Water retting process with hemp pre-treatment: effect on the enzymatic activities and microbial populations dynamic.

Proper retting process of hemp stems, in which efficient separation of cellulose fiber from the rest of the stem is promoted by indigenous microorganisms able to degrade pectin, is essential for fiber production and quality. This research aimed to investigate the effect of a pre-treatment dew retting in field of hemp stalks on the pectinolytic enzymatic activity and microbiota dynamic during lab-scale water retting process. A strong increase in the pectinase activity as well as in the aerobic and anaerobic pectinolytic concentration was observed from 14 to 21 days, especially using hemp stalks that were not subjected to a pre-retting treatment on field (WRF0 0.690 ± 0.05 U/mL). Results revealed that the microbial diversity significantly varied over time during the water retting and the development of microbiota characterizing the water retting of hemp stalks of different biosystems used in this study was affected by pre-treatment conditions in the field and water retting process and by an interaction between the two methods. Although at the beginning of the experiment a high biodiversity was recorded in all biosystems, the water retting led to a selection of microbial populations in function of the time of pre-treatment in field, especially in bacterial populations. The use of hemp stems did not subject to a field pre-treatment seems to help the development of a homogeneous and specific pectinolytic microbiota with a higher enzymatic activity in respect to samples exposed to uncontrolled environmental conditions for 10, 20, or 30 days before the water retting process. KEY POINTS: • Microbial diversity significantly varied over time during water retting. • Water retting microbiota was affected by dew pre-treatment in the field. • Retting of no pretreated hemp allows the development of specific microbiota with high enzymatic activity.

[Genome-wide screening and bioinformatics analysis of Cannabis sativa LecRLK gene family].

Lectin receptor-like kinase(LecRLK) is a class of phytokinase with lectin conserved domain, which plays an important role in plant resistance to biological and abiotic stresses, as well as plant growth and development. Cannabis sativa is an important multi-purpose plant, widely used in food, textile, medicine, and other fields. Genome-wide screening and expression analysis of the LecRLK family of C. sativa were performed in this paper, so as to provide scientific reference for functional analysis of the LecRLK family of C. sativa. Based on BLAST and HMM methods, 93 LecRLKs were identified in the whole genome of C. sativa, including 69 G types, 23 L types, and one C types. Subsequently, a series of bioinformatics analyses were performed on the LecRLK family members, and the physicochemical properties of the protein of the LecRLK family members were initially revealed. The prediction of cis-acting elements of promoters in family members showed that family members were regulated by hormones and stress response. The expression analysis showed that some family members were highly expressed in the roots, which may participate in the process of stress resistance. Several members were highly expressed in female flowers and may be involved in female flower development. This study provides a theoretical basis for further study of LecRLK gene function. Meanwhile, the expression analysis screens candidate LecRLK members who may participate in the resistance of C. sativa, which provides a theoretical basis for the subsequent selection of C. sativa varieties against resistance.

Investigation on solvent-free esterification of oleic acid by hemp tea waste-immobilized Candida rugosa lipase.

This study aimed at Candida rugosa lipase immobilization on a low-cost and readily available support. Among agro-industrial crops, hemp tea waste was chosen as the carrier because it provides higher immobilization performance than hemp flower and leaf wastes. Support characterization by ATR-FTIR, SEM and elemental analysis and the optimization of the adsorption immobilization process were performed. The lipase adsorption immobilization was obtained by soaking the support with hexane under mild agitation for 2 h and a successively incubating the enzyme for 1 h at room temperature without removing the solvent. The esterification of oleic acid with n-decanol was tested in a solvent-free system by studying some parameters that influence the reaction, such as the substrates molar ratio, the lipase activity/oleic acid ratio, reaction temperature and the presence/absence of molecular sieves. The biocatalyst showed the ability to bring the esterification reaction to equilibrium under 60 min and good reusability (maintaining 60 % of its original activity after three successive esterification reactions) but low conversion (21 %) at the optimized conditions (40 °C, 1:2 substrates molar ratio, 0.56 lipase/oleic acid ratio, without sieves). Comparing the results with those obtained by free lipase form at the same activity (1 U) and experimental conditions, slightly higher conversion (%) appeared for the free lipase. All this highlighted that probably the source of lipase for its carbohydrate-binding pocket and lid structure affected the esterification of oleic acid but certainly, the immobilization didn't induce any lipase conformational change also allowing the reuse of the catalytic material.

Influence of Fungicide Application on Rhizosphere Microbiota Structure and Microbial Secreted Enzymes in Diverse Cannabinoid-Rich Hemp Cultivars.

Microbes and enzymes play essential roles in soil and plant rhizosphere ecosystem functioning. However, fungicides and plant root secretions may impact the diversity and abundance of microbiota structure and enzymatic activities in the plant rhizosphere. In this study, we analyzed soil samples from the rhizosphere of four cannabinoid-rich hemp (Cannabis sativa) cultivars (Otto II, BaOx, Cherry Citrus, and Wife) subjected to three different treatments (natural infection, fungal inoculation, and fungicide treatment). DNA was extracted from the soil samples, 16S rDNA was sequenced, and data were analyzed for diversity and abundance among different fungicide treatments and hemp cultivars. Fungicide treatment significantly impacted the diversity and abundance of the hemp rhizosphere microbiota structure, and it substantially increased the abundance of the phyla Archaea and Rokubacteria. However, the abundances of the phyla Pseudomonadota and Gemmatimonadetes were substantially decreased in treatments with fungicides compared to those without fungicides in the four hemp cultivars. In addition, the diversity and abundance of the rhizosphere microbiota structure were influenced by hemp cultivars. The influence of Cherry Citrus on the diversity and abundance of the hemp rhizosphere microbiota structure was less compared to the other three hemp cultivars (Otto II, BaOx, and Wife). Moreover, fungicide treatment affected enzymatic activities in the hemp rhizosphere. The application of fungicides significantly decreased enzyme abundance in the rhizosphere of all four hemp cultivars. Enzymes such as dehydrogenase, dioxygenase, hydrolase, transferase, oxidase, carboxylase, and peptidase significantly decreased in all the four hemp rhizosphere treated with fungicides compared to those not treated. These enzymes may be involved in the function of metabolizing organic matter and degrading xenobiotics. The ecological significance of these findings lies in the recognition that fungicides impact enzymes, microbiota structure, and the overall ecosystem within the hemp rhizosphere.

Genome-wide identification and analysis of ascorbate peroxidase (APX) gene family in hemp (Cannabis sativa L.) under various abiotic stresses.

Ascorbate peroxidase (APX) plays a critical role in molecular mechanisms such as plant development and defense against abiotic stresses. As an important economic crop, hemp (Cannabis sativa L.) is vulnerable to adverse environmental conditions, such as drought, cold, salt, and oxidative stress, which lead to a decline in yield and quality. Although APX genes have been characterized in a variety of plants, members of the APX gene family in hemp have not been completely identified. In this study, we (1) identified eight members of the CsAPX gene family in hemp and mapped their locations on the chromosomes using bioinformatics analysis; (2) examined the physicochemical characteristics of the proteins encoded by these CsAPX gene family members; (3) investigated their intraspecific collinearity, gene structure, conserved domains, conserved motifs, and cis-acting elements; (4) constructed a phylogenetic tree and analyzed interspecific collinearity; and (5) ascertained expression differences in leaf tissue subjected to cold, drought, salt, and oxidative stresses using quantitative real-time-PCR (qRT-PCR). Under all four stresses, CsAPX6, CsAPX7, and CsAPX8 consistently exhibited significant upregulation, whereas CsAPX2 displayed notably higher expression levels under drought stress than under the other stresses. Taken together, the results of this study provide basic genomic information on the expression of the APX gene family and pave the way for studying the role of APX genes in abiotic stress.

The development of a next-generation sequencing panel targeting cannabinoid synthase genes to distinguish between marijuana and hemp.

Hemp and marijuana, both derived from Cannabis sativa L. (C. sativa), are subject to divergent legal regulations due to their different Δ9-tetrahydrocannabinol (Δ9-THC) contents. Cannabinoid synthase genes are considered the key enzymes that determine the chemical composition or chemotype of a particular cultivar. However, existing methods for crop type differentiation based on previous synthase gene theories have limitations in terms of precision and specificity, and a wider range of cannabis varieties must be considered when examining cannabis-based genetic markers. A custom next-generation sequencing (NGS) panel was developed targeting all synthase genes, including Δ9-THC acid synthase, cannabidiolic acid synthase, and cannabichromenic acid synthase, as well as the pseudogenes across diverse C. sativa samples, spanning reference hemp and marijuana, commercial hemp derivatives, and seized marijuana extracts. Interpretation of NGS data revealed a relationship between genotypes and underlying chemotypes, with the principal component analysis indicating a clear distinction between hemp and marijuana clusters. This differentiation was attributed to variations in both synthase genes and pseudogene variants. Finally, this study proposes a genetic cannabis classification method using a differentiation flow chart with novel synthase markers. The flow chart successfully differentiated hemp from marijuana with a 1.3% error rate (n = 147).

Cannabis labelling is associated with genetic variation in terpene synthase genes.

Analysis of over 100 Cannabis samples quantified for terpene and cannabinoid content and genotyped for over 100,000 single nucleotide polymorphisms indicated that Sativa- and Indica-labelled samples were genetically indistinct on a genome-wide scale. Instead, we found that Cannabis labelling was associated with variation in a small number of terpenes whose concentrations are controlled by genetic variation at tandem arrays of terpene synthase genes.

SNP in Potentially Defunct Tetrahydrocannabinolic Acid Synthase Is a Marker for Cannabigerolic Acid Dominance in Cannabis sativa L.

The regulation of cannabinoid synthesis in Cannabis sativa is of increasing research interest as restrictions around the globe loosen to allow the plant's legal cultivation. Of the major cannabinoids, the regulation of cannabigerolic acid (CBGA) production is the least understood. The purpose of this study was to elucidate the inheritance of CBGA dominance in C. sativa and describe a marker related to this chemotype. We produced two crossing populations, one between a CBGA dominant cultivar and a tetrahydrocannabinolic acid (THCA) dominant cultivar, and one between a CBGA dominant cultivar and a cannabidiolic acid (CBDA) cultivar. Chemical and genotyping analyses confirmed that CBGA dominance is inherited as a single recessive gene, potentially governed by a non-functioning allelic variant of the THCA synthase. The "null" THCAS synthase contains a single nucleotide polymorphism (SNP) that may render the synthase unable to convert CBGA to THCA leading to the accumulation of CBGA. This SNP can be reliably used as a molecular marker for CBGA dominance in the selection and breeding of C. sativa.

Transient expression of the ß-glucuronidase gene in Cannabis sativa varieties.

In plant biology, transient expression analysis plays a vital role to provide a fast method to study the gene of interest. In this study, we report a rapid and efficient method for transient expression in Cannabis sativa seedlings using Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 carrying the pCAMBIA1301 construct with uidA gene was used to transform cannabis seedlings and the GUS assay (a measurement of ß-glucuronidase activity) was used to detect the uidA expression. In the current study, we have also established a rapid germination protocol for cannabis seeds. The all three steps seed sterilization, germination and seedlings development were carried out in a 1% H2O2 solution. Transient transformation revealed that both cotyledons and young true leaves are amenable to transformation. Compared with tobacco (Nicotiana benthamiana), cannabis seedlings were less susceptible to transformation with A. tumefaciens. Susceptibility to Agrobacterium transformation also varied with the different cannabis varieties. The method established in this study has the potential to be an important tool for gene function studies and genetic improvement in cannabis.

Terpene Synthases and Terpene Variation in Cannabis sativa.

Cannabis (Cannabis sativa) resin is the foundation of a multibillion dollar medicinal and recreational plant bioproducts industry. Major components of the cannabis resin are the cannabinoids and terpenes. Variations of cannabis terpene profiles contribute much to the different flavor and fragrance phenotypes that affect consumer preferences. A major problem in the cannabis industry is the lack of proper metabolic characterization of many of the existing cultivars, combined with sometimes incorrect cultivar labeling. We characterized foliar terpene profiles of plants grown from 32 seed sources and found large variation both within and between sets of plants labeled as the same cultivar. We selected five plants representing different cultivars with contrasting terpene profiles for clonal propagation, floral metabolite profiling, and trichome-specific transcriptome sequencing. Sequence analysis of these five cultivars and the reference genome of cv Purple Kush revealed a total of 33 different cannabis terpene synthase (CsTPS) genes, as well as variations of the CsTPS gene family and differential expression of terpenoid and cannabinoid pathway genes between cultivars. Our annotation of the cv Purple Kush reference genome identified 19 complete CsTPS gene models, and tandem arrays of isoprenoid and cannabinoid biosynthetic genes. An updated phylogeny of the CsTPS gene family showed three cannabis-specific clades, including a clade of sesquiterpene synthases within the TPS-b subfamily that typically contains mostly monoterpene synthases. The CsTPSs described and functionally characterized here include 13 that had not been previously characterized and that collectively explain a diverse range of cannabis terpenes.

Thin-layer chromatography-bioautographic method for the detection of arginase inhibitors.

Arginase represents a promising therapeutic target for various pathologies including inflammatory, cardiovascular, and parasitic diseases or cancers. In the current work, we report, for the first time, about the development of a thin-layer chromatography-based bioautography which can be used to rapidly detect arginase inhibitors in complex matrices such as plant extracts. The assay is based on the detection of urea produced by arginase using the coloring reagent α-isonitrosopropiophenone, resulting in the formation of a pink background on thin-layer chromatography plates. The assay conditions were optimized in order to provide sufficient contrast between the pink colored thin-layer chromatography plate and the clearer zones generated by the presence of arginase inhibitors. Different parameters were tested, such as incubation time and temperature, atmospheric conditions, as well as substrate and enzyme concentrations. This technique makes it possible to detect 0.1 µg of a known arginase inhibitor, Nω -hydroxy-nor-Arginine, after it has been spotted, either pure or mixed with a Myrtus communis methanolic fruit extract, and the plate has been developed in an appropriate solvent. The newly developed method was used to reveal the presence of an inhibitor in hempseed cakes (Cannabis sativa L.).

Dietary Hemp Seeds More Effectively Attenuate Disorders in Genetically Obese Rats than Their Lipid Fraction.

BACKGROUND: Hemp seeds are rich in PUFAs and other bioactives that can attenuate the development of obesity-related disorders; however, the extent to which their lipid fraction is responsible for this effect is unknown. OBJECTIVE: We hypothesized that hemp seed or hemp oil supplementation can attenuate genetically determined disorders and that the former are more effective in doing so. METHODS: Lean and obese male Zucker rats, aged 8 wk, weighing 174 ± 4.2 g and 223 ± 3.8 g, respectively, were allocated to 4 groups. The lean (LC) and obese controls (OC) were fed a standard diet, whereas the other 2 obese groups were fed a modified diet in which hemp oil (4% diet; O + HO) or hemp seeds (12% diet; O + HS) were included. All diets had the same proportions of protein (18%), fat (8%), and fiber (5%) and a similar carbohydrate proportion (∼52%). Diets fed to O + HO and O + HS had similar fatty acid profiles. After 4 wk, markers of gut and liver function, antioxidant status, and lipid metabolism were measured. RESULTS: The total SCFA concentration in the cecal digesta was lower in OC (64.8 ± 4.21 µmol/g) compared with LC (78.1 ± 2.83 µmol/g) (P ≤ 0.05), whereas it was greater in O + HS (89 ± 4.41 µmol/g) compared with LC, OC, and O + HO (69.7 ± 2.68 µmol/g) (P ≤ 0.05). Plasma total cholesterol was greater in OC (6.20 ± 0.198 mmol/L) and O + HO (5.60 ± 0.084 mmol/L) compared with LC (2.71 ± 0.094 mmol/L) (P ≤ 0.05); in O + HS, the concentration did not differ from the other groups (5.16 ± 0.278 mmol/L). The liver cholesterol concentration was greater in OC (1.79 ± 0.379 mg/g) compared with the other groups (1.28-1.43 mg/g) (P ≤ 0.05). Hepatic expression of peroxisome proliferator-activated receptor γ was lower in OC (11.9 ± 0.93 units) compared with LC (17.3 ± 1.3 units) (P ≤ 0.05), whereas it was greater in O + HS (19.2 ± 1.04 units) compared with OC and O + HO (14.0 ± 1.33 units) (P ≤ 0.05). CONCLUSIONS: Dietary hemp seeds more effectively attenuate metabolic disorders in genetically obese rats than the oil extracted from them, which suggests that the lipid fraction is only partly responsible for these effects.

Structure of the Cannabis sativa olivetol-producing enzyme reveals cyclization plasticity in type III polyketide synthases.

In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three-step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 â†’ C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 â†’ C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an 'aldol switch' is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable 'aldol switch' consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue-specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high-resolution structural analysis of multiple enzyme homologues with classical site-directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes. ENZYMES: TKS, EC 2.3.1.206; OAC, EC 4.4.1.26; chalcone synthase, EC 2.3.1.74; stilbene synthase, EC 2.3.1.95; 2-PS, EC 2.3.1.-. ACCESSION NUMBERS: The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number 6GW3.

Complex Patterns of Cannabinoid Alkyl Side-Chain Inheritance in Cannabis.

The cannabinoid alkyl side-chain represents an important pharmacophore, where genetic targeting of alkyl homologs has the potential to provide enhanced forms of Cannabis for biopharmaceutical manufacture. Delta(9)-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) synthase genes govern dicyclic (CBDA) and tricyclic (THCA) cannabinoid composition. However, the inheritance of alkyl side-chain length has not been resolved, and few studies have investigated the contributions and interactions between cannabinoid synthesis pathway loci. To examine the inheritance of chemical phenotype (chemotype), THCAS and CBDAS genotypes were scored and alkyl cannabinoid segregation analysed in 210 F2 progeny derived from a cross between two Cannabis chemotypes divergent for alkyl and cyclic cannabinoids. Inheritance patterns of F2 progeny were non-Gaussian and deviated from Mendelian expectations. However, discrete alkyl cannabinoid segregation patterns consistent with digenic as well as epistatic modes of inheritance were observed among F2 THCAS and CBDAS genotypes. These results suggest linkage between cannabinoid pathway loci and highlight the need for further detailed characterisation of cannabinoid inheritance to facilitate metabolic engineering of chemically elite germplasm.

Characterization of an acidic α-galactosidase from hemp (Cannabis sativa L.) seeds and its application in removal of raffinose family oligosaccharides (RFOs).

An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ inhibited its activity. The Km and Vmax for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 µM min-1 mg-1. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.

Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativaL.

Cannabinoids are secondary natural products from the plant Cannabis sativaL. Therapeutic indications of cannabinoids currently comprise a significant area of medicinal research. We have expressed the Δ9-tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) recombinantly in Komagataella phaffii and could detect eight different products with a cannabinoid scaffold after conversion of the precursor cannabigerolic acid (CBGA). Besides five products remaining to be identified, both enzymes were forming three major cannabinoids of C. sativa - Δ9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA). In pursuit of improved enzyme properties for a biotechnological cannabinoid production, we performed site-directed mutagenesis to investigate the glycosylation pattern, the C-terminal berberine-bridge-enzyme (BBE) domain, the active site and the product specificity of both enzymes. The THCAS variant T_N89Q+N499Q (lacking two glycosylation sites) exerted about two-fold increased activity compared to wild-type enzyme. Variant T_H494C+R532C (additional disulfide bridge) exerted about 1.7-fold increased activity compared to wild-type enzyme and a shifted temperature optimum from 52 °C to 57 °C. We generated two CBDAS variants, C_S116A and C_A414V, with 2.8 and 3.3-fold increased catalytic activities for CBDA production. C_A414V additionally showed a broadened pH spectrum and a 19-fold increased catalytic activity for THCA production. These studies lay the groundwork for further research as well as biotechnological cannabinoid production.

Subcellular localization defines modification and production of Δ9-tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana.

OBJECTIVE: Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis. RESULTS: Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 ± 12 fkat g FW-1 activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat g FW-1 activity towards CBCA production). CONCLUSION: Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.

Terpene synthases from Cannabis sativa.

Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as ß-myrcene, (E)-ß-ocimene, (-)-limonene, (+)-α-pinene, ß-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

Δ9-Tetrahydrocannabinolic acid synthase: The application of a plant secondary metabolite enzyme in biocatalytic chemical synthesis.

Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) from the secondary metabolism of Cannabis sativa L. catalyzes the oxidative formation of an intramolecular CC bond in cannabigerolic acid (CBGA) to synthesize Δ(9)-tetrahydrocannabinolic acid (THCA), which is the direct precursor of Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Aiming on a biotechnological production of cannabinoids, we investigated the potential of the heterologously produced plant oxidase in a cell-free system on preparative scale. THCAS was characterized in an aqueous/organic two-liquid phase setup in order to solubilize the hydrophobic substrate and to allow in situ product removal. Compared to the single phase aqueous setup the specific activity decreased by a factor of approximately 2 pointing to a substrate limitation of CBGA in the two-liquid phase system. However, the specific activity remained stable for at least 3h illustrating the benefit of the two-liquid phase setup. In a repeated-batch setup, THCAS showed only a minor loss of specific activity in the third batch pointing to a high intrinsic stability and high solvent tolerance of the enzyme. Maximal space-time-yields of 0.121gL(-1)h(-1) were reached proving the two-liquid phase concept suitable for biotechnological production of cannabinoids.

Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.

In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+ß barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-ß-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future. DATA DEPOSITION: Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively.