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Peroxisome compartmentalization of a toxic enzyme improves alkaloid production

Nature Chemical Biology volume 17, pages 96–103 (2021)Cite this article

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Abstract

Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with cytosolic factors. We found that cytosolic expression of norcoclaurine synthase (NCS), the enzyme that catalyzes the first committed reaction in benzylisoquinoline alkaloid biosynthesis, is toxic in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer. This strategy is achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and products across the peroxisome membrane. We demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing benzylisoquinoline alkaloid titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.

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Fig. 1: Cytosolic expression of tNCS is toxic to S. cerevisiae.
Fig. 2: Compartmentalizing tNCS in the peroxisome alleviates cytotoxicity and increases product titer.
Fig. 3: Peroxisomal targeting of toxic tNCS at a very high expression level improves growth and (S)-norcoclaurine titer, but compartmentalization is incomplete.
Fig. 4: Genetic induction of peroxisome proliferation can address incomplete compartmentalization at very high expression levels of tNCS.

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Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Plasmids are available through Addgene under deposit number 78460.

References

  1. Li, F., Vijayasankaran, N., Shen, A., Kiss, R. & Amanullah, A. Cell culture processes for monoclonal antibody production. MAbs 2, 466–479 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Meadows, A. L. et al. Rewriting yeast central carbon metabolism for industrial isoprenoid production. Nature 537, 694–697 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Sauer, M. Industrial production of acetone and butanol by fermentation—100 years later. FEMS Microbiol. Lett. 363, 1–4 (2016).

    Article  CAS  Google Scholar 

  4. Eibl, R. et al. Plant cell culture technology in the cosmetics and food industries: current state and future trends. Appl. Microbiol. Biotechnol. 102, 8661–8675 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Yeates, T. O., Thompson, M. C. & Bobik, T. A. The protein shells of bacterial microcompartment organelles. Curr. Opin. Struct. Biol. 21, 223–231 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hammer, S. K. & Avalos, J. L. Harnessing yeast organelles for metabolic engineering. Nat. Chem. Biol. 13, 823–832 (2017).

    Article  CAS  PubMed  Google Scholar 

  7. Hecht, K. A., O’Donnell, A. F. & Brodsky, J. L. The proteolytic landscape of the yeast vacuole. Cell Logist. 4, e28023 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Haanstra, J. R. et al. Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes. Proc. Natl Acad. Sci. USA 105, 17718–17723 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hagel, J. M. & Facchini, P. J. Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol. 54, 647–672 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Lichman, B. R. et al. Dopamine-first mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile. FEBS J. 282, 1137–1151 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. DeLoache, W. C., Russ, Z. N. & Dueber, J. E. Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways. Nat. Commun. 7, 11152 (2016).

  12. Subramani, S. Targeting of proteins into the peroxisomal matrix. J. Membr. Biol. 125, 99–106 (1992).

    Article  CAS  PubMed  Google Scholar 

  13. Kohlwein, S. D., Veenhuis, M. & van der Klei, I. J. Lipid droplets and peroxisomes: key players in cellular lipid homeostasis or a matter of fat-store’em up or burn’em down. Genetics 193, 1–50 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Distel, B. & Kragt, A. Purification of yeast peroxisomes. in Methods in Molecular Biology, Vol. 313 (ed. Xiao, W.) https://doi.org/10.1385/1-59259-958-3:021 (Humana Press, 2006).

  15. Ehrenworth, A. M., Haines, M. A., Wong, A. & Peralta-Yahya, P. Quantifying the efficiency of Saccharomyces cerevisiae translocation tags. Biotechnol. Bioeng. 114, 2628–2636 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Antonenkov, V. D., Sormunen, R. T. & Hiltunen, J. K. The rat liver peroxisomal membrane forms a permeability barrier for cofactors but not for small metabolites in vitro. J. Cell. Sci. 117, 5633–5642 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. DeLoache, W. C. et al. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat. Chem. Biol. 11, 465–471 (2015).

    Article  CAS  PubMed  Google Scholar 

  18. Samanani, N., Liscombe, D. K. & Facchini, P. J. Molecular cloning and characterization of norcoclaurine synthase, an catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis. Plant J. 40, 302–314 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Nishihachijo, M. et al. Asymmetric synthesis of tetrahydroisoquinolines by enzymatic Pictet–Spengler reaction. Biosci. Biotechnol. Biochem. 78, 701–707 (2014).

