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Peroxisome compartmentalization of a toxic enzyme improves alkaloid production

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.

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.

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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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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.

Corresponding author

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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