代谢工程改造大肠杆菌增产酪醇  

Metabolic engineering of Escherichia coli for improving tyrosol production

作　　者：曾娇娇 余世琴 周景文[1,2] ZENG Jiaojiao;YU Shiqin;ZHOU Jingwen(Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China;Science Center for Future Foods,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,工业生物技术教育部重点实验室,江苏无锡214122 [2]未来食品科学中心(江南大学),江苏无锡214122

出　　处：《食品与发酵工业》2021年第22期8-15,共8页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2019YFA0904900)。

摘　　要：酪醇是橄榄苦苷、红景天苷和羟基酪醇等许多重要价值天然产物的前体物质,建立一个酪醇高产的平台菌株具有重要意义。由于大肠杆菌(Escherichia coli)具有繁殖速度快、易于基因操作等优点,该研究选择E.coli BL21(DE3)作为宿主菌株。通过在质粒pRSFDuet-1中应用组成型启动子P_(cspA)过量表达来自荷兰芹的芳香醛合成酶(aromatic aldehyde synthase,AAS),得到105.97 mg/L酪醇。在敲除苯丙氨酸竞争途径(pheA)、丙酮酸竞争途径(ptsG、crr、pykF)及对羟基苯乙酸(feaB)竞争途径以及解除反馈抑制(tyrR)的菌株中过量表达AAS,获得1055.36 mg/L酪醇。为了进一步强化代谢流,对E.coli来源的乙醇脱氢酶AdhE、AdhP、YahK及FrmA和酿酒酵母来源的Adh6进行了筛选。结果表明,酿酒酵母来源的Adh6表现最佳的脱氢酶活性,产生了1516.86 mg/L酪醇,产量提升了43.73%。基于启动子组合调控策略平衡代谢流,酪醇产量达到1810.46 mg/L,提升了19.40%。最后,对培养基中碳源浓度、氮源浓度以及CaCO_(3)浓度进行优化,得到2120.58 mg/L酪醇,产量提升了17.13%。研究结果为后续采用发酵法生产酪醇和相关的下游产物奠定了良好的基础。Tyrosol is an essential precursor of oleuropein,hydroxytyrosol and salidroside.Thus,it is of great importance to build a strain with high tyrosol production.In this study,Escherichia coli BL21(DE3)was chosen as the host strain,because it can reproduce quickly and it is easy to make gene manipulations.Aromatic aldehyde synthase(AAS)from Petroselinum crispum was overexpressed by a native constitutive promoter P_(cspA) with a plasmid pRSFDuet-1,resulting in 105.97 mg/L of tyrosol.Then PcAAS was overexpressed in a mutant strain with phenylalanine(pheA),pyruvate(ptsG,crr,pykF)and hydroxyphenylacetic acid(feaB)competition pathway knocked out and feedback inhibition released(tyrR),resulting in 1055.36 mg/L of tyrosol production.In order to further strengthen the metabolic flux,five alcohol dehydrogenase candidates were screened(AdhE,AdhP,YahK,FrmA from E.coli and Adh6 from Saccharomyces cerevisiae).The results showed that Adh6 from S.cerevisiae had the highest dehydrogenase activity,producing 1516.86 mg/L of tyrosol,which was improved by 43.73%.Besides,combinatorial promoter engineering of aas and adh 6 was applied to optimize the metabolic flux,as a result,1810.46 mg/L tyrosol was produced.Finally,fermentation optimizations on carbon source,nitrogen source and CaCO_(3) were conducted on shaking flasks.The titer of tyrosol increased to 2120.58 mg/L,rising by 17.13%.The results lay a good foundation for the further researches of tyrosol and related downstream products.

关 键 词：酪醇 大肠杆菌 代谢工程 组成型启动子 芳香醛合成酶 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]