    Article  CAS  PubMed  Google Scholar 

  20. Li, Y. et al. Complete biosynthesis of noscapine and halogenated alkaloids in yeast. Proc. Natl Acad. Sci. USA 115, E3922–E3931 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Karim, A. S., Curran, K. A. & Alper, H. S. Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications. FEMS Yeast Res. 13, 107–116 (2013).

    Article  CAS  PubMed  Google Scholar 

  22. Lee, M. E., Aswani, A., Han, A. S., Tomlin, C. J. & Dueber, J. E. Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay. Nucleic Acids Res. 41, 10668–10678 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth. Biol. 4, 975–986 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. Ruff, B. M., Bräse, S. & O’Connor, S. E. Biocatalytic production of tetrahydroisoquinolines. Tetrahedron Lett. 53, 1071–1074 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pesnot, T., Gershater, M. C., Ward, J. M. & Hailes, H. C. The catalytic potential of Coptis japonica NCS2 revealed—development and utilisation of a fluorescamine-based assay. Adv. Synth. Catal. 354, 2997–3008 (2012).

    Article  CAS  Google Scholar 

  26. Ramirez-Gaona, M. et al. YMDB 2.0: a significantly expanded version of the yeast metabolome database. Nucleic Acids Res. 45, D440–D445 (2017).

    Article  CAS  PubMed  Google Scholar 

  27. Walton, P. A., Hill, P. E. & Subramani, S. Import of stably folded proteins into peroxisomes. Mol. Biol. Cell 6, 675–683 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Halbach, A., Rucktächel, R., Rottensteiner, H. & Erdmann, R. The N-domain of Pex22p can functionally replace the Pex3p N-domain in targeting and peroxisome formation. J. Biol. Chem. 284, 3906–3916 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Galanie, S., Thodey, K., Trenchard, I. J., Interrante, M. F. & Smolke, C. D. Complete biosynthesis of opioids in yeast. Science 349, 1095–1100 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chan, K. M., Liu, Y. T., Ma, C. H., Jayaram, M. & Sau, S. The 2 micron plasmid of Saccharomyces cerevisiae: a miniaturized selfish genome with optimized functional competence. Plasmid 70, 2–17 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Kunau, W. H., Dommes, V. & Schulz, H. β-Oxidation of fatty acids in mitochondria, peroxisomes, and bacteria: a century of continued progress. Prog. Lipid Res. 34, 267–342 (1995).

    Article  CAS  PubMed  Google Scholar 

  32. Yofe, I. et al. Pex35 is a regulator of peroxisome abundance. J. Cell. Sci. 130, 791–804 (2017).

    CAS  PubMed  Google Scholar 

  33. Vizeacoumar, F., Torres-Guzman, J., Bouard, D., Aitchison, J. & Rachubinski, R. Pex30p, Pex31p, and Pex32p form a family of peroxisomal integral membrane proteins regulating peroxisome size and number in Saccharomyces cerevisiae. Mol. Biol. Cell 15, 665–677 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Grillitsch, K. et al. Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisited: lipidome meets proteome. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1811, 1165–1176 (2011).

    Article  CAS  Google Scholar 

  35. Ratnakumar, S. & Young, E. T. Snf1 dependence of peroxisomal gene expression is mediated by Adr1. J. Biol. Chem. 285, 10703–10714 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Baumgartner, U., Hamilton, B., Piskacek, M., Ruis, H. & Rottensteiner, H. Functional analysis of the Zn2Cys6 transcription factors Oaf1p and Pip2p. J. Biol. Chem. 274, 22208–22216 (1999).

    Article  CAS  PubMed  Google Scholar 

  37. Young, E. T., Dombek, K. M., Tachibana, C. & Ideker, T. Multiple pathways are co-regulated by the protein kinase Snf1 and the transcription factors Adr1 and Cat8. J. Biol. Chem. 278, 26146–26158 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Trzcinska-Danielewicz, J., Ishikawa, T., Miciałkiewicz, A. & Fronk, J. Yeast transcription factor Oaf1 forms homodimer and induces some oleate-responsive genes in absence of Pip2. Biochem. Biophys. Res. Commun. 374, 763–766 (2008).

    Article  CAS  PubMed  Google Scholar 

  39. Miroux, B. & Walker, J. E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins are high levels. J. Membr. Biol. 260, 289–298 (1996).

