毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸  

Modification of Aromatic Amino Acid Synthetic Pathway in Pichia pastoris to Produce Cinnamic Acid andρ-Coumaric Acid

作　　者：陈鑫洁 钱芷兰 刘启 赵清[2] 张元兴[1] 蔡孟浩[1] CHEN Xin-jie;QIAN Zhi-lan;LIU Qi;ZHAO Qing;ZHANG Yuan-xing;CAI Meng-hao(State Key Laboratory of Bioreactor Engineering,East China University of Science&Technology,Shanghai 200237,China;Plant Science Research Center,Shanghai Chenshan Botanical Garden,Shanghai Key Laboratory of Plant Functional Genomics and Resources,Shanghai 201602,China)

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]中国科学院上海辰山植物科学研究中心上海市资源植物功能基因组学重点实验室,上海201602

出　　处：《中国生物工程杂志》2021年第10期52-61,共10页China Biotechnology

基　　金：国家重点研发计划(2018YFC1706202)资助项目。

摘　　要：目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。Objective:The Pichia pastoris strain was engineered to heterologously synthesize cinnamic acid andρ-coumaric acid,which were important intermediates of flavonoid biosynthetic pathway.The biosynthetic pathway of precursors aromatic amino acids was optimized to improve the production capacity of P.pastoris.Methods:Phenylalanine ammonia lyase from Rhodotorula glutinis was expressed in P.pastoris GS115 by ethanol induced artificial transcription system,and the key enzymes or their mutants in the biosynthetic pathway of intracellular aromatic amino acids were overexpressed in the recombinant strain.Results:Heterologous expression of phenylalanine ammonia lyase could convert L-phenylalanine and L-tyrosine produced by P.pastoris into cinnamic acid(38.8 mg/L)andρ-coumaric acid(34.2 mg/L).Through overexpression of related enzymes,the yields of cinnamic acid andρ-coumaric acid reached 124.1 mg/L and 302.0 mg/L,respectively.Conclusion:Cinnamic acid andρ-coumaric acid were successfully synthesized by P.pastoris,and the biosynthetic pathway of intracellular aromatic amino acids was optimized.It shows that P.pastoris has the application potential to produce flavonoids,and it also lays a foundation for the heterologous synthesis of other aromatic amino acid derivatives or plant compounds in P.pastoris.

关 键 词：毕赤酵母 芳香族氨基酸 肉桂酸 对香豆酸 

分 类 号：Q789[生物学—分子生物学]