Fermentative Production of Thymidine by a Metabolically Engineered
            <i>Escherichia coli</i>
            Strain

Lee, Hyeon-Cheol; Kim, Jin Ha; Kim, Jin Sook; Jang, Wonhee; Kim, Sang Yong

doi:10.1128/aem.02328-08

Cited by 14 publications

(15 citation statements)

References 51 publications

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“…We found that dietary supplementation of uridine, thymidine, or cytidine, but not other nucleosides, fully restored the fertility of cdd-1/2 DKO worms ( Table 2). The rescue effect of uridine (product of CDD enzymes) or thymidine (derivative of uridine) (Lee et al 2009) is consistent with the previous finding that both CDD-1 and CDD-2 are authentic CDDs (Thompson et al 2002).…”

Section: Resultssupporting

confidence: 79%

Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling inC. elegans

et al. 2016

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Animals alter their reproductive programs to accommodate changes in nutrient availability, yet the connections between known nutrient-sensing systems and reproductive programs are underexplored, and whether there is a mechanism that senses nucleotide levels to coordinate germline proliferation is unknown. We established a model system in which nucleotide metabolism is perturbed in both the nematode Caenorhabditis elegans (cytidine deaminases) and its food (Escherichia coli); when fed food with a low uridine/thymidine (U/T) level, germline proliferation is arrested. We provide evidence that this impact of U/T level on the germline is critically mediated by GLP-1/Notch and MPK-1/MAPK, known to regulate germline mitotic proliferation. This germline defect is suppressed by hyperactivation of glp-1 or disruption of genes downstream from glp-1 to promote meiosis but not by activation of the IIS or TORC1 pathways. Moreover, GLP-1 expression is post-transcriptionally modulated by U/T levels. Our results reveal a previously unknown nucleotide-sensing mechanism for controlling reproductivity.

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Section: Resultssupporting

confidence: 79%

Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling inC. elegans

et al. 2016

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“…For cloning host E. coli XL1-Blue (Stratagene, La Jolla, CA) was used. For the enhancement of thymidine biosynthesis, a thymidineoverproducing strain E. coli BLdtug24 strain was used (Lee et al, 2009). E. coli BLdtug24 and BLdtugp24 contained two expression vectors pETD::TdNr derived on pETDuet (Novagen, Madison, WI) and pACD::DUTm derived from pACYCDuet (Novagen) including foreign genes for producing thymidine in E. coli (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…In previous studies, we facilitated the biosynthesis of thymidine in a BLdtug24 strain derived from Escherichia coli BL21 by inactivating enzymes which are involved in the salvage pathway and DNA repair system and by modifying the genetic regulation and increasing the gene dosage of key enzymes contributing to thymidine biosynthesis (Lee et al, 2009). Along with the direct pathway engineering mentioned above, altering the level of cofactors which are involved in thymidine biosynthesis can be a possible approach for further enhancing thymidine production.…”

Section: Introductionmentioning

confidence: 96%

High NADPH/NADP+ ratio improves thymidine production by a metabolically engineered Escherichia coli strain

Lee¹,

Kim²,

Jang

et al. 2010

Journal of Biotechnology

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“…We previously reported several kinds of plasmid-based production strains (5)(6)(7)(8). These strains also highlighted the development of the thymidine production process but still had many inherent problems.…”

Section: Discussionmentioning

confidence: 99%

“…Thymidine production in these strains has been further improved by random mutation and selection. A recent report described the production of thymidine by rationally engineered Escherichia coli strains, in which thymidine-biosynthesizing genes were amplified and thymidine-degrading genes were disrupted (5)(6)(7)(8). Studies of both constructed E. coli strains and Brevibacterium (and Corynebacterium) spp.…”

mentioning

confidence: 99%

Development of a Novel Plasmid-Free Thymidine Producer by Reprogramming Nucleotide Metabolic Pathways

Kim¹,

Jeong²,

Koo³

et al. 2015

Appl Environ Microbiol

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A novel thymidine-producing strain of Escherichia coli was prepared by genome recombineering. Eleven genes were deleted by replacement with an expression cassette, and 7 genes were integrated into the genome. The resulting strain, E. coli HLT013, showed a high thymidine yield with a low deoxyuridine content. DNA microarrays were then used to compare the gene expression profiles of HLT013 and its isogenic parent strain. Based on microarray analysis, the pyr biosynthesis genes and 10 additional genes were selected and then expressed in HLT013 to find reasonable candidates for enhancing thymidine yield. Among these, phage shock protein A (PspA) showed positive effects on thymidine production by diminishing redox stress. Thus, we integrated pspA into the HLT013 genome, resulting in E. coli strain HLT026, which produced 13.2 g/liter thymidine for 120 h with fedbatch fermentation. Here, we also provide a basis for new testable hypotheses regarding the enhancement of thymidine productivity and the attainment of a more complete understanding of nucleotide metabolism in bacteria. Thymidine is a commercially useful precursor in the chemical synthesis of various antiviral drugs, including stavudine and zidovudine (azidothymidine), the active ingredient in a formulation for the treatment of AIDS (1, 2). Thymidine, which is composed of 2-deoxyribose and a thymine base, has been produced by employing bacteria belonging to the genera Brevibacterium and Corynebacterium (3, 4). These strains were improved by chemical mutagenesis. Thymidine production in these strains has been further improved by random mutation and selection. A recent report described the production of thymidine by rationally engineered Escherichia coli strains, in which thymidine-biosynthesizing genes were amplified and thymidine-degrading genes were disrupted (5-8). Studies of both constructed E. coli strains and Brevibacterium (and Corynebacterium) spp. have used the same strategies, even though the productivities in those microorganisms were much lower than that in the E. coli recombinant strain (Ͻ500 mg/liter). The first shared strategy was the inactivation of the salvage pathway for preventing the degradation of thymidine. The second strategy was the enhancement of the reduction of nucleoside diphosphate (NDP) to deoxynucleoside diphosphate (dNDP). The third strategy was the enhancement of thymidylate synthase. In E. coli, these strategies could be achieved by deleting deoABCD, tdk, and udp with a rationally designed gene knockout and overexpression of phage T4 NDP reductase subunits and T4 thymidylate synthase (td). To reduce feedback inhibition, we overexpressed T4 thymidylate synthase and T4 NDP reductase subunits, instead of intrinsic enzymes of E. coli. In Brevibacterium (and Corynebacterium) spp., these strategies could be achieved by screening thymidine auxotrophs after chemical mutagenesis, hydroxyurea-resistant mutants, and fluorouraciland trimethoprim-resistant mutants (3, 4).Of the enzymes required for thymidine biosynthesis, NDP reductase and d...

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Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Cited by 14 publications

References 51 publications

Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling inC. elegans

Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling inC. elegans

High NADPH/NADP+ ratio improves thymidine production by a metabolically engineered Escherichia coli strain

Development of a Novel Plasmid-Free Thymidine Producer by Reprogramming Nucleotide Metabolic Pathways

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