Chemostat selection of Escherichia coli mutants secreting thymidine, cytosine, uracil, guanine, and thymine

Tsen, S.-D.

doi:10.1007/bf00186965

Cited by 10 publications

(12 citation statements)

References 9 publications

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“…5). We found that thymidine production using BLdtug24 was significantly improved compared with that using other strains used in previous studies, while suppressing the formation of undesirable by-products to prevent thymidine loss (33,48,49). Foreign de novo biosynthetic genes were expressed to enhance thymidine production, while both the salvage pathway genes and ung were disrupted.…”

Section: Discussionmentioning

confidence: 78%

“…Because thymidine is required only in DNA synthesis, intracellular thymidine levels are very low and are tightly controlled (40). For the production of precursors for antiviral drugs, thymidine is either biologically produced in a low yield by a few modified microorganisms or chemically synthesized through a very costly process (17,33,48,49). Thus, there is a need for developing a more efficient strain for thymidine production on a large scale.…”

mentioning

confidence: 99%

“…Several different microorganisms have been modified for thymidine production, including E. coli, Brevibacterium helvolum, and Corynebacterium ammoniagenes, by classical mutagenesis methods, and they were selected based on their capacity to grow on toxic thymidine analogues (30,33,48,49). In these studies, feedback inhibition-resistant variants of thymidine biosynthetic enzymes were obtained by random mutation, and high-producing variants were selected.…”

mentioning

confidence: 99%

“…The most optimum B. helvolum strain obtained by this procedure produced 500 mg liter Ϫ1 of thymidine by batch fermentation (33). However, engineered B. helvolum and E. coli mutants also produced thymine, deoxyuridine, and uracil, which are unfavorable for thymidine production since it increases costs during the purification process (30,33,48,49). Furthermore, these thymidine-producing strains have residual thymidine degradation activities, resulting in decreased productivities.…”

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confidence: 99%

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Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Lee¹,

Kim²,

Kim³

et al. 2009

Appl Environ Microbiol

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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 Nglycosylase) 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 ϳ1.2-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.

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Section: Discussionmentioning

confidence: 78%

mentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Lee¹,

Kim²,

Kim³

et al. 2009

Appl Environ Microbiol

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“…byproducts can accumulate due to metabolic or regulational change (Tsen, 1994). Hence, to accumulate a large amount of deoxycytidine, cells must be resistant to feedback regulation and metabolically modified.…”

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confidence: 99%

Deoxycytidine production by metabolically engineered Corynebacterium ammoniagenes

et al. 2011

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A number of essential oils from Mongolian aromatic plants are claimed to have antimicrobial activities. The essential oil of Dracocephalum foetidum, a popular essential oil used in Mongolian traditional medicine, was examined for its antimicrobial activity. Eight human pathogenic microorganisms including B. subtilis, S. aureus, M. lutens, E. hirae, S. mutans, E. coli, C. albicans, and S. cerevisiae were examined. The essential oil of Dracocephalum foetidum exhibited strong antimicrobial activity against most of the pathogenic bacteria and yeast strains that were tested; by both the agar diffusion method and the minimum inhibitory concentration (MIC) assay (MIC range was 26-2592 microg/ml). Interestingly, Dracocephalum foetidum even showed antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. We also analyzed the chemical composition of the oil by GC-MS and identified several major components, including n-Mentha-1,8-dien-10-al, limonene, geranial, and neral.

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Evolutionary Engineering of Industrially Important Microbial Phenotypes

Sauer

2001

Advances in Biochemical Engineering/Biotechnology

181

164

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Chemostat selection of Escherichia coli mutants secreting thymidine, cytosine, uracil, guanine, and thymine

Cited by 10 publications

References 9 publications

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Deoxycytidine production by metabolically engineered Corynebacterium ammoniagenes

Evolutionary Engineering of Industrially Important Microbial Phenotypes

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