کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
31514 | 44807 | 2015 | 11 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration](/preview/png/31514.png)
• 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%.
Journal: Metabolic Engineering - Volume 29, May 2015, Pages 86–96