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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
BIOspektrum Open Access 09 June 2022
Nature Communications Open Access 16 March 2022
BIOspektrum Open Access 13 February 2022
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Li, F., Vijayasankaran, N., Shen, A., Kiss, R. & Amanullah, A. Cell culture processes for monoclonal antibody production. MAbs 2, 466–479 (2010).
Meadows, A. L. et al. Rewriting yeast central carbon metabolism for industrial isoprenoid production. Nature 537, 694–697 (2016).
Sauer, M. Industrial production of acetone and butanol by fermentation—100 years later. FEMS Microbiol. Lett. 363, 1–4 (2016).
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).
Yeates, T. O., Thompson, M. C. & Bobik, T. A. The protein shells of bacterial microcompartment organelles. Curr. Opin. Struct. Biol. 21, 223–231 (2011).
Hammer, S. K. & Avalos, J. L. Harnessing yeast organelles for metabolic engineering. Nat. Chem. Biol. 13, 823–832 (2017).
Hecht, K. A., O’Donnell, A. F. & Brodsky, J. L. The proteolytic landscape of the yeast vacuole. Cell Logist. 4, e28023 (2014).
Haanstra, J. R. et al. Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes. Proc. Natl Acad. Sci. USA 105, 17718–17723 (2008).
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).
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).
DeLoache, W. C., Russ, Z. N. & Dueber, J. E. Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways. Nat. Commun. 7, 11152 (2016).
Subramani, S. Targeting of proteins into the peroxisomal matrix. J. Membr. Biol. 125, 99–106 (1992).
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).
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).
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).
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).
DeLoache, W. C. et al. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat. Chem. Biol. 11, 465–471 (2015).
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).
Nishihachijo, M. et al. Asymmetric synthesis of tetrahydroisoquinolines by enzymatic Pictet–Spengler reaction. Biosci. Biotechnol. Biochem. 78, 701–707 (2014).
Li, Y. et al. Complete biosynthesis of noscapine and halogenated alkaloids in yeast. Proc. Natl Acad. Sci. USA 115, E3922–E3931 (2018).
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).
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).
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).
Ruff, B. M., Bräse, S. & O’Connor, S. E. Biocatalytic production of tetrahydroisoquinolines. Tetrahedron Lett. 53, 1071–1074 (2012).
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).
Ramirez-Gaona, M. et al. YMDB 2.0: a significantly expanded version of the yeast metabolome database. Nucleic Acids Res. 45, D440–D445 (2017).
Walton, P. A., Hill, P. E. & Subramani, S. Import of stably folded proteins into peroxisomes. Mol. Biol. Cell 6, 675–683 (1995).
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).
Galanie, S., Thodey, K., Trenchard, I. J., Interrante, M. F. & Smolke, C. D. Complete biosynthesis of opioids in yeast. Science 349, 1095–1100 (2015).
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).
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).
Yofe, I. et al. Pex35 is a regulator of peroxisome abundance. J. Cell. Sci. 130, 791–804 (2017).
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).
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).
Ratnakumar, S. & Young, E. T. Snf1 dependence of peroxisomal gene expression is mediated by Adr1. J. Biol. Chem. 285, 10703–10714 (2010).
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).
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).
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).
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).
Outeiro, T. F. & Lindquist, S. Yeast cells provide insight into α-synuclein biology and pathobiology. Science 302, 1772–1775 (2003).
Ju, S. et al. A yeast model of FUS/TLS-dependent cytotoxicity. PLoS Biol. 9, e1001052 (2011).
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).
Ajikumar, P. K. et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330, 70–74 (2010).
Brochado, A. R. et al. Improved vanillin production in baker’s yeast through in silico design. Microb. Cell Fact. 9, 1–15 (2010).
Li, S. C. & Kane, P. M. The yeast lysosome-like vacuole: endpoint and crossroads. Biochim. Biophys. Acta Mol. Cell Res. 1793, 650–663 (2009).
Dragosits, M. & Mattanovich, D. Adaptive laboratory evolution—principles and applications for biotechnology. Microb. Cell Fact. 12, 1–17 (2013).
Bentley, G. J. et al. Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440. Metab. Eng. 59, 64–75 (2020).
Meijer, W. H. et al. Peroxisomes are required for efficient penicillin biosynthesis in Penicillium chrysogenum. Appl. Environ. Microbiol. 76, 5702–5709 (2010).
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).
Weng, H., Endo, K., Li, J., Kito, N. & Iwai, N. Induction of peroxisomes by butyrate-producing probiotics. PLoS ONE 10, 1–11 (2015).
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.
J.E.D. declares competing financial interests in the form of a pending patent application, US application no. 62/094,877.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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.
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.
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.
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.
About this article
Cite this article
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
This article is cited by
Nature Communications (2022)
Nature Reviews Microbiology (2022)
Systems Microbiology and Biomanufacturing (2022)
Current advances in the biotechnological synthesis of betulinic acid: new findings and practical applications
Systems Microbiology and Biomanufacturing (2022)