Dataset Information

Deoxycytidine production by a metabolically engineered Escherichia coli strain.

ABSTRACT:

Background

Rational engineering studies for deoxycytidine production were initiated due to low intracellular levels and tight regulation. To achieve high-level production of deoxycytidine, a useful precursor of decitabine, genes related to feed-back inhibition as well as the biosynthetic pathway were engineered. Additionally, we predicted the impact of individual gene expression levels on a complex metabolic network by microarray analysis. Based on these findings, we demonstrated rational metabolic engineering strategies capable of producing deoxycytidine.

Results

To prepare the deoxycytidine producing strain, we first deleted 3 degradation enzymes in the salvage pathway (deoA, udp, and deoD) and 4 enzymes involved in the branching pathway (dcd, cdd, codA and thyA) to completely eliminate degradation of deoxycytidine. Second, purR, pepA and argR were knocked out to prevent feedback inhibition of CarAB. Third, to enhance influx to deoxycytidine, we investigated combinatorial expression of pyrG, T4 nrdCAB and yfbR. The best strain carried pETGY (pyrG-yfbR) from the possible combinatorial plasmids. The resulting strain showed high deoxycytidine yield (650 mg/L) but co-produced byproducts. To further improve deoxycytidine yield and reduce byproduct formation, pgi was disrupted to generate a sufficient supply of NADPH and ribose. Overall, in shake-flask cultures, the resulting strain produced 967 mg/L of dCyd with decreased byproducts.

Conclusions

We demonstrated that deoxycytidine could be readily achieved by recombineering with biosynthetic genes and regulatory genes, which appeared to enhance the supply of precursors for synthesis of carbamoyl phosphate, based on transcriptome analysis. In addition, we showed that carbon flux rerouting, by disrupting pgi, efficiently improved deoxycytidine yield and decreased byproduct content.

SUBMITTER: Kim JS

PROVIDER: S-EPMC4491880 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Publications

Deoxycytidine production by a metabolically engineered Escherichia coli strain.

Kim Jin-Sook JS Koo Bong-Seong BS Hyun Hyung-Hwan HH Lee Hyeon-Cheol HC

Microbial cell factories 20150707

<h4>Background</h4>Rational engineering studies for deoxycytidine production were initiated due to low intracellular levels and tight regulation. To achieve high-level production of deoxycytidine, a useful precursor of decitabine, genes related to feed-back inhibition as well as the biosynthetic pathway were engineered. Additionally, we predicted the impact of individual gene expression levels on a complex metabolic network by microarray analysis. Based on these findings, we demonstrated rationa ...[more]

PMID: 26148515

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Dataset Information

Deoxycytidine production by a metabolically engineered Escherichia coli strain.

Background

Results

Conclusions

Publications

Deoxycytidine production by a metabolically engineered Escherichia coli strain.

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