Engineering strategies for the fermentative production of plant alkaloids in yeast

Abstract

Microbial hosts engineered for the biosynthesis of plant natural products offer enormous potential as powerful discovery and production platforms. However, the reconstruction of these complex biosynthetic schemes faces numerous challenges due to the number of enzymatic steps and challenging enzyme classes associated with these pathways, which can lead to issues in metabolic load, pathway specificity, and maintaining flux to desired products. Cytochrome P450 enzymes are prevalent in plant specialized metabolism and are particularly difficult to express heterologously. Here, we describe the reconstruction of the sanguinarine branch of the benzylisoquinoline alkaloid pathway in Saccharomyces cerevisiae, resulting in microbial biosynthesis of protoberberine, protopine, and benzophenanthridine alkaloids through to the end-product sanguinarine, which we demonstrate can be efficiently produced in yeast in the absence of the associated biosynthetic enzyme. We achieved titers of 676 μg/L stylopine, 548 μg/L cis-N-methylstylopine, 252 μg/L protopine, and 80 μg/L sanguinarine from the engineered yeast strains. Through our optimization efforts, we describe genetic and culture strategies supporting the functional expression of multiple plant cytochrome P450 enzymes in the context of a large multi-step pathway. Our results also provided insight into relationships between cytochrome P450 activity and yeast ER physiology. We were able to improve the production of critical intermediates by 32-fold through genetic techniques and an additional 45-fold through culture optimization.

Keywords: Cytochrome P450; Plant natural products; Synthetic biology; Yeast.

Figure 1

Engineered pathway for sanguinarine biosynthesis in yeast from (R, S)-norlaudanosoline. Red arrows indicate reactions catalyzed by a plant cytochrome P450 enzyme. Structural class of the metabolites are indicated as green, protoberberine; blue, protopine; purple, benzophenanthridine. Ps6OMT, P. somniferum 6-O-methyltransferase; PsCNMT, P. somniferum coclaurine N-methyltransferase; Ps4’OMT, P. somniferum 4’-O-methyltransferase; PsBBE, P. somniferum berberine bridge enzyme; CPR, cytochrome P450 reductase; CFS, cheilanthifoline synthase; STS, stylopine synthase; TNMT, tetrahydroprotoberberine N-methyltransferase; MSH, cis-N-methylstylopine 14-hydroxylase; P6H, protopine 6-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase.

Figure 2

Optimization of (S)-cheilanthifoline production through genetic strategies. (A) Schematic representing genetic strategies used to optimize cheilanthifoline production, including expression method (plasmids or integrated), promoter, species variants, and CPR variants. All strains contain Ps6OMT, PsCNMT, Ps4’OMT, and PsBBE chromosomally integrated. (B) Cheilanthifoline production in yeast strains as a function of CFS and CPR variants. Variants of CFS from E. californica (EcCFS), A. mexicana (AmCFS), and P. somniferum (PsCFS) were expressed from low-copy plasmids in yeast strains with an integrated cytochrome P450 reductase enzyme (CPR) from either the native yeast (CSY953) or various plant sources (A. thaliana, CSY844; E. californica, CSY850; P. somniferum, CSY985). Inset, cheilanthifoline production with EcCFS and ATR1 as a function of expression method, normalized to production from the low-copy plasmid. (C) Confocal microscopy analysis of EcCFS C-terminally tagged with GFP on high-copy (top) or low-copy (bottom) plasmids coexpressed with ER marker Kar2-DsRed-HDEL in CSY844. Wild-type ER (no heterologous P450 expressed) is shown for comparison (right). Percentages indicate portion of the yeast population that are GFP positive under the indicated expression condition. Scale bars are 4 µm. Images are representative of at least 3 independent experiments. (D) Cheilanthifoline production in yeast strains engineered to express EcCFS from a low-copy plasmid under the control of different promoters in CSY844. Data are normalized to production from P_GPD. (B, D) Metabolite production is determined by LC-MS analysis of culture media after indicated strains were grown for 96 h. Data is reported as the mean ± s.d. of at least 3 independent experiments.

Figure 3

Optimization of stylopine production through genetic and culture strategies. (A) Stylopine production in yeast strains as a function of the combination of the species variants of CFS and STS. CFS and STS variants were expressed from separate low-copy plasmids in CSY844. (B) Stylopine production in CSY904 grown under various culture conditions. 30 °C: 30 °C growth temperature; 25 °C: 25 °C growth temperature. 2 Stages: cultured at 25 °C with an initial growth phase followed by a production phase; Galactose: grown as described in 2 stages with 2% galactose used as the carbon source during the production phase. Metabolite production is determined by LC-MS analysis of culture media after indicated strains were grown for 96 h. Data is reported as the mean ± s.d. of at least 3 independent experiments.

Figure 4

Microbial production of cis-N-methylstylopine, protopine, and sanguinarine in optimized culture conditions. (A) Production of cis-N-methylstylopine, protopine, and sanguinarine yeast strains under optimized growth conditions. CSY968 contains PsTNMT integrated and CSY969 contains both PsTNMT and PsMSH integrated. P6H variants were expressed from a low-copy plasmid in CSY969. Metabolite production is determined by LC-MS analysis of culture media after indicated strains were grown for 96 h (CSY968) or 10 d (CSY969, CSY969 with P6H). Data is reported as the mean ± s.d. of at least 3 independent experiments. (B) LC-MS analysis of growth media of CSY969 with EcP6H. Traces are shown for a no EcP6H enzyme control strain (left) and an engineered strain expressing EcP6H (right). EICs for compounds corresponding to protopine (354 m/z), dihydrosanguinarine (334 m/z), and sanguinarine (332 m/z) are shown. A sanguinarine standard is included for comparison. (C) MS2 spectra for the sanguinarine standard (left) and the 332 EIC peak produced from the engineered yeast strain (right). LC-MS traces and fragmentation patterns are representative of at least 3 independent experiments.