Production of Cannabidiol Acid Synthase by Chassis <i>Pichia pastoris</i> and of Its Catalytic Activity Analysis

NIU Tingli; TIAN Yuan; ZHANG Liguo; ZHANG Shuquan; LI Zhijiang; KANG Yue; CHI Jian; GAO Baochang; DAI Lingyan

doi:10.13386/j.issn1002-0306.2022120201

Volume 44 Issue 20

Oct. 2023

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Science and Technology of Food Industry > 2023 > 44(20): 135-142. > DOI: 10.13386/j.issn1002-0306.2022120201

NIU Tingli, TIAN Yuan, ZHANG Liguo, et al. Production of Cannabidiol Acid Synthase by Chassis Pichia pastoris and of Its Catalytic Activity Analysis[J]. Science and Technology of Food Industry, 2023, 44(20): 135−142. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022120201.

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Production of Cannabidiol Acid Synthase by Chassis Pichia pastoris and of Its Catalytic Activity Analysis

1.
College of Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing 163319, China
2.
Institute of Plant Chemistry, Daqing Branch of Heilongjiang Academy of Sciences, Daqing 163316, China
3.
Institute of Industrial Crop, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
4.
College of Food Science, Heilongjiang Bayi Agricultural University, Daqing 163319, China

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Received Date: December 25, 2022
Available Online: August 02, 2023

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Abstract

Abstract

In order to obtain the higher expression of cannabinoid acid synthase (CBDAS) through the microbial chassis, the recombinant enzyme for the in vitro synthesis of cannabinoid (CBD) was studied. The CBDAS gene was firstly cloned from cannabis leaves with higher content of CBD, its basic physicochemical properties of proteins were then analyzed by bioinformatics methods. After construction of the recombinant plasmid pPIC9K-CBDAS and transformation into Pichia pastoris, conditions of the recombinant protein CBDAS were optimized and its catalytic activities were finally analyzed. The results indicated that the constructed pPIC9K-CBDAS fusion protein expression system could be successfully expressed in P. pastoris. Under conditions of 1% of methanol addition, pH6.0 of medium, 48 h of induction, the maximum expression was obtained. At 4 h reaction, the product of cannabigerol acid (CBGA) was significantly increased, in which attributed to catalysis of substrate CBGA by the recombinant CBDAS. In the following, contents of CBD were significantly increased at 8 h (P<0.05) and those of CBDA and CBD were reached to the maximum at 12 h. After reactions at 12 h in the crude extract of cannabis leaf and the standardized CBGA, CBDAS could catalyze to product 60.64 and 20.12 ng/mL of CBDA, 128.01 and 207.87 ng/mL of CBD, respectively, in which CBDAS indicated a stronger catalytic activity. In conclusion, heterologous recombinant expression of cannabis CBDAS was achieved through yeast chassis, which provided active enzymes for the efficient synthesis of CBDA and CBD.
- Cannabis sativa L.,
- cannabidiol acid synthase,
- cannabidiol,
- heterologous expression,
- conditional optimization

