NIDA IRP Technology Development Initiative Featured Paper

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast

Published in Nature (2019)

Authors

Xiaozhou Luo, Michael A Reiter, Leo d’Espaux, Jeff Wong, Charles M Denby, Anna Lechner, Yunfeng Zhang, Adrian T Grzybowski, Simon Harth, Weiyin Lin, Hyunsu Lee, Changhua Yu, John Shin, Kai Deng, Veronica T Benites, George Wang, Edward E K Baidoo, Yan Chen, Ishaan Dev, Christopher J Petzold, Jay D Keasling

Paper presented by Dr. Nicholas Beacher and selected by the NIDA TDI Paper of the Month Committee

Publication Brief Description

Yeast have been utilized for thousands of years to generate alcohol from simple sugars. Modern advancements in bioengineering have enabled researchers to harness similar processes and generate other substances. Luo & Reiter et. al. created publicly available strains of bioengineered yeasts (yCAN53, yCAN40, yCAN31, and others) capable of synthesizing compounds associated with cannabis from the simple sugar (galactose). Included in these compounds is an array of precursor molecules (e.g., CBGa) as well as compounds (e.g. THCa & CBDa) that convert to THC and CBD with heat (i.e., when smoked or vaporized). Feeding additional acids (e.g., hexanoic acid, heptanoic acid, 6-heptynoic acid, etc.) to the different yeast strains resulted in modified c3 side chains of THCa (yCAN40) and CBDa (yCAN31) which produced a host of novel and unexplored compounds. The paper details the methodology for creating the yeast over multiple generations and rationale for each stage. This work may aid the study of substances that only exist in small quantities in the cannabis plant (e.g., CBGa) while also identifying synthetic cannabinoids that do not exist in nature. The resulting compounds could also be explored in the context of addiction and pharmacotherapies using cell and animal models.

Luo, Xiaozhou; Reiter, Michael A; d'Espaux, Leo; Wong, Jeff; Denby, Charles M; Lechner, Anna; Zhang, Yunfeng; Grzybowski, Adrian T; Harth, Simon; Lin, Weiyin; Lee, Hyunsu; Yu, Changhua; Shin, John; Deng, Kai; Benites, Veronica T; Wang, George; Baidoo, Edward E K; Chen, Yan; Dev, Ishaan; Petzold, Christopher J; Keasling, Jay D

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast Journal Article

In: Nature, vol. 567, no. 7746, pp. 123–126, 2019, ISSN: 1476-4687.

Abstract | Links

@article{pmid30814733,

title = {Complete biosynthesis of cannabinoids and their unnatural analogues in yeast},

author = {Xiaozhou Luo and Michael A Reiter and Leo d'Espaux and Jeff Wong and Charles M Denby and Anna Lechner and Yunfeng Zhang and Adrian T Grzybowski and Simon Harth and Weiyin Lin and Hyunsu Lee and Changhua Yu and John Shin and Kai Deng and Veronica T Benites and George Wang and Edward E K Baidoo and Yan Chen and Ishaan Dev and Christopher J Petzold and Jay D Keasling},

url = {https://pubmed.ncbi.nlm.nih.gov/30814733/},

doi = {10.1038/s41586-019-0978-9},

issn = {1476-4687},

year  = {2019},

date = {2019-03-01},

urldate = {2019-03-01},

journal = {Nature},

volume = {567},

number = {7746},

pages = {123--126},

abstract = {Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ-tetrahydrocannabinolic acid, cannabidiolic acid, Δ-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.},

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pubstate = {published},

tppubtype = {article}

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Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ-tetrahydrocannabinolic acid, cannabidiolic acid, Δ-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.