基于嗜热链球菌半乳糖代谢调控机制的靶向基因组改造

Streptococcus thermophilus is the main strain for making yogurt, but it is unable to metabolize galactose and expel this sugar into the medium during lactose fermentation. As results, some fermented dairy products contain significant residual amounts of galactose. The presence of galactose was harmful to the individuals suffering from the rare disease galactosemia. Therefore, the availability of starter strains with improved galactose utilization capacity is desirable to develop high-quality dairy products. It was reported that the galactose-positive strains (Gal+) were resulted from the spontaneous mutation of the promoter sequence of galKTE gene or galR gene in the genome, suggesting that the change of promoter sequence of galKTE gene or galR was available for galactose utilization in Streptococcus thermophilus. Here, this project was focused on the galKET gene or galR gene promoter sequence as a target sequence, and randomly mutation it using degenerate primers to synthetize various promoter sequences; Meanwhile, rational design of transcriptional activation regulating protein dGalR by mutation the active site in the DNA binding domain of the regulatory protein GalR; then detection of the binding ability of the dGalR to different promoters by EMSA or ITC in vitro so as to identify the effective target promoter. Construction of the Gal+ derivatives by targeted editing the galKET gene or galR gene promoter and galR gene in the genome of S. thermophilus based on the the molecular tools available for lactic acid bacteria; subsequently analysis of the transcription level of galKET gene, enzymic activity of Leloir pathway and the effects of galactose metabolites on yogurt quality. The ultimate aim of this project is to improve the galactose utilization in Streptococcus thermophilus and provide excellent starter strains for high quality yogurt manufacture.

嗜热链球菌是生产酸奶的主要菌株，但它不利用半乳糖并将乳糖分解的半乳糖分泌至胞外，导致产品半乳糖含量高，危害人体健康。因此，提高半乳糖利用能力是选育发酵剂的重要课题。目前已报道利用半乳糖的嗜热链球菌（Gal+）突变株均源于半乳糖代谢基因galKTE或调节基因galR启动子的自发突变，其效率低。本项目拟以galKTE或galR启动子为靶点，利用简并引物随机突变其启动子；同时结合生物信息学预测调节蛋白GalR的DNA结合域并改造，理性设计调节蛋白dGalR；通过EMSA和ITC技术，体外检测dGalR与不同启动子的结合能力，寻找有效突变靶点。在此基础上，对嗜热链球菌基因组上galKTE或galR启动子和galR基因靶点定点编辑，构建Gal+突变株；分析该Gal+突变株galKTE基因转录水平、代谢流向及对酸奶品质影响。其目标通过遗传改造提高嗜热链球菌利用半乳糖能力，为选育优良发酵剂菌株奠定基础。

嗜热链球菌是重要的发酵剂菌株，广泛应用于酸奶和硬质奶酪的生产。但是该菌株只利用乳糖中的葡萄糖，而将半乳糖分泌至胞外，导致产品残糖量过高，不利于机体健康。基于此，本项目围绕如何提高嗜热链球菌的半乳糖利用能力或将其转化成塔格糖、低聚半乳糖等问题开展了研究：首先在嗜热链球菌基因组上鉴定到携带有利用半乳糖的基因簇galKTE，揭示了该菌株不利用半乳糖不是没有编码利用半乳糖的基因，而是在正常生长条件下基因簇galKTE受控于调控蛋白GalR的控制，使其低水平表达。为了解除GalR抑制，本项目通过设计兼并引物，随机突变galKTE启动子，构建启动子文库，获得了强启动子P59；利用同源重组，将强启动子P59替换掉S.thermophilus ST1基因组galKTE基因簇原来的启动子Pgal，结果galKTE基因簇的转录水平下调3.5倍，galR基因表达上调4.7倍，进一步抑制了半乳糖的利用，发现galR-galK启动子序列的变化不利于半乳糖利用。因此，利用强启动子P5通过质粒pSec，高效表达了galK和galKTE基因，获得了利用半乳糖的重组菌S.thermophilus ST1/galK和S.thermophilus ST1/galKTE；突破了国外通过自然筛选获得Gal+菌株的专利保护。.鉴于遗传修饰嗜热链球菌的安全性以及GalR蛋白自身调控机制的限制，本项目没有进一步改造调控蛋白GalR，而是选择了利用半乳糖的植物乳杆菌WCFS1，将其与嗜热链球菌和德氏乳杆菌共发酵制备酸奶，结果发现植物乳杆菌的加入极大地降低了酸奶中总糖含量，尽管游离半乳糖浓度降低不明显，这是因为植物乳杆菌在分解乳糖的同时释放出更多的半乳糖。接着又利于代谢工程手段将阿拉伯糖异构酶引物植物乳杆菌，使半乳糖减少与塔格糖生产偶联，由此增强了酸奶的功能性；同时我们还筛选了β-半乳糖苷酶转糖基活性高的嗜热链球菌菌株，并通过定点突变提高半乳糖苷酶转糖基活性，将半乳糖转化成低聚半乳糖，增强了酸奶的益生功能。以上研究结果，为生产高品质强化性酸奶提供技术参考。