离子转运蛋白调控钝齿棒杆菌离子和pH稳态促进L-精氨酸合成

The ion transporter endows ion-and pH-homeostasis enhancing L-arginine synthesis in Corynebacterium crenatum

L-精氨酸是一种半必需氨基酸,广泛应用于食品、制药、饲料等行业。【目的】当前对L-精氨酸生产菌株的研究,极少涉及离子转运领域。在本研究中,发现在发酵时适量添加外源K+有利于促进钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5合成L-精氨酸。【方法】在C.crenatum SYPA5-5发酵培养基外源添加0.5 g/L和2.5 g/L的K3PO4,取对数期发酵样品进行转录组数据分析,挖掘出K+转运相关的阳离子转运ATP酶CTAP1以及单价阳离子/H+逆转运蛋白Mrp1A,研究其在C.crenatum SYPA5-5快速合成L-精氨酸阶段,对菌株生长及L-精氨酸合成的影响。【结果】对基因ctap1和mrp1分别进行敲除和过表达,深入研究突变株对L-精氨酸合成的影响。研究发现同时过表达离子转运蛋白CTAP1和Mrp1A更有利于胞内离子、pH稳态和渗透压调节,最终提高L-精氨酸的产量。在补料分批发酵中分别过表达Mrp1A、CTAP1以及同时过表达Mrp1A和CTAP1的菌株L-精氨酸产量分别达到61.4 g/L、63.9 g/L和65.3 g/L,产率分别为0.383 g/g、0.392 g/g和0.395 g/g,比C.crenatum SYPA5-5分别提高了34.9%、38.0%和39.1%。【结论】CTAP1是特异性的K+转运ATP酶,可以将培养基中的K+运输到胞内。同时Mrp1A可将胞内K+和Na+等单价阳离子运输到胞外,将胞外H+运输至胞内,中和胞内L-精氨酸所导致的碱性环境,从而维持胞内pH稳定。CTAP1和Mrp1A的研究为解析离子转运机制和L-精氨酸合成之间的联系奠定了基础。

L-arginine is a semi-essential amino acid that is widely used in food, pharmacy and feed industries. In recent years, the metabolic engineering of L-arginine producing strains is rarely involved in the field of ion transport. [Objective] In this study, we found that adding appropriate K+ in the medium was beneficial to Corynebacterium crenatum SYPA5-5 to increase L-arginine production. [Methods] Transcriptome of C. crenatum SYPA5-5 was analyzed under the concentrations of 0.5 g/L and 2.5 g/L K3PO4. We selected significant monovalent cation/H+ antiporter Mrp1A and cation transport ATPase CTAP1 to investigate the relation among K+ transport, growth and L-arginine synthesis in C. crenatum SYPA5-5. [Results] The genes mrp1 and ctap1 were deleted and overexpressed to analyze their effects on the production of L-arginine in C. crenatum SYPA5-5. The overexpression of Mrp1A and CTAP1 was beneficial to ion homeostasis, pH tolerance and osmoregulation, ultimately increasing L-arginine production. In fed-batch fermentation, L-arginine production of 5-5(mrp1), 5-5(ctap1) and 5-5(mrp1ctap1) strains reached 61.4 g/L, 63.9 g/L and 65.3 g/L. The yield reached 0.383, 0.392 and 0.395 g/g, which were 34.9%, 38.0% and 39.1% increase compared with C. crenatum SYPA5-5, respectively. [Conclusions] The CTAP1 is K+ transporter that can transport K+ from extracellular to intracellular. Meanwhile, the Mrp1A can transport K+ and Na+ to the extracellular, and the extracellular H+ was transported into the cell, thus maintaining the intracellular pH stability. The study of Mrp1A and CTAP1 provide a foundation for understanding the relationship between ion transport mechanisms and L-arginine synthesis.