title: Rational design of a synthetic Entner-Doudoroff pathway for improved and controllable NADPH regeneration.
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%.
authors.affiliation: Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802, United States.
authors.forename: Chiam Yu
authors.initials: CY
authors.lastname: Ng
authors.affiliation: Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802, United States.
authors.forename: Iman
authors.initials: I
authors.lastname: Farasat
authors.affiliation: Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802, United States.
authors.forename: Costas D
authors.initials: CD
authors.lastname: Maranas
authors.affiliation: 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.
authors.forename: Howard M
authors.initials: HM
authors.lastname: Salis
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000096:biosynthesis* / Q000235:genetics; D009876:Operon; D017355:Zymomonas / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D009249:NADP; D005947:Glucose
keywords: Biophysical models; Entner–Doudoroff; Genome engineering; NADPH; Pathway engineering; Synthetic biology