利用重组胞苷转移酶酿酒酵母生物合成胞二磷胆碱的工艺研究

Cloning of Cholinephosphate Cytidylyltransferase in Saccharomyces Cerevisiae and Biosynthesis Technology of Cytidine Diphosphatecholine

课题涉及一种过表达磷酸胆碱胞苷转移酶(Cholinephosphate cytidylyltransferase,EC 2.7.7.15,CCT)酿酒酵母基因工程菌的构建和应用。该菌株是将酿酒酵母来源的编码CCT酶基因cct与p YES2.0-Kanmx载体构建重组质粒,然后将其转化于酿酒酵母DFH中,构建基因工程菌QZ-016。通过过表达反应关键酶CCT酶而提高胞二磷胆碱(CDP-C)的转化率和产量。经过优化YPD培养基后,该菌株的菌浓PMV可达70.2 g/L,可应用于以CMP和CP为原料,生物转化制造CDP-C,反应7 h摩尔转化率可高达73.1%。该菌株的产率高,应用于生产CDP-C,具有成本低、制造能耗低、反应周期短等优点。

To achieve one yeast strain which can highly produce CDP-C,this research clarified the construction and application of a new genetic engineering yeast strain which over-expressed cholinephosphate cytidylyl transferase（ EC 2. 7. 7. 15,CCT）. To build the genetic engineering strain named QZ-016,the cct gene which encoded the CCT enzyme in S. cere was transferred to express vector p YES 2. 0-Kanmx. The recombinant plasmid was then transformed to S. cere DFH. The conversion rate and yield of cytidine diphosphatecholine（ CDP-C） was increased by the over-expression of CCT enzyme. After optimizing the YPD culture medium,the yeast concentration（ PMV） of the strain was 70. 2 g/L,which can be applied to biosynthesize CDP-C with CMP and CP as raw materials.CDP-C conversion rate of 7 h Moore can be as high as 73. 1%. To achieve efficient production of CDP-C,this genetic engineering yeast strain will be applied which has the advantages of lower cost,lower energy consumption,shorter reaction cycle and so on.Through this subject,the overexpressing of enzyme CCT in S. cere DFH can apparently enhance the yield of CDP-C and increase the PMV of strain DFH. This subject greatly improved the production efficiency,and provided a new way of thinking for the industrialization of the CDP-C. However,in order to obtain the best strains,the analysis of the flux distribution of CDP-C in the process of reaction、the glycolytic pathway、ATP regeneration way and the relationship between predicted strains of physiological characteristics need to be carried out deeply on the starting strain orientation transformation.