表达外源谷氨酰胺转胺酶基因的重组乳酸乳球菌抵抗多重环境胁迫的生理机制

Lactococcus lactis which is widely used as a starter in dairy industries, is also an efficient probiotic bacteria and the delivery of antigens or therapeutic proteins to human mucosal surfaces. In the applying process, L. lactis often encounters various environmental stresses, including oxidative stress, gastric acid stress and bile stress, which has a negative influence on cell activity. In a previous study, the tg gene from Streptoverticillium mobaraense that encodes the transglutaminase mature protein was cloned into L.lactis subsp. cremoris NZ9000,we found that compared with strain NZ9000 harboring pNZ8148 (the control strain) , strain NZ9000 harboring pFL010 ( the recombinant strain) had a remarkably improved resistance to oxidative stress, gastric acid stress and bile stress. To clarify the mechanism of the recombinant strain's significant ability to confront environmental stresses, we plan to carry out the research work mainly from two aspects,i.e.,intracellular metabolic pathway and extracellular cell wall. That is, to determine if some activated intracellular resistant system protects the recombinant strain from the stress, differential proteomics technology will be applied to analyze changes in the proteomic profile of the two strains responding to oxidative stress, gastric acid stress and bile stress. In addition, to clarify if the thickened cell wall is responsible for stress resistance of the recombinant strain, the tg gene from S. mobaraense will be cloned into another appropriate lactic acid bacteria strain. Then changes in the cell wall thickness of the selected strain will be observed by the transmission electron microscopy and comparative study on the stress resistance of the new recombinant strain and its control stain will be done. The result will help to L. lactic cells taken into human bodies remain active to ensure the maximum utilization of their health care functions at the same time offering maximum protection. And L lactis is the model strain of lactic acid bacteria, the result will also help to improve the stress resistant capability of other lactic acid bacteria.

乳酸乳球菌作为益生菌和活菌疫苗方面的研究已经广泛开展，其在应用过程中常会面临环境胁迫（氧、胃酸和胆盐胁迫），直接影响其发挥功效。申请者在前期研究中发现在乳酸乳球菌NZ9000中表达源自茂原链轮丝菌WSH-Z2的谷氨酰胺转胺酶(TG)能显著提高宿主菌抗氧、胃酸及胆盐胁迫的能力。本课题拟从胞内代谢、胞外细胞壁两方面对重组菌抵抗环境胁迫的生理机制进行研究。即应用差异蛋白质组学研究重组菌和对照菌在胁迫条件下胞内蛋白谱的差异，探明重组菌胁迫抗性是否与胞内某抗胁迫系统的激活有关；在另一合适的乳酸菌菌株中表达tg基因，观察重组菌株细胞壁变化以及其对胁迫条件的抗性，探明重组菌胁迫抗性是否与胞外增厚的细胞壁形成的"保护层"有关。研究结果将有助于在保证安全性的前提下，使人体摄取的乳酸乳球菌能在体内最大限度地发挥作用。同时，乳酸乳球菌是乳酸菌的模式种，研究结果也将有助于改善其他乳酸菌的抗胁迫性能。

乳酸乳球菌作为重要的微生物已广泛应用于食品工业、生物化工和医药工业，但其在应用过程中不可避免地会受到各种环境胁迫，影响其生物活性和生产效率。项目组在前期研究中发现，与对照菌株乳酸乳球菌NZ9000(pNZ8148)相比，表达外源谷氨酰胺转氨酶(TG酶)的乳酸乳球菌NZ9000重组菌株对氧、胃酸及胆盐胁迫的抗性显著提升，本项目从胞内代谢、细胞壁两方面对此现象的机理进行了研究。研究结果发现，发酵液pH会显著影响乳酸乳球菌NZ9000菌株胞内积累血红素的能力。当发酵液恒定保持在pH5.5时，乳酸乳球菌NZ9000(pNZ8148)对胞外血红素的吸收能力增强，对数末期每mg胞内蛋白中可积累最高值为14.44 μg的血红素，显著高于pH 5.0 (10.58 μg/mg蛋白)、pH 6.0 (12.29 μg/mg蛋白)和pH 5.5（2.34 μg/mg 蛋白）条件下的胞内血红素量。胞内血红素浓度的增加促进了细胞有氧呼吸的进行，在通气及血红素存在条件下，控制发酵液pH为5.5时，乳酸乳球菌NZ9000(pNZ8148)的最高生物量和生物量转化得率均显著高于其他三种pH条件下的。同时，发酵液恒定保持在pH5.5也最有利于提高菌体对H2O2胁迫的抗性。根据已取得的结果，项目组认为，重组菌由于表达了产生碱性物质（NH3）的TG酶，发酵液pH值比对照菌的高，更易从培养基中吸收血红素，有氧呼吸的高效进行提高了其抗氧胁迫的能力。本研究首次表明，发酵液pH对乳酸乳球菌的有氧呼吸效率存在显著影响，这有助于建立乳酸乳球菌的高密度培养方法，从而提高该菌种的工业生产性能。另一方面，研究发现，在培养基中添加TG酶可以显著提高乳酸乳球菌NZ9000(pNZ8148)菌株对多重环境（胆盐、胃液、抗生素、NaCl和低温胁迫）的抗性。透射电镜观察发现，在培养基中添加终浓度为9 U/ml的TG酶的乳酸乳球菌NZ9000(pNZ8148)细胞壁是未经TG处理的1.9倍，表明该菌株可利用增厚的细胞壁所形成的“保护层”抵抗多重环境胁迫。进一步研究发现，这种多重胁迫抗性的表型是菌株特异性的，即当细菌细胞壁肽聚糖中含有TG酶的作用底物-谷氨酰胺和赖氨酸时，TG酶处理就会增强细胞对环境胁迫的抗性。该研究结果有助于在保证安全性的前提下，使人体摄取的乳酸菌在体内最大限度地发挥作用。