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Complete biosynthesis of cannabinoids and their unnatural analogues in yeast

DOI:10.1038/s41586-019-0978-9
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Michael Reiter at ETH Zurich
Michael Reiter
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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.
Olivetolic acid production of yCAN03 All LC–MS chromatograms were selected for the theoretical m/z values of the respective compounds of interest (Extended Data Table 3). a, yCAN03 produced olivetolic acid. An additional peak was observed for the byproduct HTAL (same m/z as olivetolic acid), which has previously been reported. No zymolyase was added during extraction. Chromatography gradient for cannabinoid analogues was used. b, Mass spectrum of HTAL. Mass accuracy for observed m/z at a given retention time (RT) is reported in parts per million (p.p.m.).
Olivetolic acid production of yCAN03 All LC–MS chromatograms were selected for the theoretical m/z values of the respective compounds of interest (Extended Data Table 3). a, yCAN03 produced olivetolic acid. An additional peak was observed for the byproduct HTAL (same m/z as olivetolic acid), which has previously been reported. No zymolyase was added during extraction. Chromatography gradient for cannabinoid analogues was used. b, Mass spectrum of HTAL. Mass accuracy for observed m/z at a given retention time (RT) is reported in parts per million (p.p.m.).
… 
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.
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.
… 
In vitro activity of NphB and HlPT 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. a, Purified NphB catalysed the condensation of GPP and olivetolic acid to CBGA when incubated with 5 mM olivetolic acid and 5 mM GPP at room temperature for 24 h. The enzyme produced at least one other isomer of CBGA, which is consistent with previous reports¹⁸. Boiling NphB (NphB ΔT) abolished activity. b, Microsomal fractions were prepared from yCAN21, yCAN22, and yCAN23 that expressed HlPT1L-T (1L-T), HlPT1L-T and HlPT2-T (1L-T/2-T), and HlPT2-T (2-T), respectively. Incubation with 5 mM olivetolic acid and 5 mM GPP for 24 h at 30 °C yielded CBGA as well as several isomers. CBGA production was not observed when incubating HlPT1L-T and HlPT2-T with their native substrates phlorisovalerophenone and dimethylallyl diphosphate (neg).
In vitro activity of NphB and HlPT 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. a, Purified NphB catalysed the condensation of GPP and olivetolic acid to CBGA when incubated with 5 mM olivetolic acid and 5 mM GPP at room temperature for 24 h. The enzyme produced at least one other isomer of CBGA, which is consistent with previous reports¹⁸. Boiling NphB (NphB ΔT) abolished activity. b, Microsomal fractions were prepared from yCAN21, yCAN22, and yCAN23 that expressed HlPT1L-T (1L-T), HlPT1L-T and HlPT2-T (1L-T/2-T), and HlPT2-T (2-T), respectively. Incubation with 5 mM olivetolic acid and 5 mM GPP for 24 h at 30 °C yielded CBGA as well as several isomers. CBGA production was not observed when incubating HlPT1L-T and HlPT2-T with their native substrates phlorisovalerophenone and dimethylallyl diphosphate (neg).
… 
Divarinolic acid and CBGVA production All LC–MS chromatograms were selected for the theoretical m/z values of the respective compounds of interest (Extended Data Table 3). a, Compared with a strain that lacks the olivetolic acid synthesis pathway (yCAN14), yCAN32 produced an additional compound with an m/z value corresponding to divarinolic acid (DA). The retention time of divarinolic acid is reduced relative to olivetolic acid, owing to the reduced hydrophobicity that is conferred by the shorter 5-propyl side chain. No zymolyase was added during extraction. Chromatography gradient for cannabinoid analogues was used. b, Mass spectrum of divarinolic acid. Mass accuracy for observed m/z at a given retention time is reported in parts per million. c, As expected, the prenylated variant of divarinolic acid, CBGVA, was also detected. d, Mass spectrum of CBGVA, with mass accuracy reported in parts per million.
Divarinolic acid and CBGVA production All LC–MS chromatograms were selected for the theoretical m/z values of the respective compounds of interest (Extended Data Table 3). a, Compared with a strain that lacks the olivetolic acid synthesis pathway (yCAN14), yCAN32 produced an additional compound with an m/z value corresponding to divarinolic acid (DA). The retention time of divarinolic acid is reduced relative to olivetolic acid, owing to the reduced hydrophobicity that is conferred by the shorter 5-propyl side chain. No zymolyase was added during extraction. Chromatography gradient for cannabinoid analogues was used. b, Mass spectrum of divarinolic acid. Mass accuracy for observed m/z at a given retention time is reported in parts per million. c, As expected, the prenylated variant of divarinolic acid, CBGVA, was also detected. d, Mass spectrum of CBGVA, with mass accuracy reported in parts per million.
