Journal
METABOLIC ENGINEERING
Volume 29, Issue -, Pages 86-96Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2015.03.001
Keywords
Entner-Doudoroff; NADPH; Pathway engineering; Genuine engineering; Biophysical models; Synthetic biology
Categories
Funding
- Department of Energy [DE-SC10822882]
- Air Force Office of Scientific Research [FA9550-14-1-0089]
- Office of Naval Research [N00014-13-1-0074]
- DARPA [N66001-12-C-4017]
- NSF Career Award [CBET-1253641]
- DARPA Young Faculty Award [N66001-10-1-4019]
- Directorate For Engineering
- Div Of Chem, Bioeng, Env, & Transp Sys [1253641] Funding Source: National Science Foundation
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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%. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
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