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References

[1]	ANDRE C M, HAUSMAN J F, GUERRIERO G. Cannabis sativa: The plant of the thousand and one molecules[J]. Frontiers in Plant Science,2016,7:19.
[2]	BONINI S A, PREMOLI M, TAMBARO S, et al. Cannabis sativa: A comprehensive ethnopharmacological review of a medicinal plant with a long history[J]. Journal of Ethnopharmacology,2018,227:300−315. doi: 10.1016/j.jep.2018.09.004
[3]	ROSENBERG, E C, PATRA, P H, WHALLEY, B J. Therapeutic effects of cannabinoids in animal models of seizures, epilepsy, epileptogenesis, and epilepsy-related neuroprotection[J]. Epilepsy and Behavior, 2017, 70(Pt B): 319-327.
[4]	CROMBIE L, PONSFORD R, SHANI A, et al. Photochemical production of cannabicyclol from cannabichromene[J]. Tetrahedron Letters,1968,55:5771−5772.
[5]	MORIMOTO S, KOMATSU K, TAURA F, et al. Purification and characterization of cannabichromenic acid synthase from Cannabis sativa[J]. Phytochemistry,1998,49(6):1525−1529. doi: 10.1016/S0031-9422(98)00278-7
[6]	AMIN M R, ALI D W. Pharmacology of medical cannabis[J]. Advances in Experimental Medicine and Biology,2019,1162:151−165.
[7]	RUSSO E, GUY G W. A tale of two cannabinoids: The therapeutic rationale for combining tetrahydrocannabinol and cannabidiol[J]. Medical Hypotheses,2006,66(2):234−246. doi: 10.1016/j.mehy.2005.08.026
[8]	LI H, LIU Y, TIAN D, et al. Overview of cannabidiol (CBD) and its analogues: Structures, biological activities, and neuroprotective mechanisms in epilepsy and Alzheimer's disease[J]. European Journal of Medicinal Chemistry,2020,192:112−163.
[9]	NACHNANI R, RAUP-KONSAVAGE W M, VRANA K E. The pharmacological case for cannabigerol[J]. Journal of Pharmacology and Experimental Therapeutics,2021,376(2):204−212. doi: 10.1124/jpet.120.000340
[10]	GASTON T E, FRIEDMAN D. Pharmacology of cannabinoids in the treatment of epilepsy[J]. Epilepsy and Behavior,2017,70:313−318. doi: 10.1016/j.yebeh.2016.11.016
[11]	PISANTI S, MALFITANO A M, CIAGLIA E, et al. Cannabidiol: State of the art and new challenges for therapeutic applications[J]. Pharmacology and Therapeutics,2017,175:133−150. doi: 10.1016/j.pharmthera.2017.02.041
[12]	宁康, 董林林, 李孟芝, 等. 非精神活性药用大麻的应用及开发[J]. 中国实验方剂学杂志,2020,26(8):228−240. [NING K, DONG L L, LI M Z, et al. Application and development of non-psychoactive medicinal cannabis[J]. Chinese Journal of Experimental Formulary,2020,26(8):228−240. [NING K, DONG L L, LI M Z, et al. Application and development of non-psychoactive medicinal cannabis[J]. Chinese Journal of Experimental Formulary, 2020, 26(8): 228-240.
[13]	常丽, 李建军, 黄思齐, 等. 植物大麻活性成分及其药用研究概况[J]. 生命的化学,2018,38(2):273−280. [CHANG L, LI J J, HUANG S Q, et al. Overview of research on the active ingredients of plant cannabis and their medicinal uses[J]. The Chemistry of Life,2018,38(2):273−280. [CHANG L, LI J J, HUANG S Q, et al. Overview of research on the active ingredients of plant cannabis and their medicinal uses[J]. The Chemistry of Life, 2018, 38(2): 273-280.
[14]	MOHAMMADZADEH R, KARBALAEI M, SOLEIMANPOUR S, et al. Practical methods for expression of recombinant protein in the Pichia pastoris system[J]. Current Protocols,2021,1(6):155−161.
[15]	RASCHMANOVÁ H, WENINGER A, KNEJZLÍK Z, et al. Engineering of the unfolded protein response pathway in Pichia pastoris: Enhancing production of secreted recombinant proteins[J]. Applied Microbiology and Biotechnology,2021,5(11):4397−4414.
[16]	TÜRKANOĞLU Ö A, YıLMAZ S, INAN M. Pichia pastoris promoters[J]. Methods in Molecular Biology,2019,1923:97−112.
[17]	MASTROPIETRO G, AW R, POLIZZI K M. Expression of proteins in Pichia pastoris[J]. Methods in Enzymology,2021,660:53−80.
[18]	DALY R, HEARN M T. Expression of heterologous proteins in Pichia pastoris: A useful experimental tool in protein engineering and production[J]. Journal of Molecular Recognition,2005,18(2):119−138. doi: 10.1002/jmr.687