… 
Production of cannabinoid analogues a, Pathway assayed for incorporation of different fatty acid precursors into the corresponding olivetolic acid and CBGA analogues. b, Ion-extracted chromatograms of the engineered strain (yCAN31) grown in the presence of butanoic acid (VII), nonanoic acid (VIII), 5-phenylpentanoic acid (IX), 6-phenylhexanoic acid (X) and 7-phenylheptanoic acid (XI); peaks corresponding to their respective olivetolic acid and HTAL analogues were detected. Detected analogue peaks shifted in retention time depending on their change in hydrophobicity relative to olivetolic acid and CBGA standards. Feeding 1 mM hexanoic acid to yCAN30 yielded neither olivetolic acid nor CBGA (neg (I)). Supplementation of the medium with butanoic acid did not increase production of divarinolic acid or CBGVA over baseline levels. Feeding phenyl-substituted acids yielded only either the corresponding HTAL analogues or olivetolic acid analogues. All LC–MS chromatograms were selected for the theoretical m/z values of the respective compounds of interest (Extended Data Table 3).
+5
Production of cannabinoid analogues a, Pathway assayed for incorporation of different fatty acid precursors into the corresponding olivetolic acid and CBGA analogues. b, Ion-extracted chromatograms of the engineered strain (yCAN31) grown in the presence of butanoic acid (VII), nonanoic acid (VIII), 5-phenylpentanoic acid (IX), 6-phenylhexanoic acid (X) and 7-phenylheptanoic acid (XI); peaks corresponding to their respective olivetolic acid and HTAL analogues were detected. Detected analogue peaks shifted in retention time depending on their change in hydrophobicity relative to olivetolic acid and CBGA standards. Feeding 1 mM hexanoic acid to yCAN30 yielded neither olivetolic acid nor CBGA (neg (I)). Supplementation of the medium with butanoic acid did not increase production of divarinolic acid or CBGVA over baseline levels. Feeding phenyl-substituted acids yielded only either the corresponding HTAL analogues or olivetolic acid analogues. All LC–MS chromatograms were selected for the theoretical m/z values of the respective compounds of interest (Extended Data Table 3).
… 
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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l−1 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-T–CsPT7-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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Catharanthine can be coupled with vindoline to synthesize vinblastine and vincristine, which have been used clinically as potent anticancer drugs. However, the structural complexity and low abundance in nature hamper bulk chemical synthesis and plant extraction, leading to a limited supply and a high cost of this plant natural product. Here, we engineer the methylotrophic yeast Pichia pastoris for complete biosynthesis of catharanthine from simple carbon sources. Through the selection of stable integration sites, screening of biosynthetic pathway enzymes with higher activity and/or specificity, amplification of flux-limiting enzyme encoding genes, rewiring of cellular metabolism and process optimization, we achieve de novo biosynthesis of catharanthine with a titre as high as 2.57 mg l−1, which also represents the most complicated molecule heterologously synthesized in a non-model microorganism. Our study establishes P. pastoris as a cell factory for producing plant natural products with complex biosynthetic pathways. The synthesis of catharanthine, the direct precursor of anticancer drugs vinblastine and vincristine, is challenging due to its structural complexity. Here synthetic biology enables the construction of a Pichia pastoris cell factory for the biosynthesis and potentially scalable production of catharanthine from simple carbon sources.
A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae
Article
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Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production.
The inheritance of chemical phenotype in Cannabis sativa L. (V): regulation of the propyl-/pentyl cannabinoid ratio, completion of a genetic model
Article
Full-text available
In order to complete a genetic model for the inheritance of chemotype in Cannabis, this paper explores the regulation of the propyl-/pentyl cannabinoid ratio. Plants almost pure in compounds with a C5 side chain are by far the most common, and such a chemotype can be considered a wild-type condition. Mutant progenitors with higher levels of the rarer cannabinoid THC-C3 (tetrahydrocannabivarin) were identified. Their propyl cannabinoid proportion in the total cannabinoid fraction (PC3) ranged from 14 to 69 %, which, through selective inbreeding, could be increased to highly specific lineage maxima. Inbred plants with maximised PC3 derived from the different progenitors, were then crossed with a pure C5 wild type and the PC3 distribution patterns of the F2s examined. Distinct patterns, compatible with oligogenic and polygenic segregation appeared. It was hypothesised that the PC3 regulating loci of the six source progenitors would be at least partially different, complementary, and additive in their phenotypical effect. So, high PC3 offspring from the different lineages were mutually crossed. Inbred lines derived from multi-cross hybrid combinations reached unprecedented PC3 levels of up to 96 % which supports the hypothesis. For the regulation of C3/C5 ratios, a model of a multiple locus A 1–A 2–…A n is proposed, with the pentyl- and propyl cannabinoid pathway being enhanced by alleles A pe1−n and A pr1−n, respectively.
W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis
Article
Full-text available
  • Apr 2016
This article presents W-IQ-TREE, an intuitive and user-friendly web interface and server for IQ-TREE, an efficient phylogenetic software for maximum likelihood analysis. W-IQ-TREE supports multiple sequence types (DNA, protein, codon, binary and morphology) in common alignment formats and a wide range of evolutionary models including mixture and partition models. W-IQ-TREE performs fast model selection, partition scheme finding, efficient tree reconstruction, ultrafast bootstrapping, branch tests, and tree topology tests. All computations are conducted on a dedicated computer cluster and the users receive the results via URL or email. W-IQ-TREE is available at http://iqtree.cibiv.univie.ac.at. It is free and open to all users and there is no login requirement.
ETE 3: Reconstruction, Analysis, and Visualization of Phylogenomic Data
Article
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The Environment for Tree Exploration (ETE) is a computational framework that simplifies the reconstruction, analysis, and visualization of phylogenetic trees and multiple sequence alignments. Here, we present ETE v3, featuring numerous improvements in the underlying library of methods, and providing a novel set of standalone tools to perform common tasks in comparative genomics and phylogenetics. The new features include (i) building gene-based and supermatrix-based phylogenies using a single command, (ii) testing and visualizing evolutionary models, (iii) calculating distances between trees of different size or including duplications, and (iv) providing seamless integration with the NCBI taxonomy database. ETE is freely available at http://etetoolkit.org
Cannabinoids for Medical Use: A Systematic Review and Meta-analysis
Article
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Cannabis and cannabinoid drugs are widely used to treat disease or alleviate symptoms, but their efficacy for specific indications is not clear. To conduct a systematic review of the benefits and adverse events (AEs) of cannabinoids. Twenty-eight databases from inception to April 2015. Randomized clinical trials of cannabinoids for the following indications: nausea and vomiting due to chemotherapy, appetite stimulation in HIV/AIDS, chronic pain, spasticity due to multiple sclerosis or paraplegia, depression, anxiety disorder, sleep disorder, psychosis, glaucoma, or Tourette syndrome. Study quality was assessed using the Cochrane risk of bias tool. All review stages were conducted independently by 2 reviewers. Where possible, data were pooled using random-effects meta-analysis. Patient-relevant/disease-specific outcomes, activities of daily living, quality of life, global impression of change, and AEs. A total of 79 trials (6462 participants) were included; 4 were judged at low risk of bias. Most trials showed improvement in symptoms associated with cannabinoids but these associations did not reach statistical significance in all trials. Compared with placebo, cannabinoids were associated with a greater average number of patients showing a complete nausea and vomiting response (47% vs 20%; odds ratio [OR], 3.82 [95% CI, 1.55-9.42]; 3 trials), reduction in pain (37% vs 31%; OR, 1.41 [95% CI, 0.99-2.00]; 8 trials), a greater average reduction in numerical rating scale pain assessment (on a 0-10-point scale; weighted mean difference [WMD], -0.46 [95% CI, -0.80 to -0.11]; 6 trials), and average reduction in the Ashworth spasticity scale (WMD, -0.36 [95% CI, -0.69 to -0.05]; 7 trials). There was an increased risk of short-term AEs with cannabinoids, including serious AEs. Common AEs included dizziness, dry mouth, nausea, fatigue, somnolence, euphoria, vomiting, disorientation, drowsiness, confusion, loss of balance, and hallucination. There was moderate-quality evidence to support the use of cannabinoids for the treatment of chronic pain and spasticity. There was low-quality evidence suggesting that cannabinoids were associated with improvements in nausea and vomiting due to chemotherapy, weight gain in HIV infection, sleep disorders, and Tourette syndrome. Cannabinoids were associated with an increased risk of short-term AEs.
A Heteromeric Membrane-Bound Prenyltransferase Complex from Hop Catalyzes Three Sequential Aromatic Prenylations in the Bitter Acid Pathway
Article
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Bitter acids (α-type and β-type) account for more than 30% of the fresh weight of hop (Humulus lupulus L.) glandular trichomes and are well-known for their contribution to the bitter taste of beer. These multi-prenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast system for the functional identification of aromatic prenyltransferase genes (PT). Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific cDNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Step-wise mutation of the Asp-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L only catalyzed the first prenylation step; PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two hybrid system, reciprocal co-immunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop.
Data Structures for Statistical Computing in Python
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  • Jan 2010
—In this paper we are concerned with the practical issues of working with data sets common to finance, statistics, and other related fields. pandas is a new library which aims to facilitate working with these data sets and to provide a set of fundamental building blocks for implementing statistical models. We will discuss specific design issues encountered in the course of developing pandas with relevant examples and some comparisons with the R language. We conclude by discussing possible future directions for statistical computing and data analysis using Python.
Engineering yeasts as platform organisms for cannabinoid biosynthesis
Article
  • Jul 2017
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.
Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa L
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.