热带假丝酵母β-紫罗兰酮工程菌的构建及其发酵优化

Construction and fermentation optimization ofβ-ionone engineered strain in Candida tropicalis

β-紫罗兰酮是一种来源于植物的不规则单萜化合物,具有良好的驱虫、抗菌、抗癌和抗氧化等生物活性,广泛应用于农业、食品、医药和香精香料等行业。热带假丝酵母是潜在的萜类高效合成底盘细胞。为了提高β-紫罗兰酮产量,该研究在课题组前期构建的β-紫罗兰酮工程菌的基础上,通过酶的亚细胞区室组合,构建了一株β-紫罗兰酮合成关键基因PhCCD1,同时在其胞质、过氧化物酶体和脂滴表达的β-紫罗兰酮工程菌PCL-01;然后,对其摇瓶发酵培养基装液量、培养基碳氮比、发酵温度和辅因子Fe^(2+)浓度等条件进行优化;最后进行5 L发酵罐小试。工程菌PCL-01的β-紫罗兰酮摇瓶产量为152.4 mg/L,较出发菌株增加50%;经过摇瓶发酵优化,β-紫罗兰酮产量为185.8 mg/L,提升22%;最后,在5 L发酵罐上可达403.9 mg/L。该研究对于其他萜类物质合成的菌株代谢改造具有借鉴意义,亦可为其他菌种发酵性能的研究提供参考价值。

β-ionone is a monoterpene compound found in plants that exhibits various biological activities,including deworming,antibacterial,anticancer,and antioxidant properties.It is commonly utilized in agriculture,food,medicine,and flavor industries.Candida tropicalis is a synthetic chassis cell with the potential for efficient terpenoid production.To enhance the yield ofβ-ionone,based on theβ-ionone engineered strain previously constructed by our research group,this study co-expressed PhCCD1,the key enzyme ofβ-ionone synthesis,in the cytoplasm,peroxisomes,and lipid droplets through subcellular compartment combination of enzymes.This resulted in the generation ofβ-ionone engineered strain PCL-01.Subsequently,this study optimized the conditions for shaking flask fermentation,including the liquid amount in the medium,the carbon-to-nitrogen ratio,the fermentation temperature,and the concentration of cofactor Fe^(2+).Finally,this study conducted a small-scale test in the 5 L bioreactor.The shake flask yield ofβ-ionone for the engineered strain PCL-01 was 152.4 mg/L,representing a 50%increase compared to the starting strain.After optimizing shake flask fermentation,the yield increased by 22%to 185.8 mg/L.Ultimately,in a 5 L bioreactor,the yield reached 403.9 mg/L.This study is significant for the metabolic transformation of other strains in terpenoid synthesis.It also provides valuable references for researching the fermentation performance of other bacterial species.