Overproduction of thymidine by recombinant brevibacterium helvolum amplified with thymidine monophosphate phosphohydrolase gene from bacteriophage PBS2.
A microbial fermentation process could be used to produce thymidine biologically but many of the enzymes related to nucleotide biosynthesis are highly regulated. To overcome the complex regulation steps, an analogue mutant of Brevibacterium helvolum resistant to fluorouracil, hydroxyurea, and trimethoprim was constructed. This mutant accumulated 380 mg thymidine 1(-1) in 16 h in shake-flask culture. However, the accumulation of thymidine monophosphate (TMP) inside the cells suggested a low activity of nucleotidase which degrades TMP to thymidine. This limitation was overcome by cloning the TMP phosphohydrolase (TMPase) gene of the unusual bacteriophage, PBS2. As a result, TMP in recombinant cells decreased from 230 micromol g(-1) cell to 20 micromol g(-1) cell with accumulation of 500 mg thymidine l(-1) in the medium.
Lee
Hyeon Cheol
HC
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Ahn
Jung Mi
JM
Lee
Sang Nam
SN
Kim
Jung Hoe
JH
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001951:Brevibacterium / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial; D005285:Fermentation; D005472:Fluorouracil / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D009154:Mutation; D009708:Nucleotidases / Q000235:genetics* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013936:Thymidine / Q000096:biosynthesis* / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013938:Thymidine Monophosphate; D014295:Trimethoprim; D009708:Nucleotidases; D010744:Phosphoric Monoester Hydrolases; C023163:thymidylate 5'-phosphatase; D005472:Fluorouracil; D013936:Thymidine; D006918:Hydroxyurea