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Metab Eng. 2015 May;29:86-96. doi: 10.1016/j.ymben.2015.03.001. Epub 2015 Mar 10.

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

Author information

1
Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802, United States.
2
Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802, United States; Department of Biological Engineering, Pennsylvania State University, University Park, PA 16802, United States. Electronic address: salis@psu.edu.

Abstract

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:

Biophysical models; Entner–Doudoroff; Genome engineering; NADPH; Pathway engineering; Synthetic biology

PMID:
25769287
DOI:
10.1016/j.ymben.2015.03.001
[Indexed for MEDLINE]

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