高效合成NISIN乳酸乳球菌的简化基因组构建及其优化

As the L. lactis has many advantages such as the smaller genome, higher biosafety, more effective metabolization and higher protein expression ability, the construction of the simplified lactis genome could be very meaningful. In this proposal,the whole genome of lactis F44 with high nisin yield will be sequenced and the metabolism networks will be constructed. Upon the networks, the simplified lactis genome will be constructed through knocking out the secondary metabolites genes, IS sequences, and redundant sequences. Some genes for the lactic acid tolerance would be expressed appropriately in the simplified genome strain in order to overcome the problem that the lactis like the neutral environment and the nisin could be more stable in the acid environment. Meanwhile the nisin biosynthesis genes would be expressed appropriately to further improve the nisin production. We aim to get a lactis strain with the simplified genome, effective nisin production and high lactic acid tolerance. And this strain could be the chassis lactis strain with high biosafety for the expression of heterologous proteins

综合考虑乳酸乳球菌基因组小、生物安全性高、代谢旺盛、蛋白表达能力强等优点，本课题尝试进行构建乳酸乳球菌底盘微生物的探索性研究。本课题拟进行nisin高产菌株乳酸乳球菌F44的全基因组测序，构建该菌的基因组尺度代谢网络，在此基础上通过敲除其他次生代谢产物合成基因、敲除IS序列、敲除冗余序列的办法简化基因组，初步构建得到基因组缩小的乳酸乳球菌。针对菌株偏好中性pH值，而nisin在酸性pH值下稳定的问题，尝试适度表达部分耐酸基因，提高菌株的耐酸性；同时适度表达nisin合成相关基因，进一步提高nisin的生产效率。本课题预期可以获得高产nisin的小基因组的耐酸乳酸乳球菌，同时为构建一个具备高生物安全性的、可以高效表达酸性外源蛋白的乳酸乳球菌底盘奠定基础。

课题进行 nisin 高产菌株乳酸乳球菌 F44 及多株耐酸菌株的全基因组测序,构建了基因组尺度代谢网络。进行了酸应激下的转录组及蛋白质组分析，确定了多个与耐酸相关的转录因子。在此基础上通过敲除多个耐酸调控的基因，了解了这些基因的功能，并获得了缩小基因组的乳酸乳球菌。针对菌株偏好中性 pH值, 而 nisin 在酸性 pH值下稳定的问题, 表达了多个耐酸基因, 提高了菌株的耐酸性和nisin产量; 同时适度表达 nisin合成的关键基因nisI，提高了nisin的耐受性和nisin的产量。另外，课题对nisin 高产菌株乳酸乳球菌F44 小RNA进行深度测序，表达一系列耐酸基因及小RNA，确定了其作用靶点。并且从代谢角度，通过碳代谢分流提高菌株能量供应提高菌株的耐酸性，构建高产nisin的耐酸乳酸乳球菌。利用脱脂米糠和豆粕作为培养基部分原料对乳酸乳球菌生产nisin的工艺进行研究。