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Deoxycytidine production by metabolically engineered Corynebacterium ammoniagenes
Yun-Bom Lee1, Hong Baek, Sang-Kyum Kim
1Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Gyunggi, 449-791, Republic of Korea.
Journal of Microbiology (Seoul, Korea)
|March 4, 2011
Summary
Metabolically engineered Corynebacterium ammoniagenes N424 to overproduce deoxycytidine. Multiple genetic modifications, including resistance to hydroxyurea and 5-fluorouracil, significantly increased deoxycytidine yields by over 2,000-fold compared to the wild type.
Area of Science:
- Microbiology
- Metabolic Engineering
- Biotechnology
Background:
- Deoxycytidine is a crucial nucleoside with therapeutic applications.
- Efficient microbial production of deoxycytidine is essential for meeting demand.
- Previous methods for deoxycytidine isolation faced limitations in yield and purity.
Purpose of the Study:
- To develop a metabolically engineered strain of Corynebacterium ammoniagenes for high-yield deoxycytidine production.
- To identify and implement key genetic modifications to enhance deoxycytidine biosynthesis.
- To optimize fermentation conditions for maximizing deoxycytidine accumulation.
Main Methods:
- Random mutagenesis to introduce inosine auxotrophy (ino-) and hydroxyurea resistance (HU(r)).
- Introduction of thymine auxotrophy (thy(-)) to block dCTP conversion to TMP.
- Selection for resistance to 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC) to enhance carbamoyl phosphate synthase activity and relieve pyrimidine nucleoside repression.
Main Results:
- Engineered strains showed stepwise increases in deoxycytidine production.
- Mutant IM7 (ino-, HU(r), thy(-)) produced 81.3 mg/L deoxycytidine.
- Final strain IC14-C6 (ino-, HU(r), thy(-), 5-FU(r), 5-FC(r)) achieved 226.3 mg/L deoxycytidine, a >2,000-fold increase over the wild type.
- The final strain accumulated negligible amounts of other pyrimidines.
Conclusions:
- Metabolic engineering strategies, including auxotrophy and resistance selections, are effective for enhancing deoxycytidine production in C. ammoniagenes.
- The developed strain IC14-C6 represents a significant advancement in microbial deoxycytidine synthesis.
- This engineered strain holds potential for industrial-scale production of deoxycytidine.
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