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Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration

Paper ID Volume ID Publish Year Pages File Format Full-Text
31514 44807 2015 11 PDF Available
Title
Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration
Abstract

•We re-engineered the Entner–Doudoroff pathway from Zymomonas mobilis to rapidly regenerate NADPH in Escherichia coli.•Operons expressing the 5-enzyme pathway were designed by the Operon Calculator and integrated into the E. coli genome.•Measurements using enzyme assays, a NADPH-binding fluorescent reporter, and a NADPH-dependent biosynthesis pathway.•The pathway was optimized by combining the RBS Library Calculator with MAGE genome mutagenesis.

NADPH is an essential cofactor for the biosynthesis of several high-value chemicals, including isoprenoids, fatty acid-based fuels, and biopolymers. Tunable control over all potentially rate-limiting steps, including the NADPH regeneration rate, is crucial to maximizing production titers. We have rationally engineered a synthetic version of the Entner–Doudoroff pathway from Zymomonas mobilis that increased the NADPH regeneration rate in Escherichia coli MG1655 by 25-fold. To do this, we combined systematic design rules, biophysical models, and computational optimization to design synthetic bacterial operons expressing the 5-enzyme pathway, while eliminating undesired genetic elements for maximum expression control. NADPH regeneration rates from genome-integrated pathways were estimated using a NADPH-binding fluorescent reporter and by the productivity of a NADPH-dependent terpenoid biosynthesis pathway. We designed and constructed improved pathway variants by employing the RBS Library Calculator to efficiently search the 5-dimensional enzyme expression space and by performing 40 cycles of MAGE for site-directed genome mutagenesis. 624 pathway variants were screened using a NADPH-dependent blue fluorescent protein, and 22 were further characterized to determine the relationship between enzyme expression levels and NADPH regeneration rates. The best variant exhibited 25-fold higher normalized mBFP levels when compared to wild-type strain. Combining the synthetic Entner–Doudoroff pathway with an optimized terpenoid pathway further increased the terpenoid titer by 97%.

Keywords
Entner–Doudoroff; NADPH; Pathway engineering; Genome engineering; Biophysical models; Synthetic biology
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Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 29, May 2015, Pages 86–96
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