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

Paper ID Volume ID Publish Year Pages File Format Full-Text
24173 43503 2010 9 PDF Available
Title
High NADPH/NADP+ ratio improves thymidine production by a metabolically engineered Escherichia coli strain
Abstract

Thymidine is a commercially important precursor in the production of antiviral drugs, including azidothymidine for the treatment of AIDS. In order to produce thymidine in a large scale, we previously developed a thymidine-overproducing Escherichia coli strain BLdtug24 by engineering pathways. To further enhance thymidine yield, we increased the availability of a cofactor (NADPH) in thymidine biosynthesis by disrupting phosphoglucose isomerase in BLdtug24 to construct BLdtugp24. Additionally, NAD+ kinase or soluble transhydrogenase was overexpressed in BLdtugp24, which can reroute glucose metabolic flow from the EMP pathway to the PP pathway, to construct BLdtugp34N or BLdtugp34U, respectively. In chemostat cultures, BLdtugp24 had an increased NADPH availability and a 4-fold enhancement in thymidine yield for glucose compared with BLdtug24. BLdtugp34N and BLdtugp34U had increased thymidine yields for glucose by 1.2- and 2-fold compared with BLdtugp24, respectively. The NADPH/NADP+ ratios at steady-state had overall positive correlations with thymidine yields in these strains. Real-time RT-PCR analysis revealed that the transcriptional regulations of NAD(P)H-related enzymes and transhydrogenases affected the redox balance and shifted reaction equilibrium toward increasing NADPH. In fed-batch fermentation, BLdtugp34U with the highest NADPH/NADP+ ratio in chemostat experiment produced 1.9 g l−1 of thymidine with 29.7 mg l−1 h−1 of thymidine productivity.

Keywords
EMP pathway, Embden–Meyerhof–Parnas pathway; PP pathway, pentose phosphate pathway; zwf, glucose 6-phosphate dehydrogenase; gnd, 6-phosphogluconate dehydrogenase; icd, isocitrate dehydrogenase; mae, BNADP+-dependent malic enzyme; udhA, soluble pyridine tr
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High NADPH/NADP+ ratio improves thymidine production by a metabolically engineered Escherichia coli strain
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 149, Issues 1–2, 20 August 2010, Pages 24–32
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us