钝齿棒杆菌氮素调节因子AmtR_GlnK在L-精氨酸合成中的调控机理研究

L-arginine, as a conditionally essential amino acid in human nutrition, has numerous applications in food flavor and pharmaceutical. Its chemical formula is C6H14N4O2 and the high nitrogen content is 32.1% of arginine. Due to the highest nitrogen to carbon ratio among the 21 proteinogenic amino acids, the arginine catabolism serves on the one hand to mobilize nitrogen storages. Therefore, arginine metabolism plays a key role in nitrogen assimilation and distribution.. Corynebacterium crenatum SYPA is an aerobic and industrial L-arginine producer. Some efficient strategies and elevations have been used to improve the L-arginine producing ability in Corynebacterium crenatum SYPA. As a result, the L-arginine concentration of the batch fermentation reached a high level of 76.4 g/L in the recombinant strain. However, over the past 10 years, research mainly on arginine metabolic engineering has generated significant progress in our understanding of the carbon metabolism in arginine biosynthesis, whereas several crucial regulation questions on the nitrogen metabolism remain unanswered. The present project highlights challenges for research on the regulation mechanism of the regulator AmtR_GlnK in nitrogen matabolism and arginine biosynthesis in C. crenatum SYPA. This project will aim to understand the connection of the nitrogen regulatory networks and the arginine biosynthesis pathway. The mechanisms involve in AmtR control regulating gene expression and protein activity are repression of transcription, GlnK protein-complex formation and protein modification by adenylylation in C. crenatum. For this purpose, a powerful combination of bioinformatics, transcriptome, and proteome analyses as well as flux and metabolome analyses will be necessary, leading towards a systems biology approach and a holistic view on the C. crenatum cell. Furthermore, by developing metabolic engineering and modular pathway engineering methods, the strain will be adapted the metabolic pathway of nitrogen regulation to increase the arginine production.

L-精氨酸是一种半必需氨基酸，其氮（N）素占量高达32.1%，是天然氨基酸中N:C比例最高的氨基酸。L-精氨酸的合成是细胞N源利用的一个重要途径，L-精氨酸合成效率与细胞N源吸收与利用效率密切相关。钝齿棒杆菌SYPA是项目组具有独立自主知识产权的精氨酸生产菌株，项目组已针对精氨酸合成途径中碳代谢进行了系统代谢工程改造。本项目拟在前期工作基础上，通过生物信息学、组学分析、EMSA、Pulldown等方法深入研究钝齿棒杆菌SYPA中AmtR在N源吸收、利用与L-精氨酸合成中的转录调控和GlnK信号转导蛋白的后转录调控机制，解析AmtR_GlnK在L-谷氨酸、L-谷氨酰胺和L-精氨酸合成代谢途径中的协同调控机理，利用系统代谢工程和模块化策略对AmtR_GlnK调控目标代谢网络进行精细调控，实现胞内L-谷氨酸和L-谷氨酰胺的高效合成与利用，进一步提高L-精氨酸生产效率。

L-精氨酸的合成与细胞氮源吸收与利用效率密切相关，本研究以高产L-精氨酸的钝齿棒杆菌(Corynebacterium crenatum)作为出发菌株，通过转录组和差异蛋白组系统研究N源调节因子AmtR_GlnK在钝齿棒杆菌中N源吸收利用途径及L-精氨酸合成代谢中的调控机制。在敲除AmtR菌株中整合表达tacM启动子和铵转运蛋白AmtB增强NH4+吸收，同时串联表达氮代谢相关基因glnA-gltB-gltD，精氨酸产量为70.4 g·L-1。通过GST pull-down和ITC实验证实鉴定出在非光和细菌谷氨酸棒杆菌中PII信号转导蛋白（GlnK）可与L-精氨酸合成关键限速酶N-乙酰谷氨酸激酶(NAGK)具有相互作用，可缓解NAGK受L-精氨酸的反馈抑制。结合分子模拟及定点突变鉴定复合体中GlnK F11、R47和K85以及NAGK N258和R261是PII-NAGK关键结合位点。结果表明GlnK不但能通过上调L-精氨酸合成关键基因的转录水平，还能通过缓解NAGK受L-精氨酸的抑制，最终促进L-精氨酸的合成。对双功能尿苷酰转移/去除酶 GlnD 进行整合突变，将 H414和 D415位点突变为两个丙氨酸 AA；发现减弱 GlnD 尿苷酰去除酶的活性后，胞内尿苷酰化的 GlnK-UMP 增加，GlnK-UMP 与氮转录调控因子 AmtR 结合，转运至胞内的 NH4+浓度提高，促使 L-精氨酸产量显著提高；通过系统代谢工程适配各模块强度增加胞内谷氨酰胺合成可显著提高重要前体氨甲酰磷酸的合成，重组菌株ARG-CP4在5 L发酵罐中的L-精氨酸产量比SYPA5-5提高了54.5%，显著提高L-精氨酸的产量和铵利用率，为微生物发酵法生产L-精氨酸提供了优势菌株。.在Applied Environment Microbiology、Applied Microbiology and Biotechnology和微生物学报等期刊发表学术论文17篇；参加国际国内学术会议2 人次，发表学术会议论文2 篇；相关成果申请国际PCT专利并获美国授权1项，申请国家发明专利5项，授权国家发明专利4项。培养硕士研究生5名，联合培养博士研究生2名。