Production of thymidine by metabolically engineered brevibacterium helvolumBrevibacterium helvolum을 이용한 β-thymidine의 생산

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Thymidine is a pyrimidine nucleoside that is used as an intermediate in the synthesis of azidothymidine, an active ingredient in a formulation for the treatment of AIDS. Thymidine is currently produced by a multi-step chemical reaction. A microbial fermentation process could be used to produce thymidine biologically, but many of the enzymes related to nucleotide biosynthesis are highly regulated. In order to overcome the complex regulation steps, an analogue mutant resistant to fluorouracil, hydroxyurea, and trimethoprim was constructed. The culture medium from a 16-hour flask culture of this mutant contained 382.7 mg/l of thymidine, in addition to several nucleosides, amino acids, and urea. However, the accumulation of thymidine monophosphate (TMP) inside the cells suggested a low activity level of TMP phosphohydrolase, which degrades TMP to produce thymidine. This limitation was overcome by cloning the TMP phosphohydrolase gene of the unusual bacteriophage SP8 into the pEKEx1 expression vector and expressing the enzyme in analogue mutants. Bacteriophage SP8, a phage whose genome contains hydroxymethyl uracil instead of thymine, productively infects B. subtilis strains and expresses TMP phosphohydrolase after infection. Novel TMP phosphohydrolase of SP8 was identified and cloned by using 2D-electrophoresis, MS/MS sequencing and in vitro transcription / translation. SP8 TMP phosphohydrolase is free of complex nucleotide regulation and has a high TMP hydrolysis activity. As results, the level of TMP in the transformant harboring pEKEx::TMPase was substantially decreased and thymidine was accumulated at 499.1 mg/l concentrations in the culture medium.
Kim, Jung-Hoeresearcher김정희researcher
한국과학기술원 : 생물과학과,
Issue Date
231023/325007  / 000975312

학위논문(박사) - 한국과학기술원 : 생물과학과, 2003.8, [ xi, 81 p. ]


recombinant Brevibacterium; thymidine; SP8 TMP phosphohydrolase; SP8 TMP 포스포하드롤라제; 브레비박테리움; 싸이미딘

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