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Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the β-oxidation reversal

Journal Article · · Metabolic Engineering
 [1];  [1];  [1];  [1];  [1]
  1. University of South Florida, Tampa, FL (United States)
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Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Here, considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.
Research Organization:
Montana State University, Bozeman, MT (United States)
Sponsoring Organization:
US National Science Foundation (NSF); USDOE
Grant/Contract Number:
EE0008499
OSTI ID:
2478675
Alternate ID(s):
OSTI ID: 2369739
Journal Information:
Metabolic Engineering, Journal Name: Metabolic Engineering Vol. 82; ISSN 1096-7176
Publisher:
ElsevierCopyright Statement
Country of Publication:
United States
Language:
English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Combinatorial Biosynthesis
Iterative metabolic pathways
Orthogonal control of gene expression
RBOX
TriO