    CAS  Google Scholar 

  40. Outeiro, T. F. & Lindquist, S. Yeast cells provide insight into α-synuclein biology and pathobiology. Science 302, 1772–1775 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ju, S. et al. A yeast model of FUS/TLS-dependent cytotoxicity. PLoS Biol. 9, e1001052 (2011).

  42. Martin, V. J., Pitera, D. J., Withers, S. T., Newman, J. D. & Keasling, J. D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796–802 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Ajikumar, P. K. et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330, 70–74 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Brochado, A. R. et al. Improved vanillin production in baker’s yeast through in silico design. Microb. Cell Fact. 9, 1–15 (2010).

    Article  CAS  Google Scholar 

  45. Li, S. C. & Kane, P. M. The yeast lysosome-like vacuole: endpoint and crossroads. Biochim. Biophys. Acta Mol. Cell Res. 1793, 650–663 (2009).

    Article  CAS  Google Scholar 

  46. Dragosits, M. & Mattanovich, D. Adaptive laboratory evolution—principles and applications for biotechnology. Microb. Cell Fact. 12, 1–17 (2013).

    Article  Google Scholar 

  47. Bentley, G. J. et al. Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440. Metab. Eng. 59, 64–75 (2020).

    Article  CAS  PubMed  Google Scholar 

  48. Meijer, W. H. et al. Peroxisomes are required for efficient penicillin biosynthesis in Penicillium chrysogenum. Appl. Environ. Microbiol. 76, 5702–5709 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Veenhuis, M., Keizer, I. & Harder, W. Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media. Arch. Microbiol. 120, 167–175 (1979).

    Article  CAS  Google Scholar 

  50. Weng, H., Endo, K., Li, J., Kito, N. & Iwai, N. Induction of peroxisomes by butyrate-producing probiotics. PLoS ONE 10, 1–11 (2015).

    Google Scholar 

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Acknowledgements

We thank members of the Dueber laboratory for valuable assistance and feedback throughout this project, particularly Z. Russ for training and K. Siu for useful discussions; S. Dupuis for construction of the Pex22–RFP strain; and members of the Wenjun Zhang laboratory for assistance with LC–MS, particularly W. Skyrud and A. Del Rio Flores. This work is supported by NSF grant MCB 1818307 and by the Center for Cellular Construction, an NSF Science and Technology Center, under grant agreement DBI-1548297.

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Author notes

  1. Jennifer A. Samson

    Present address: Sound Agriculture, Emeryville, CA, USA

  2. Brian Choi

    Present address: Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA

  3. These authors contributed equally: Parbir S. Grewal, Jennifer A. Samson.

Authors and Affiliations

  1. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, USA

    Parbir S. Grewal & Brian Choi

  2. Department of Bioengineering, University of California, Berkeley, CA, USA

    Jennifer A. Samson, Jordan J. Baker & John E. Dueber

  3. UC Berkeley and UCSF Graduate Program in Bioengineering, University of California, Berkeley, CA, USA

    Jordan J. Baker

  4. Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    John E. Dueber

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Contributions

P.S.G., J.A.S., J.J.B. and J.E.D. designed the research. P.S.G. performed BIA production experiments, growth curves, microscopy, fluorescence measurements, metabolite feeding experiments and cloning. J.A.S. performed growth curves, microscopy, PDV experiments and cloning. J.J.B. performed microscopy, preliminary TF fluorescence measurements and cloning. B.C. assisted with cloning and preliminary experiments. P.S.G., J.A.S. and J.J.B. analyzed the results. J.E.D. supervised the research. P.S.G., J.A.S., J.J.B. and J.E.D. wrote the manuscript.

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Correspondence to John E. Dueber.

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J.E.D. declares competing financial interests in the form of a pending patent application, US application no. 62/094,877.

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Extended data

Extended Data Fig. 1 NCS homologues and truncated variants.

a, Truncation strategy for NCS homologues from Papaver somniferum (Ps), Thalictrum flavum (Tf), and Coptis japonica (Cj). Signal sequence (Sig seq) and Bet v1 domains were identified using the SMART domain analysis tool. b, (S)-Norcoclaurine production by each NCS variant at 72 hours. Gray bar shows our original NCS from Papaver somniferum17. Error bars represent mean ± s.d. of three biological replicates.

Extended Data Fig. 2 Dopamine and norcoclaurine are not toxic to S. cerevisiae at relevant concentrations.

