Enhancement of thymidine production in E. coli by eliminating repressors regulating the carbamoyl phosphate synthetase operon

Koo, Bong Seong; Hyun, Hyung Hwan; Kim, Sang Yong; Kim, Chan Hwa; Lee, Hyeon-Cheol

doi:10.1007/s10529-010-0413-7

Cited by 17 publications

(17 citation statements)

References 19 publications

(22 reference statements)

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“…We previously worked to develop an optimal thymidine-producing strain of E. coli by expressing foreign genes, increasing NADPH/NADP ratios, and inactivating carAB repressors (8). Based on this work, we prepared an improved strain for industrial production using BLdtugRPA (see Table S1 in the supplemental material).…”

Section: Resultsmentioning

confidence: 99%

“…1). Under the same culture conditions, we compared HLT011 to BLdtug24 (5) and BLdtugRPA24 (8), which were previously constructed. Even though the expression levels of the integrated genes in our strains were not higher than plasmid-based expression, the thymidine production yield of HLT011 was the highest of any strains studied to date.…”

Section: Resultsmentioning

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%

“…The two promoters of carAB shared several transcription factors in their regulation, PurR (16), PepA (17), and ArgR (16,18). The disruption of these three repressors (purR, pepA, and argR, respectively) is an efficient engineering strategy to increase thymidine productivity (8).…”

mentioning

confidence: 99%

“…Based on this experience and knowledge, we prepared a plasmid-free strain for industrial production using BLdtugRPA (8). Even though a thymidine-producing E. coli strain was successfully developed as a plasmid-free strain with a lower burden of protein overexpression, it is possible that the deletions in the genomic regions not directly related to metabolite biosynthesis (ung, purR, pepA, argR, and rpoS) cause unwanted changes in cell physiology and growth retardation.…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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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The New Strategy of Breeding Cytidine Excessive Biosynthesis Mutants by pyr Operon Rearrangement of Bacillus amyloliquefaciens

Liu

Fang

et al. 2015

Lecture Notes in Electrical Engineering

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Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15

Fang

Xie

et al. 2012

Biotechnol Lett

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Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-D-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735 mg cytidine/l using glucose as substrate, which was approx. 128 % higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2 %) slightly and increased glucose uptake (72 %). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli.

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Enhancement of thymidine production in E. coli by eliminating repressors regulating the carbamoyl phosphate synthetase operon

Cited by 17 publications

References 19 publications

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

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

The New Strategy of Breeding Cytidine Excessive Biosynthesis Mutants by pyr Operon Rearrangement of Bacillus amyloliquefaciens

Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15

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