Engineering biosynthesis of the anticancer alkaloid noscapine in yeast

Abstract

Noscapine is a potential anticancer drug isolated from the opium poppy Papaver somniferum, and genes encoding enzymes responsible for the synthesis of noscapine have been recently discovered to be clustered on the genome of P. somniferum. Here, we reconstitute the noscapine gene cluster in Saccharomyces cerevisiae to achieve the microbial production of noscapine and related pathway intermediates, complementing and extending previous in planta and in vitro investigations. Our work provides structural validation of the secoberberine intermediates and the description of the narcotoline-4'-O-methyltransferase, suggesting this activity is catalysed by a unique heterodimer. We also reconstitute a 14-step biosynthetic pathway of noscapine from the simple alkaloid norlaudanosoline by engineering a yeast strain expressing 16 heterologous plant enzymes, achieving reconstitution of a complex plant pathway in a microbial host. Other engineered yeasts produce previously inaccessible pathway intermediates and a novel derivative, thereby advancing protoberberine and noscapine related drug discovery.

Figure 1. Overview of the biosynthetic pathway of noscapine.

Biosynthetic pathways from norlaudanosoline, 12, to noscapine, 1, proposed according to in situ virus-induced gene silencing experiments in P. somniferum (green), in vitro characterization (blue) and pathway reconstitution in yeast (yellow). Solid colour: proposed major biosynthetic pathway to noscapine; faded colour: putative side reaction due to enzyme promiscuity; solid arrow: characterized enzymatic step; dashed arrow: unverified enzymatic step.

Figure 2. LC–MS analysis of yeast strains fed with canadine.

(a) Extracted ion chromatogram (EIC) of m/z⁺=370 of (i) 3-producing yeast strain and (ii) strain expressing PsTNMT, AtATR1 and CYP82Y1. (b) EIC of m/z⁺=386 of (i) 4-producing yeast strain, (ii) strain expressing PsTNMT, AtATR1, CYP82Y1 and CYP82X1 and (iii) strain expressing PsTNMT, AtATR1, CYP82Y1 and CYP82X2. (c) EIC of m/z⁺=428 of (i) 5-producing yeast strain, (ii) strain expressing PsTNMT, AtATR1, CYP82Y1, CYP82X2 and PsAT1 and (iii) 7-producing strain. (d) EIC of m/z⁺=444 of (i) 6-producing yeast strain and (ii) strain expressing PsTNMT, AtATR1, CYP82Y1, CYP82X1, CYP82X2 and PsAT1. (e) EIC of m/z⁺=402 of (i) 7-producing yeast strain and (ii) strain expressing PsTNMT, AtATR1, CYP82Y1, CYP82X2, PsAT1, CYP82X1 and PsCXE1. (f) EIC of m/z⁺=400 of (i) 8-producing yeast strain and (ii) strain expressing PsTNMT, AtATR1, CYP82Y1, CYP82X2, PsAT1, CYP82X1, PsCXE1 and PsSDR1. (g) EIC of m/z⁺=458 of (i) 7-producing yeast strain, and 7-producing strain expressing (ii) PsMT2, (iii) PsMT3, (iv) Ps6OMT, (v) PsMT2 and PsMT3, (vi) PsMT2 and Ps6OMT. (h) EIC of m/z⁺=416 of (i) 8-producing yeast strain, and 8-producing strain expressing (ii) PsMT2 and PsSDR1, (iii) PsMT3, (iv) Ps6OMT, (v) PsMT2, PsMT3 and PsSDR1, (vi) PsMT2, Ps6OMT and SDR1. (i) EIC of m/z⁺=414 of (i) noscapine standard, (ii) 9-producing yeast strain, and 9-producing strain expressing (iii) PsMT2, (iv) PsMT3, (v) PsMT2 and PsMT3, (vi) PsMT2 and Tf6OMT, (vii) PsMT2 and Ps6OMT. All traces are representative of at least three biological replicates for each engineered yeast strain.

Figure 3. Optimization of the activities for three pathway-specific cytochrome P450s.

