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

【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.