James T. Payne, Timothy R. Valentic, and Christina D. Smolke

PNAS December 21, 2021 118 (51) e2112520118

APPLIED BIOSCIENCE

Significance

This work demonstrates microbial biosynthesis of bisbenzylisoquinoline (bisBIA) alkaloids. We show that several didomain epimerases can function in yeast to epimerize the nonnative substrate N-methylcoclaurine, an essential step in bisBIA biosynthesis. The N-methylcoclaurine epimerase activity was increased 10-fold by combining individual reductase and oxidase domains from different plant species. Strain engineering and optimization of media and growth conditions increased the bisBIA titer over 10,000-fold. We show that strains can be engineered to primarily produce one bisBIA product by selection of the cytochrome P450 variant that couples the monomer BIA subunits. We then leverage our bisBIA biosynthetic strain as a platform for the screening of other plant enzymes to produce two additional plant natural products de novo in a heterologous host.

Abstract

Benzylisoquinoline alkaloids (BIAs) are a diverse class of medicinal plant natural products. Nearly 500 dimeric bisbenzylisoquinoline alkaloids (bisBIAs), produced by the coupling of two BIA monomers, have been characterized and display a range of pharmacological properties, including anti-inflammatory, antitumor, and antiarrhythmic activities. In recent years, microbial platforms have been engineered to produce several classes of BIAs, which are rare or difficult to obtain from natural plant hosts, including protoberberines, morphinans, and phthalideisoquinolines. However, the heterologous biosyntheses of bisBIAs have thus far been largely unexplored. Here, we describe the engineering of yeast strains that produce the Type I bisBIAs guattegaumerine and berbamunine de novo. Through strain engineering, protein engineering, and optimization of growth conditions, a 10,000-fold improvement in the production of guattegaumerine, the major bisBIA pathway product, was observed. By replacing the cytochrome P450 used in the final coupling reaction with a chimeric variant, the product profile was inverted to instead produce solely berbamunine. Our highest titer engineered yeast strains produced 108 and 25 mg/L of guattegaumerine and berbamunine, respectively. Finally, the inclusion of two additional putative BIA biosynthesis enzymes, SiCNMT2 and NnOMT5, into our bisBIA biosynthetic strains enabled the production of two derivatives of bisBIA pathway intermediates de novo: magnocurarine and armepavine. The de novo heterologous biosyntheses of bisBIAs presented here provide the foundation for the production of additional medicinal bisBIAs in yeast.

See: https://www.pnas.org/content/118/51/e2112520118

Figure 1: Biosynthetic pathway for de novo bisBIA biosynthesis. (A) Biosynthetic pathway from fed sugar to the bisBIA products berbamunine and guattegaumerine. The color of the arrows represents the species of origin of the enzyme employed: light gray, unmodified native yeast enzyme; dark gray, modified native yeast enzyme; green, enzyme of plant origin; orange, enzyme of bacterial origin; purple, enzyme of mammalian origin. (B) De novo production of (S)-N-methylcoclaurine in engineered yeast strains. Strains were cultured in synthetic complete media with 2% dextrose at 30 °C for 96 h before LC-MS/MS analysis of the growth media using the MRM transitions reported in SI Appendix, Table S5. (C) Production of the bisBIA guattegaumerine in a yeast strain expressing BsCYP80A1 and fed exogenous (R)-N-methylcoclaurine. Strains were cultured in selective media (YNB-Ura) with 2% dextrose at 30 °C for 96 h before being pelleted, resuspended in an equal volume of 200 µM (R)-NMC in 50 mM HEPES at pH 7.6, and incubated an additional 72 h at 30 °C before LC-MS/MS analysis of the media using the MRM transitions reported in SI Appendix, Table S5. (D) De novo production of (R)-N-methylcoclaurine in strain CSY1326 expressing PsCPR and different DRS-DRR enzymes off of low-copy plasmids. Strains were cultured in selective media (YNB-DO) with 2% dextrose at 30 °C for 96 h before extraction, purification, and analysis by normal-phase chiral chromatography. Data for B and C are presented as mean values ± the SD of three biologically independent samples. Asterisks represent Student’s two-tailed t test: *P < 0.05, **P < 0.01. Exact P values are given in SI Appendix, Table S6. Source data underlying B and C are provided in Dataset S1.