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.
journal_name
J Biotechnoljournal_title
Journal of biotechnologyauthors
Zirpel B,Kayser O,Stehle Fdoi
10.1016/j.jbiotec.2018.07.031subject
Has Abstractpub_date
2018-10-20 00:00:00pages
17-26eissn
0168-1656issn
1873-4863pii
S0168-1656(18)30569-8journal_volume
284pub_type
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