| Title |
Deoxycytidine Production by Metabolically Engineered Corynebacterium ammoniagenes |
| Author |
Yun-Bom Lee, Hong Baek, Sang-Kyum Kim, and Hyung-Hwan Hyun* |
| Address |
Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Gyunggi 449-791, Republic of Korea |
| Bibliography |
Journal of Microbiology, 49(1),53-57, 2011,
|
| DOI |
|
| Key Words |
Corynebacterium ammoniagenes, deoxycytidine, mutation, metabolic engineering, NTG, dCTP deaminase |
| Abstract |
Corynebacterium ammoniagenes N424 was metabolically modified to isolate overproducers of deoxycytidine. Inosine auxotrophy (ino-) was initially introduced to prevent the flow of PRPP (phosphoribosyl pyrophosphate) into the purine biosynthetic pathway by random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. Following that, mutants possessing hydroxyurea resistance (HUr) were isolated to increase the activity of ribonucleoside diphosphate reductase, which catalyzes the reduction of ribonucleoside diphosphate to deoxyribonucleoside
diphosphate. Then, in order to block the flow of dCTP into the TMP biosynthetic pathway via dUTP, thymine auxotrophy (thy-) was introduced into the mutant IH30 with ino- and HUr. The resulting mutant IM7, possessing the characteristics of ino-, HUr, and thy-, was deficient in dCTP deaminase and produced significantly higher amounts of deoxycytidine (81.3 mg/L) compared to its mother strain IH30 (6.2 mg/L). Deoxycytidine productivity was further enhanced by isolating the mutant IU19, which was resistant to 5-fluorouracil, an inhibitor of carbamoyl phosphate synthase. This enzyme catalyzed the synthesis of carbamoyl phosphate from glutamine, HCO3
-, and ATP. 5-Fluorouracil also inhibited aspartate transcarbamoylase, catalyzeing the condensation of carbamoyl phosphate and aspartate. Finally, 5-fluorocytosine
resistance (FCr) was introduced into the mutant strain IU19 to relieve the repression caused by accumulation of pyrimidine nucleosides. The mutant strain IC14-C6 possessing all the five characteristics described above produced 226.3 mg/L of deoxycytidine, which was at least 2,000 fold higher compared to the wild type,
and accumulated only a negligible amount of other pyrimidines under shake flask fermentation. |
