This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, H. C.
Right arrow Articles by Kim, S. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, H. C.
Right arrow Articles by Kim, S. Y.
Agricola
Right arrow Articles by Lee, H. C.
Right arrow Articles by Kim, S. Y.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, April 2009, p. 2423-2432, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.02328-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain{triangledown}

Hyeon Cheol Lee,1* Jin Ha Kim,1 Jin Sook Kim,1 Wonhee Jang,2 and Sang Yong Kim1

BioNgene Co., Ltd., 10-1, 1 Ka, Myungryun-Dong, Jongro-Ku, Seoul 110-521,1 Department of Life Science, Dongguk University, 3-26 Pil-Dong, Chung-Ku, Seoul 100-715, Republic of Korea2

Received 10 October 2008/ Accepted 14 February 2009

Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter–1 of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA N-glycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ~1.2-fold (740.3 mg liter–1). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.

* Corresponding author. Mailing address: BioNgene Co., Ltd., 10-1, 1 Ka, Myungryun-Dong, Jongro-Ku, Seoul 110-521, Republic of Korea. Phone: 82-2-747-9796. Fax: 82-2-747-0750. E-mail: churry{at}biongene.com

{triangledown} Published ahead of print on 27 February 2009.

Applied and Environmental Microbiology, April 2009, p. 2423-2432, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.02328-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»