阻断消耗途径提高毕赤酵母工程菌S-腺苷甲硫氨酸产量  

Blocking consumption pathway increases production of S-adenosyl-L-methionine by Pichia pastoris

作　　者：苏慧颖 蒙世拯 赵欣欣 钱江潮[2] 储炬[2] 秦秀林[1] SU Huiying;MENG Shizheng;ZHAO Xinxin;QIAN Jiangchao;CHU Ju;QIN Xiulin(State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,Guangxi Research Center for Microbial and Enzyme Engineering Technology,College of Life Science and Technology,Guangxi University,Nanning 530004,Guangxi,China;State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China)

机构地区：[1]广西大学生命科学与技术学院、亚热带农业生物资源保护与利用国家重点实验室、广西微生物与酶工程技术研究中心,广西南宁530004 [2]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《微生物学通报》2023年第2期441-453,共13页Microbiology China

基　　金：国家自然科学基金(31300076);广西壮族自治区自然科学基金(2019GXNSFAA245001,2018GXNSFAA281005,2017GXNSFAA198136)。

摘　　要：【背景】S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM)作为所有生物体内的重要中间代谢物,不仅可作为膳食补充剂,还具有良好的临床应用价值。【目的】将毕赤酵母重组菌GS115/DS16的SAM消耗途径阻断,进一步提高SAM的产量。【方法】分别敲除毕赤酵母重组菌GS115/DS16的S-腺苷同型半胱氨酸水解酶基因sah1、S-腺苷甲硫氨酸脱羧酶基因spe2和L-甲硫氨酰tRNA合酶基因msm1,构建工程菌G/Dsah、G/Dspe和G/Dmsm。检测3个工程菌的生长和SAM产量,以及L-Met添加量对SAM积累的影响。【结果】与出发菌GS115/DS16相比,工程菌G/Dsah、G/Dspe和G/Dmsm的单位菌体SAM产量分别提高了29.3%、55.6%和24.8%,其生长无显著差异。L-Met添加量优化后(0.06%),G/Dsah和G/Dmsm单位菌体的SAM产量分别提高了26.4%和28.9%。【结论】构建的毕赤酵母工程菌可用于SAM的工业化生产,该代谢工程策略可用于改进其他化学品的生产。[Background]S-Adenosyl-L-methionine(SAM)is an important intracellular metabolite that can be used as a dietary supplement and to treat a variety of diseases.[Objective]To enhance SAM production for industrial application by blocking SAM consumption pathways in the recombinant Pichia pastoris strain GS115/DS16.[Methods]The genes associated with the metabolism of SAM,sah1(encoding S-adenosyl-L-homocysteine hydrolase),spe2(encoding S-adenosylmethionine decarboxylase),and msm1(encoding mitochondrial methylthio-tRNA synthase),were knocked out in SAM-producing strain GS115/DS16.Accordingly,the engineered strains G/Dsah,G/Dspe,and G/Dmsm were constructed.The cell growth and SAM production of the three engineered strains were investigated.Additionally,the effect of methionine addition on SAM accumulation was studied.[Results]The knock-out did not affect cell growth,whereas it increased SAM production by 29.3%,55.6%,and 24.8%in G/Dsah,G/Dspe,and G/Dmsm,respectively,compared with the parental strain GS115/DS16.When L-Met addition was decreased from 0.10%to 0.06%,the SAM production increased by 26.4%and 28.9%in G/Dsah and G/Dmsm,respectively.[Conclusion]Therefore,the engineered P.pastoris strains can be utilized in industrial production of SAM in a cost-effective manner,and the strategy can also be employed for improving the production of other chemicals.

关 键 词：S-腺苷甲硫氨酸 毕赤酵母 S-腺苷甲硫氨酸脱羧酶 S-腺苷同型半胱氨酸水解酶 L-甲硫氨酰tRNA合酶 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]