All strains produce dopamine due to expression of CYP76AD5 and DODC. Norcoclaurine (NCCL) was supplemented in the growth media from 0.1 to 100 mg/L. Additional dopamine was supplemented at 25 mg/L to match the production level previously reported17. Only expression of tNCS causes slower growth. Error bars represent mean ± s.d. of seven (No supplement, black line) or eight (all others) biological replicates.

Extended Data Fig. 3 OD data from (S)-norcoclaurine shake flask fermentation experiment.

tNCS-VioE-ePTS1 and -dead_ePTS1 constructs were expressed using the strong pTDH3 promoter on a CEN6/ARS4 plasmid. Error bars represent mean ± s.d. of four biological replicates.

Extended Data Fig. 4 OD data from (S)-reticuline shake flask fermentation experiment.

tNCS-ePTS1 and -dead_ePTS1 constructs, along with 6OMT, CNMT, NMCH, and 4’OMT, were expressed on a CEN6/ARS4 plasmid. Error bars represent mean ± s.d. of four biological replicates.

Extended Data Fig. 5 OD data from 2μ (S)-norcoclaurine shake flask fermentation experiment.

tNCS-VioE-ePTS1 and -dead_ePTS1 constructs were expressed using the strong pTDH3 promoter on a 2µ plasmid. Error bars represent mean ± s.d. of four biological replicates.

Extended Data Fig. 6 Expression of engineered transcription factors activates promoters in the peroxisome proliferation network.

Promoters were used to drive expression of yellow fluorescent protein (YFP). Promoters pFDH1 and pACS1 are targets of the ADR1 transcription factor, pFAA2 is a target of the OAF1/PIP2 transcription factors, and pPOX1 is a target of both transcription factor types37,38. Promoters pTDH3, pTEF1, and pRPL18B are previously characterized constitutive promoters23. In addition to YFP, strains contained either a LEU2 marker only (No TF) or ADR1c, OAF1c, and PIP2c, plus a LEU2 marker (+TF). Error bars represent mean ± s.d. of eight biological replicates. a, Linear scale. b, Log scale.

Extended Data Fig. 7 Dynamics of peroxisomal protection of UbiY-YFP-ePTS1 from degradation.

a, Fluorescence. b, Fluorescence normalized by OD600. Yellow fluorescent protein (YFP) was expressed with an N-terminal degradation signal (UbiY) in the presence or absence of constitutively-active transcription factors ADR1c/OAF1c/PIP2c (+TF). All strains contain the peroxisomal targeting signal ePTS1 fused to YFP. Constructs transformed into a wildtype background strain will target (UbiY-)YFP-ePTS1 protein to the peroxisome (perox). Cytosolic expression of (UbiY-)YFP-ePTS1 (cyto) is achieved by use of a pex5Δ background strain, which is import-deficient due to knockout of the cytosolic receptor protein Pex5p. Error bars represent mean ± s.d. of twelve biological replicates.

Extended Data Fig. 8 Expression of engineered transcription factors increases protection of peroxisomally-targeted UbiY-YFP-ePTS1 (zoomed-out version of Fig. 4c).

Fluorescence microscopy showing cells without (left) or with (right) expression of constitutively-active transcription factors ADR1c, OAF1c, PIP2c (+TF). Both strains contain YFP fused to the UbiY degradation signal on the N-terminus and the peroxisomal targeting signal ePTS1 on the C-terminus. YFP channel brightness was increased identically across both images to allow better visualization of peroxisomes from the UbiY-YFP strain. Scale bars = 50 µm. Experiment was repeated four times with similar results.

Extended Data Fig. 9 (S)-Norcoclaurine titer, OD600, and OD-normalized titer from transcription factor overexpression experiment.

All strains contain the upstream BIA pathway plus 2µ pTDH3-tNCS-ePTS1. The +TF strain also contains ADR1c, OAF1c, and PIP2c. Error bars represent mean ± s.d. of eight biological replicates.

Extended Data Fig. 10 Expression of engineered transcription factors is linked to slower growth.

All strains contain the upstream BIA pathway. Strains without transcription factor overexpression (WT) contain a LEU2 marker. Strains with transcription factor overexpression (TF) contain ADR1c, OAF1c, PIP2c, and a LEU2 marker. Error bars represent mean ± s.d. of twelve biological replicates.

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Grewal, P.S., Samson, J.A., Baker, J.J. et al. Peroxisome compartmentalization of a toxic enzyme improves alkaloid production. Nat Chem Biol 17, 96–103 (2021). https://doi.org/10.1038/s41589-020-00668-4

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