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Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the ß-oxidation reversal.
Lee, Seung Hwan; Hu, Yang; Chou, Alexander; Chen, Jing; Gonzalez, Ramon.
Afiliação
  • Lee SH; Department of Chemical, Biological, and Materials Engineering, University of South Florida, Tampa, FL, USA.
  • Hu Y; Department of Chemical, Biological, and Materials Engineering, University of South Florida, Tampa, FL, USA.
  • Chou A; Department of Chemical, Biological, and Materials Engineering, University of South Florida, Tampa, FL, USA.
  • Chen J; Department of Chemical, Biological, and Materials Engineering, University of South Florida, Tampa, FL, USA.
  • Gonzalez R; Department of Chemical, Biological, and Materials Engineering, University of South Florida, Tampa, FL, USA.
Metab Eng ; 82: 262-273, 2024 Mar.
Article em En | MEDLINE | ID: mdl-38387675
ABSTRACT
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 ß-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. 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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Escherichia coli / Engenharia Metabólica Idioma: En Revista: Metab Eng Assunto da revista: ENGENHARIA BIOMEDICA / METABOLISMO Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Escherichia coli / Engenharia Metabólica Idioma: En Revista: Metab Eng Assunto da revista: ENGENHARIA BIOMEDICA / METABOLISMO Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos