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

Zirpel, Bastian; Kayser, Oliver; Stehle, Felix

doi:10.1016/j.jbiotec.2018.07.031

Cited by 78 publications

(98 citation statements)

References 29 publications

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“…A purported active THCAS from CBD-dominant fiber-type hemp was later shown to be a cannabichromenic acid synthase (CBCAS) (Kojoma et al, 2006;Laverty et al, 2019). Lastly, in vitro expression of wild-type CBDAS leads to production of CBDA:THCA in a ratio very close to 20:1 at optimal pH (Zirpel, Kayser, & Stehle, 2018). Future breeding efforts should be informed by this promiscuous activity.…”

Section: Ccp-1 Cannabinoid Chemotype Assaymentioning

confidence: 99%

Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L.

et al. 2020

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Hemp (Cannabis sativa L.) is an emerging dioecious crop grown primarily for grain, fiber, and cannabinoids. There is good evidence for medicinal benefits of the most abundant cannabinoid in hemp, cannabidiol (CBD). For CBD production, female plants producing CBD but not tetrahydrocannabinol (THC) are desired. We developed and validated high‐throughput PACE (PCR Allele Competitive Extension) assays for C. sativa plant sex and cannabinoid chemotype. The sex assay was validated across a wide range of germplasm and resolved male plants from female and monoecious plants. The cannabinoid chemotype assay revealed segregation in hemp populations, and resolved plants producing predominantly THC, predominantly CBD, and roughly equal amounts of THC and CBD. Cultivar populations that were thought to be stabilized for CBD production were found to be segregating phenotypically and genotypically. Many plants predominantly producing CBD accumulated more than the current US legal limit of 0.3% THC by dry weight. These assays and data provide potentially useful tools for breeding and early selection of hemp.

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Section: Ccp-1 Cannabinoid Chemotype Assaymentioning

confidence: 99%

Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L.

et al. 2020

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“…The enzyme consists of 545 amino acids and has a monomeric structure divided into two domains by a flavin adenine dinucleotide (FAD) binding pocket, in which the FAD molecule is covalently bound to the enzyme at positions H114 and C176 (Shoyama et al 2012). Covalent binding of the FAD molecule is essential for the activity of THCA synthase (Sirikantaramas et al 2004;Zirpel et al 2018b). A disulfide bond is formed between C37 and C99 of domain I, and at least six Nglycosylation sites were detected.…”

Section: Tetrahydrocannabinolic Acid Synthasementioning

confidence: 99%

“…Y484 is essential for the catalytic activity of THCAS, and the amino acids H292 and Y417 are suggested to be important for substrate stabilization or binding (Shoyama et al 2012). Protein engineering of THCAS resulted in a 2-fold increased activity of THCAS variant N89Q/N499Q and a 1.7-fold increased activity of THCAS variant H494C/R532C, aiming to add a disulfide bond (Zirpel et al 2018b).…”

Section: Tetrahydrocannabinolic Acid Synthasementioning

confidence: 99%

Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast

Thomas

Schmidt

Kayser

2020

Appl Microbiol Biotechnol

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Heterologous biosynthesis of tetrahydrocannabinolic acid (THCA) in yeast is a biotechnological process in Natural Product Biotechnology that was recently introduced. Based on heterologous genes from Cannabis sativa and Streptomyces spp. cloned into Saccharomyces cerevisiae, the heterologous biosynthesis was fully embedded as a proof of concept. Low titer and insufficient biocatalytic rate of most enzymes require systematic optimization of recombinant catalyst by protein engineering and consequent C-flux improvement of the yeast chassis for sufficient precursor (acetyl-CoA), energy (ATP), and NADH delivery. In this review basic principles of in silico analysis of anabolic pathways towards olivetolic acid (OA) and cannabigerolic acid (CBGA) are elucidated and discussed to identify metabolic bottlenecks. Based on own experimental results, yeasts are discussed as potential platform organisms to be introduced as potential cannabinoid biofactories. Especially feeding strategies and limitations in the committed mevalonate and olivetolic acid pathways are in focus of in silico and experimental studies to validate the scientific and commercial potential as a realistic alternative to the plant Cannabis sativa. Key points • First time critical review of the heterologous process for recombinant THCA/CBDA production and critical review of bottlenecks and limitations for a bioengineered technical process • Integrative approach of protein engineering, systems biotechnology, and biochemistry of yeast physiology and biosynthetic cannabinoid enzymes • Comparison of NphB and CsPT aromatic prenyltransferases as rate-limiting catalytic steps towards cannabinoids in yeast as platform organisms

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“…The importance of protein post-translational modifications (PTMs) in Cannabis are, to our knowledge, relatively unknown, outside of results suggesting glycosylation on the THCA synthase protein [19]. Current strategies for identifying PTMs from shotgun proteomics data require the addition of dynamic modifications.…”

Section: Post-translational Modification Identification and Analysismentioning

confidence: 99%

The Cannabis Proteome Draft Map Project

Jenkins

Orsburn

2020

IJMS

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Recently we have seen a relaxation of the historic restrictions on the use and subsequent research on the Cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. We report herein our progress on constructing a comprehensive draft map of the Cannabis proteome. To date we have identified over 17,000 potential protein sequences. Unfortunately, no annotated genome of Cannabis plants currently exists. We present a method by which “next generation” DNA sequencing output and shotgun proteomics data can be combined to produce annotated FASTA files, bypassing the need for annotated genetic information altogether in traditional proteomics workflows. The resulting material represents the first comprehensive annotated protein FASTA for any Cannabis plant. Using this annotated database as reference we can refine our protein identifications, resulting in the confident identification of 13,000 proteins with putative function. Furthermore, we demonstrate that post-translational modifications play an important role in the proteomes of Cannabis flower, particularly lysine acetylation and protein glycosylation. To facilitate the evolution of analytical investigations into these plant materials, we have created a portal to host resources developed from our proteomic and metabolomic analysis of Cannabis plant material as well as our results integrating these resources.

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

Cited by 78 publications

References 29 publications

Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L.

Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L.

Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast

The Cannabis Proteome Draft Map Project

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