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J Biotechnol. 2018 Oct 20;284:17-26. doi: 10.1016/j.jbiotec.2018.07.031. Epub 2018 Jul 24.

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

Author information

1
Department of Technical Biochemistry, TU Dortmund University, Emil-Figge Str. 66, 44227 Dortmund, Germany.
2
Department of Technical Biochemistry, TU Dortmund University, Emil-Figge Str. 66, 44227 Dortmund, Germany. Electronic address: oliver.kayser@tu-dortmund.de.

Abstract

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.

KEYWORDS:

CBDA synthase; Cannabichromenic acid; Cannabidiolic acid; Komagataella phaffii; Site-directed mutagenesis; THCA synthase; Δ(9)-tetrahydrocannabinolic acid

PMID:
30053500
DOI:
10.1016/j.jbiotec.2018.07.031
[Indexed for MEDLINE]

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