苜蓿中华根瘤菌腺苷咕啉醇酰胺磷酸的合成机理

Vitamin B12 is an important chemical derived from certain bacteria and archaea. De novo pathway of vitamin B12 includes the aerobic pathway and anaerobic pathway. We have recently characterized the biosynthetic pathway of the intermediate adenosylcobinamide-phosphate in the aerobic-pathway-utilizing Rhodobacter capsulatus. However, how adenosylcobinamide-phosphate is formed in other bacteria is still unclear. Therefore, L-threonine kinase, L-Threonine-O-3-phosphate decarboxylase and adenosylcobinamide-phosphate synthase in the biosynthetic pathway of adenosylcobinamide-phosphate from a non-photosynthetic Sinorhizobium meliloti 320 will be characterized via in vitro and in vivo assay in this proposed project. Afterwards, adenosylcobinamide-phosphate and vitamin B12 can be formed with the involvement of (R)-1-Amino-2-propanol O-2-Phosphate in Escherichia coli chassis cells which synthesize adenosylcobyric acid. And then vitamin B12 production of recombinant strains will be improved via tuning the metabolic flux of (R)-1-Amino-2-propanol O-2-Phosphate branch. This project will elucidate the synthesis mechanism of adenosylcobinamide-phosphate in non-photosynthetic bacteria and will be of great importance in engineering of industrial strains for vitamin B12 production.

维生素B12是来源于某些细菌或古细菌的重要化学品。维生素B12从头合成途径分为好氧与厌氧合成途径。我们近期在利用好氧途径的Rhodobacter capsulatus中鉴定出其中间体腺苷咕啉醇酰胺磷酸的合成途径，但是在其它细菌中腺苷咕啉醇酰胺磷酸如何生成至今仍不清晰。本项目拟通过体外与体内实验验证鉴定一种非光合细菌Sinorhizobium meliloti 320腺苷咕啉醇酰胺磷酸合成途径的L-苏氨酸激酶、L-苏氨酸磷酸脱羧酶和腺苷咕啉醇酰胺磷酸合成酶。在此基础上，在合成腺苷钴啉胺酸的大肠杆菌底盘细胞中利用(R)-1-氨基-2-丙醇O-2-磷酸作为底物合成腺苷咕啉醇酰胺磷酸和维生素B12。然后通过调节(R)-1-氨基-2-丙醇O-2-磷酸支路的代谢流来提高重组菌的维生素B12产量。本项目不仅能阐明非光合细菌中间体腺苷咕啉醇酰胺磷酸的合成机制，也对维生素B12生产菌株的改造具有重要意义。

维生素B12是来源于某些细菌或古细菌的重要化学品。维生素B12从头合成途径分为好氧与厌氧合成途径。好氧合成途径中腺苷咕啉醇酰胺磷酸如何生成具有争议。本项目通过体外与体内实验验证鉴定一种非光合细菌Sinorhizobium meliloti 320腺苷咕啉醇酰胺磷酸合成途径的L-苏氨酸磷酸脱羧酶和腺苷咕啉醇酰胺磷酸合成酶。通过多层级代谢工程策略构建了高产氢咕啉酸的生产菌株，通过筛选酶并对酶的表达强度进行调节解除了合成途径中CobB和CobNST参与的代谢瓶颈。在此基础上，在合成腺苷咕啉醇酰胺磷酸的大肠杆菌底盘细胞中利用腺苷钴啉胺酸和(R)-1-氨基-2-丙醇O-2-磷酸合成腺苷咕啉醇酰胺磷酸和维生素B12，证明了好氧与厌氧途径中从钴（II）啉酸a，c-二酰胺到腺苷钴胺素的步骤是相同的。然后通过调节(R)-1-氨基-2-丙醇O-2-磷酸支路的代谢流来提高重组菌的维生素B12产量。本项目不仅能阐明非光合细菌中间体腺苷咕啉醇酰胺磷酸的合成机制，也对维生素B12生产菌株的改造具有重要意义。