[19]	KARBALAEI M, REZAEE S A, FARSIANI H. Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins[J]. Journal of Cellular Physiology,2020,235(9):5867−5881. doi: 10.1002/jcp.29583
[20]	ZHENG X, ZHANG Y, ZHANG X, et al. Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins[J]. Microbial Cell Factories,2019,18(207):1−12.
[21]	THOMAS V, LEIGH G, ROBIN W, et al. Effect of plasmid design and type of integration event on recombinant protein expression in Pichia pastoris[J]. Applied and Environmental Microbiology,2018,84(6):2712−2717.
[22]	LI J S, CHEN L, SUN L, et al. Optimization of the secretory expression of recombinant human C-reactive protein in Pichia pastoris[J]. 3 Biotech,2017,7(5):1−8.
[23]	ELLIS S B, BRUST P F, KOUTZ P J, et al. Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris[J]. Molecular and Cellular Biology,1985,5(5):1111−1121.
[24]	赵明明, 李星颖, 江文康, 等. 猫血清白蛋白在毕赤酵母中的分泌表达[J]. 中国畜牧兽医,2022,49(3):897−903. [ZHAO M M, LI X Y, JIANG W K, et al. Secretory expression of feline serum albumin in Pichia pastoris[J]. China Veterinary Animal Husbandry,2022,49(3):897−903. ZHAO M M, LI X Y, JIANG W K, et al. Secretory expression of feline serum albumin in Pichia pastoris[J]. China Veterinary Animal Husbandry, 2022, 49(3): 897-903.
[25]	刘微, 戴凌燕, 苗青, 等. 一种经济高效的酵母菌落PCR模板DNA制备方法的建立[J]. 中国生物制品学杂志,2022,35(7):848−852. [LIU W, DAI L Y, MIAO Q, et al. Establishment of an economical and efficient method for preparing yeast colony PCR template DNA[J]. Chinese Journal of Biological Products,2022,35(7):848−852. LIU W, DAI L Y, MIAO Q, et al. Establishment of an economical and efficient method for preparing yeast colony PCR template DNA[J]. Chinese Journal of Biological Products, 2022, 35(7): 848-852.
[26]	卢可心. 产5-氨基乙酰丙酸重组毕赤酵母的构建及发酵研究[D]. 杭州: 浙江大学, 2022 LU K X. Studies on construction and fermentation of a recombinant Pichia pastoris 5-aminolevulinic acid[D]. Hangzhou: Zhejiang University, 2022.
[27]	蔡欣月. 转录因子AtVIP1介导IAA与ABA信号途径调控高粱侧根发育的分子机制初探[D]. 大庆: 黑龙江八一农垦大学, 2020 CAI X Y. The molecular mechanism of AtVIP1 mediates IAA and ABA signaling pathways to regulate lateral root development in sorghum-preliminary study[D]. Daqing: Heilongjiang Bayi Agricultural University, 2020.
[28]	TAURA F, SIRIKANTARAMAS S, SHOYAMA Y, et al. Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa[J]. FEBS Letters,2007,581(16):2929−2934. doi: 10.1016/j.febslet.2007.05.043
[29]	常丽, 唐慧娟, 李建军, 等. 大麻CBDA1基因的生物信息学分析[J]. 安徽农业科学,2017,45(29):144−148. [CHANG L, TANG H J, LI J J, et al. Bioinformatics analysis of CBDA1 gene in Cannabis sativa[J]. Anhui Agricultural Science,2017,45(29):144−148. CHANG L, TANG H J, LI J J, et al. Bioinformatics analysis of CBDA1gene in Cannabis sativa[J]. Anhui Agricultural Science, 2017, 45(29): 144-148.
[30]	姚昌阳, 谢兴欢, 蒋思婧, 等. 裂解多糖单加氧酶在毕赤酵母中的异源表达[J]. 湖北大学学报(自然科学版),2019,41(6):561−566. [YAO C Y, XIE X H, JIANG S J, et al. Heterologous expression of lysolysaccharide monooxygenase in Pichia pastoris[J]. Journal of Hubei University (Natural Science Edition),2019,41(6):561−566. YAO C Y, XIE X H, JIANG S J, et al. Heterologous expression of lysolysaccharide monooxygenase in Pichia pastoris[J]. Journal of Hubei University (Natural Science Edition), 2019, 41(6): 561-566.
[31]	关波, 金坚, 李华钟. 改良毕赤酵母分泌表达外源蛋白能力的研究进展[J]. 微生物学报,2011,51(7):851−857. [GUAN B, JIN J, LI H Z. Research progress on the ability of improved Pichia pastoris to secrete and express foreign proteins[J]. Journal of Microbiology,2011,51(7):851−857. GUAN B, JIN J, LI H Z. Research progress on the ability of improved Pichia pastoris to secrete and express foreign proteins[J]. Journal of Microbiology, 2011, 51(7): 851-857.

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