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Complete biosynthesis of cannabinoids and their unnatural analogues in yeast

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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, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-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.
CsPT4-T characterization a, CsPT4-T localized to the purified microsomal fraction. All LC–MS chromatograms were selected for the theoretical m/z values of the respective compounds of interest (Extended Data Table 3). Chromatography gradient for cannabinoid analogues was used. Signals were compared to genuine standards. We incubated boiled microsomal fraction (mic ΔT), soluble fraction (sol) and microsomal fraction (mic) of yCAN14 in the presence of 500 µM olivetolic acid and 500 µM GPP for 1 h at 30 °C and observed GOT activity only in the microsomal fraction. b, CsPT4-T olivetolic acid kinetics. Using nonlinear regression to fit the Michaelis–Menten kinetic model for varied olivetolic acid (0.25 µM–0.56 mM) and constant GPP (1.67 mM) concentrations revealed a Km(olivetolic acid) = 6.73 ± 0.26 µM (mean ± s.d.) (n = 4 technically independent samples; measurements were plotted individually). c, CsPT4-T GPP kinetics. Eadie–Hofstee linearization of Michaelis–Menten model showed non-Michaelis–Menten type behaviour for CsPT4-T for varied GPP (0.25 µM–1.67 mM) and constant olivetolic acid (1.67 mM) concentrations. Measurements do not fall on a line as would be expected (R², coefficient of determination; n = 3 technically independent samples; measurements were plotted individually). d, Phylogenetic tree of Cannabis and related prenyltransferases. The numbers indicate the bootstrap value (%) from 1,000 replications. Grey dashed lines depict branch offsets to accommodate labels. The scale bar shows the amino acid substitution ratio. Prenyltransferases cluster by biosynthetic pathway. CsPT4 catalyses CBGA production. The function of the other Cannabis prenyltransferases remains unknown.
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LETTER https://doi.org/10.1038/s41586-019-0978-9
Complete biosynthesis of cannabinoids and their
unnatural analogues in yeast
Xiaozhou Luo1,15, Michael A. Reiter1,2,15, Leo d’Espaux3,12, Jeff Wong3,12, Charles M. Denby1,13, Anna Lechner4,5,14,
Yunfeng Zhang1,6, Adrian T. Grzybowski1, Simon Harth3, Weiyin Lin3, Hyunsu Lee3,7, Changhua Yu3,5, John Shin3,4,
Kai Deng8,9, Veronica T. Benites3, George Wang3, Edward E. K. Baidoo3, Yan Chen3, Ishaan Dev3,4, Christopher J. Petzold3 &
Jay D. Keasling1,3,4,5,10,11*
Cannabis sativa L. has been cultivated and used around the globe
for its medicinal properties for millennia1. Some cannabinoids,
the hallmark constituents of Cannabis, and their analogues
have been investigated extensively for their potential medical
applications2. Certain cannabinoid formulations have been
approved as prescription drugs in several countries for the
treatment of a range of human ailments3. 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 cannabinoids4, and their structural
complexity, which limits bulk chemical synthesis. Here we report
the complete biosynthesis of the major cannabinoids cannabigerolic
acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-
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
pathway5. We also introduced the Cannabis genes that encode
the enzymes involved in the biosynthesis of olivetolic acid6,
as well as the gene for a previously undiscovered enzyme with
geranylpyrophosphate:olivetolate geranyltransferase activity and the
genes for corresponding cannabinoid synthases
7,8
. 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 potency9. 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.
We began our generation of the cannabinoid-producing yeast by
focusing first on the production of olivetolic acid (Fig.1), an initial
intermediate in the cannabinoid biosynthetic pathway. Two Cannabis
enzymes, a tetraketide synthase (C. sativa TKS; CsTKS)
10
and an olive-
tolic acid cyclase (CsOAC)
6
, have been reported to produce olivetolic
acid from hexanoyl-CoA and malonyl-CoA. To produce olivetolic acid
in yeast, we introduced a CsTKS and CsOAC expression cassette into
S. cerevisiae to generate strain yCAN01 (Extended Data Table1). The
strain produced 0.2mgl
1
olivetolic acid from galactose (Fig.2a), con-
sistent with the fact that S. cerevisiae maintains low intracellular levels
of hexanoyl-CoA
11
. To increase the supply of hexanoyl-CoA, we fed the
yCAN01 strain with 1mM hexanoic acid, which can be converted to
hexanoyl-CoA by an endogenous acyl activating enzyme (AAE), and
observed a sixfold increase in olivetolic acid production (1.3mgl
1
).
A known byproduct of TKS, hexanoyl triacetic acid lactone (HTAL)6,
was also detected (Extended Data Fig.1).
To optimize the conversion of hexanoic acid to hexanoyl-CoA, we
introduced CsAAE1, an AAE from Cannabis that is thought to catalyse
this step in planta, into yCAN01
12
. When fed with 1mM hexanoic acid,
the resulting strain (yCAN02) showed a twofold increase in olivetolic
acid titre (3.0mgl
1
) compared with yCAN01 (Fig.2a). To produce
hexanoyl-CoA from galactose and complete the olivetolic acid path-
way, we introduced a previously reported hexanoyl-CoA pathway into
yCAN015. The resulting strain (yCAN03) produced 1.6mgl1 olive-
tolic acid (Fig.2a).
Cannabigerolic acid (CBGA)—the precursor to Δ9-
tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and
numerous other cannabinoids—is produced from olivetolic acid and
the mevalonate-pathway intermediate geranyl pyrophosphate (GPP)
by a geranylpyrophosphate:olivetolate geranyltransferase (GOT). GOT
activity was detected in Cannabis extracts two decades ago13, and a
Cannabis GOT (CsPT1) was patented ten years later
14
. To test CsPT1
in vivo, we constructed a GPP-overproducing strain (yCAN10) with
an upregulated mevalonate pathway15 and a mutant version of the
endogenous farnesyl pyrophosphate synthase ERG20 (ERG20(F69W/
N127W) that preferentially produces GPP over FPP
16
. However, we
were unable to observe any GOT activity when we expressed CsPT1,
or any truncations of it, in yCAN10.
To identify an enzyme with GOT activity that would function in
yeast, we searched for candidate prenyltransferase enzymes from
Cannabis and other organisms. These included NphB, a soluble pre-
nyltransferase from Streptomyces sp.17 that displayed GOT activity in
vitro
18
, and HlPT1L and HlPT2, two prenyltransferases involved in bit-
ter acid biosynthesis in Humulus lupulus (a close relative of Cannabis)
19
.
In addition, we mined published Cannabis transcriptomes20 (http://
medicinalplantgenomics.msu.edu) for GOT candidates. We searched
full-length transcripts using the Basic Local Alignment Search Tool
(BLAST) against CsPT1, HlPT1L and HlPT2, and on the basis of this
chose six Cannabis enzymes (CsPT2–CsPT7).
For functional expression of the six Cannabis and two H. lupulus
prenyltransferases in yeast, we removed predicted N-terminal plastid-
targeting sequences21,22, resulting in CsPT2-TCsPT7-T and
HlPT1L-Tand HlPT2-T, respectively. Each GOT candidate was then
introduced into yCAN10, and the resulting strains (yCAN12–yCAN20)
1California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA. 2Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland. 3Biological
Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. 4Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA.
5Department of Bioengineering, University of California, Berkeley, CA, USA. 6Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China. 7Department of
Chemistry, University of California, Berkeley, CA, USA. 8Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA. 9Biotechnology and Bioengineering Department,
Sandia National Laboratories, Livermore, CA, USA. 10Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark. 11Center for Synthetic Biochemistry,
Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technologies, Shenzhen, China. 12Present address: Demetrix, Inc., Emeryville, CA, USA. 13Present address: Berkeley Brewing
Science, Inc., Berkeley, CA, USA. 14Present address: Genomatica, Inc., San Diego, CA, USA. 15These authors contributed equally: Xiaozhou Luo, Michael A. Reiter. *e-mail: keasling@berkeley.edu
There are amendments to this paper
7 MARCH 2019 | VOL 567 | NATURE | 123
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Δ⁹-tetrahydrocannabinolic acid (THCA) is a plant derived secondary natural product from the plant Cannabis sativa l. The discovery of the human endocannabinoid system in the late 1980s resulted in a growing number of known physiological functions of both synthetic and plant derived cannabinoids. Thus, manifold therapeutic indications of cannabinoids currently comprise a significant area of research. Here we reconstituted the final biosynthetic cannabinoid pathway in yeasts. The use of the soluble prenyltransferase NphB from Streptomyces sp. strain CL190 enables the replacement of the native transmembrane prenyltransferase cannabigerolic acid synthase from C. sativa. In addition to the desired product cannabigerolic acid, NphB catalyzes an O-prenylation leading to 2-O-geranyl olivetolic acid. We show for the first time that the bacterial prenyltransferase and the final enzyme of the cannabinoid pathway tetrahydrocannabinolic acid synthase can both be actively expressed in the yeasts Saccharomyces cerevisiae and Komagataella phaffii simultaneously. While enzyme activities in S. cerevisiae were insufficient to produce THCA from olivetolic acid and geranyl diphosphate, genomic multi-copy integrations of the enzyme’s coding sequences in K. phaffii resulted in successful synthesis of THCA from olivetolic acid and geranyl diphosphate. This study is an important step toward total biosynthesis of valuable cannabinoids and derivatives and demonstrates the potential for developing a sustainable and secure yeast bio-manufacturing platform.
Article
The Δ9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of Δ9-tetrahydrocannabinolic acid (THCA) using whole cells. Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml(-1) (intracellular) and 44 pkat ml(-1) (extracellular) after 192 h of cultivation at 15 °C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l(-1)) THCA could be produced by 10.5 gCDW l(-1) before enzyme activity was lost. Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production.