【作者】 张彬；

【导师】 王启明；

【作者基本信息】 河北大学 ， 工程硕士（专业学位）， 2024， 硕士

【摘要】 大麻二酚（CBD）是来自植物大麻的一种天然化合物,不仅对人体的各种代谢、免疫和生理功能有积极的调节作用,而且具备减少癫痫发作和抗炎特性,有极大的研究价值。在大麻的腺毛分泌腔中,可以积累100多种大麻素,大麻二酚是含量丰富的大麻素之一。大麻二酚并非直接在植物大麻中合成,而是经过一系列步骤从大麻素的中心前体大麻萜酚酸（CBGA）催化生成前体大麻二酚酸（CBDA）,最终通过对CBDA的非酶脱羧处理形成CBD。通过植物大麻获取CBD存在生产周期长、产量低、产品不纯等问题。异源宿主生产可能是一种有效的替代方式。近年来,酿酒酵母作为代谢工程的宿主已被广泛用于生产有价值的化合物,尤其是在萜类化合物的生产中表现突出。本研究中,首先采用Ⅱ型CRISPR（成簇规则间隔短回文重复序列）Cas9基因编辑技术对酿酒酵母进行基因改造,将过表达异戊烯转移酶（CsPT4）、截断型异戊烯转移酶（CsPT4t）和大麻二酚酸合成酶（CBDAS）基因整合进酿酒酵母构建重组菌株。并对重组菌株进行发酵及体外催化验证该三种酶在酿酒酵母中表达后的功能活性,得到生成153pmol/L/OD/h大麻萜酚酸产物的酿酒酵母重组菌株CEN02。在此基础上又将截断型异戊烯转移酶CsPT4t基因、大麻二酚酸合酶CBDAS基因和定点突变后的MVA途径关键基因ERG20^F96W-N127W以及来自植物大麻的外源己酰-COA途径关键基因CsTKS和CsOAC分步导入同一个酿酒酵母菌株内,尝试在酿酒酵母内合成大麻萜酚酸（CBGA）和大麻二酚酸（CBDA）,将构建的重组菌株CEN08进行发酵及产物萃取后得到1.05 mg/L的大麻萜酚酸产物。最后在酿酒酵母内合成大麻萜酚酸产物的基础上对甲羟戊酸MVA途径中的关键基因ERG8、ERG19、ERG12、IDI 1、ERG10、ERG13进行过表达,以及对HMG1基因进行截断改造为HMG1t,通过增强MVA的代谢流量增强重组菌株大麻萜酚酸的合成,构建的重组菌株进行发酵萃取后得到1.9 mg/L的大麻萜酚酸产物,是未进行MVA代谢途径增强重组菌株的1.8倍。更多还原

【Abstract】 Cannabidiol（CBD）is a natural compound from the plant Cannabis sativa,which not only has a positive regulatory effect on various metabolic,immune and physiological functions in the human body,but also possesses seizure reduction and anti-inflammatory properties that are of great research value.In the secretory cavity of the glandular hairs of cannabis,more than 100 cannabinoids can be accumulated,and cannabidiol is one of the abundant cannabinoids.Cannabidiol is not synthesized directly in plant cannabis,but rather undergoes a series of steps to catalytically generate precursor cannabidiolic acid（CBDA）from Cannabigerolic acid（CBGA）,and ultimately CBD is formed through non-enzymatic decarboxylation of CBDA.Obtaining CBD from plant cannabis is associated with a long production cycle,low yields,and impurity in the product.Heterologous host production may be an effective alternative.In recent years,Saccharomyces cerevisiae has been widely used as a metabolically engineered host for the production of valuable compounds,with particular prominence in the production of terpenoids.In this study,the genes of overexpressed isoprenyltransferase（CsPT4）,truncated isoprenyltransferase（CsPT4t）,and cannabidiolate synthase（CBDAS）were firstly genetically modified in Saccharomyces cerevisiae by using a type-Ⅱ CRISPR（clusters of regularly interspaced short palindromic repeats）Cas9 gene editing technology to construct a recombinant strain by integrating the genes of overexpressed isoprenyltransferase（CsPT4）,truncated isoprenyltransferase（CsPT4t）and cannabidiolate synthase（CBDAS）into Saccharomyces cerevisiae.The recombinant strain was also subjected to fermentation and in vitro catalytic verification of the functional activities of the three enzymes after expression in Saccharomyces cerevisiae,and the recombinant strain CEN02 of Saccharomyces cerevisiae,which generates 153 pmol/L/OD/h Cannabigerolic acid product,was obtained.On this basis,the truncated isoprenyltransferase CsPT4t gene,the cannabidiolate synthase CBDAS gene,and the key genes of the MVA pathway,ERG20^F96W-N127W,as well as the key genes of the exogenous hexanoyl-COA pathway from the plant Cannabis sativa,CsTKS and CsOAC,were introduced into the same Saccharomyces cerevisiae strain in a step-by-step manner,and attempted to synthesize Cannabigerolic acid（CBGA）and cannabidiolic acid（CBDA）in Saccharomyces cerevisiae.Cannabis terpene phenolic acid（CBGA）and Cannabidiolic acid（CBDA）were synthesized in Saccharomyces cerevisiae.1.05mg/L of Cannabigerolic acid product was obtained after fermentation and product extraction of the constructed recombinant strain CEN08.Finally,based on the synthesis of Cannabigerolic acid products in Saccharomyces cerevisiae,overexpression of key genes in the mevalonate MVA pathway,ERG8,ERG19,ERG12,IDI 1,ERG10,and ERG13,as well as truncation of the HMG1 gene and transformation to HMG1t,enhanced Cannabigerolic acid synthesis in recombinant strains by augmenting the metabolic flux of MVA.The constructed recombinant strain was fermented and extracted to obtain 1.9 mg/L of Cannabigerolic acid product,which was 1.8 times higher than that of the recombinant strain without MVA metabolic pathway enhancement.更多还原