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

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。

Abstract:

[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.