PAS variants expressed in P. pastoris. The vacuole was targeted by eGFP (PEP4sp-eGFP), while PAS variants were fused with mCherry and their localization was analyzed by comparing the green (500/540 nm) and red (570/670 nm) fluorescence. Yeast cells were cultured in YPM medium until mid-log phase and subject to confocal microscopy analysis (OLYMPUS IX83-FV3000). The results represent the mean ± s.d. of biological triplicates (n = 3).

Source data

Extended Data Fig. 5 Growth curves of P. pastoris strains, wild-type strain (CAN1) and catharanthine-producing strains (CAN7, CAN11, and CAN17).

Growth curves of P. pastoris strains, wild-type strain (CAN1) and catharanthine-producing strains (CAN7, CAN11, and CAN17). A single colony of P. pastoris was pre-cultured in YPAD medium until saturation and then inoculated into YPAM medium with 2% inoculum. OD600 was measured every 4 h. The results represent the mean ± s.d. of biological triplicates (n = 3).

Source data

Extended Data Fig. 6 Stability evaluation of P. pastoris CAN17.

Stability evaluation of P. pastoris CAN17. Strain CAN17 was cultured in non-selective YPAM medium for ~72 generations, and then spread into YPAD plates. 10 colonies were randomly picked and evaluated for fermentative production of catharanthine (with methanol as the sole carbon source). The production of catharanthine in all clones as well as their consistency in catharanthine titer indicated the stability of the catharanthine-producing P. pastoris strain.

Source data

Extended Data Fig. 7 Fed-batch fermentation profiles of methanol concentration, dissolved oxygen (DO), and pH after induction.

Fed-batch fermentation profiles of methanol concentration, dissolved oxygen (DO), and pH after induction. Methanol (a) or methanol/mannitol (b) was fed into the bioreactor as carbon sources for fermentative production of catharanthine. After induction for 30 h, 100 mL 5-fold concentrated YPA medium was supplemented to the bioreactors as additional nitrogen source.

Source data

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–5 and Tables 1–9.

Reporting Summary

Supplementary Data 1

DNA coding sequences of the catharanthine biosynthetic pathway genes.

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Gao, J., Zuo, Y., Xiao, F. et al. Biosynthesis of catharanthine in engineered Pichia pastoris. Nat. Synth 2, 231–242 (2023). https://doi.org/10.1038/s44160-022-00205-2

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