(a) Optimization of CYP82Y1 activity for the synthesis of 4 through varying N-terminal tag, promoter and growth temperature. (b) Optimization of CYP82X2 activity for the synthesis of 5 through varying promoter and gene copy number. (c) Optimization of CYP82X1 activity for the synthesis of 7 through varying promoter. (d) Noscapine titre analysed from engineered strains harbouring different sets of expression cassettes. CSYN10: AtATR1, PsTNMT, CYP82X2 and PsAT1 expressed from the chromosome; CYP82Y1, PsMT2, PsMT3, CYP82X1, PsCXE1 and PsSDR1 expressed from a low-copy plasmid (Supplementary Data 1). CSYN11: AtATR1, PsTNMT, CYP82X2 and PsAT1 expressed from the chromosome; CYP82Y1, PsMT2, PsMT3, CYP82X1, PsCXE1, PsSDR1 and CYP82X2 expressed from a low-copy plasmid (Supplementary Data 1). Bars represent mean values±1 s.d. of three biological replicates, and the error bars represent the s.d. of the replicates. CYP82Y1A is the MSH N-terminus swapped CYP82Y1.

Figure 4. Identification of the narcotoline-4′-O-methyltransferase as the PsMT2/PsMT3 heterodimer.

(a) EIC MRM using noscapine's highest characteristic precursor ion/product ion transition (414→220) of in vitro assays containing (i) no enzyme (control), (ii) PsMT2 homodimer, (iii) PsMT3 homodimer and (iv) PsMT2/PsMT3 heterodimer. All traces are in the same scale and are representative of three biological replicates for each enzyme. (b) Western blot analysis of the purified and concentrated (1) PsMT2 homodimer, (2) PsMT3 homodimer and (3) PsMT2/PsMT3 heterodimer with (i) Anti-T7 tag antibody and (ii) Anti-6X His tag antibody. Gels and blots are representative of two biological replicates. (c) SEC analyses of the PsMT2/PsMT3 heterodimer. The left inset is the calibration curve of the protein standards, with the RT on the x axis, and the molecular weight in log scale on the y axis. The right insert is the SDS–PAGE analysis of (M) ladder and (S) the purified and concentrated PsMT2/PsMT3 heterodimer. The molecular weight of the His-tagged PsMT2 is 40.09 kDa, the T7-tagged PsMT3 is 38.70 kDa. The calculated molecular weight of the PsMT2/PsMT3 complex is ∼80 kDa, calculated based on at least three replicates. The error bars represent the s.d. of three biological replicates.

Figure 5. LC–MS analysis of yeast strains synthesizing noscapine from norlaudanosoline.

(a) MS/MS spectra of noscapine standard (black) and medium of the noscapine-producing strain CSYN16 (Supplementary Data 1; red). (b) Noscapine titre analysed from engineered strains harbouring different sets of expression cassettes. CSYN15: AtATR1, Ps6OMT, Ps4′OMT, PsCNMT, PsBBE, TfS9OMT, CjCAS, CYP82Y1, CYP82X2, PsAT1, PsSDR1, PsTNMT and PsMT2 expressed from the chromosome; CYP82X1 and PsCXE1 expressed from a low-copy plasmid (Supplementary Data 1). CSYN16: AtATR1, Ps6OMT, Ps4'OMT, PsCNMT, PsBBE, PsS9OMT, CjCAS, CYP82Y1, CYP82X2, PsAT1, PsSDR1, PsTNMT, PsMT2, CYP82X1, PsCXE1 and PsMT3 expressed from the chromosome (Supplementary Data 1). CSYN18: AtATR1, Ps6OMT, Ps4′OMT, PsCNMT, PsBBE, TfS9OMT, CjCAS, CYP82Y1, CYP82X2, PsAT1, PsSDR1, PsMT2, PsTNMT, CYP82X1, PsCXE1 and PsMT3 expressed from the chromosome; CYP82X2, PsS9OMT expressed from a low-copy plasmid (Supplementary Data 1). (c) EIC of m/z⁺=288 (black) and 414 (red) of noscapine-producing yeast strain CSYN18. Bars represent mean values±1 s.d. of five biological replicates, and the error bars represent the s.d. of the replicates.