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Enzymatic pathways have evolved uniquely preferred protein expression stoichiometry in living cells, but our ability to predict the optimal abundances from basic properties remains underdeveloped. Here, we report a biophysical, first-principles model of growth optimization for core mRNA translation, a multi-enzyme system that involves proteins with a broadly conserved stoichiometry spanning two orders of magnitude. We show that predictions from maximization of ribosome usage in a parsimonious flux model constrained by proteome allocation agree with the conserved ratios of translation factors. The analytical solutions, without free parameters, provide an interpretable framework for the observed hierarchy of expression levels based on simple biophysical properties, such as diffusion constants and protein sizes. Our results provide an intuitive and quantitative understanding for the construction of a central process of life, as well as a path toward rational design of pathway-specific enzyme expression stoichiometry.
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Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the β-oxidation reversal
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 syn thesis 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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- Award ID(s):
- 1916854
- PAR ID:
- 10567324
- Publisher / Repository:
- Elsevier-ScienceDirect
- Date Published:
- Journal Name:
- Metabolic engineering
- Volume:
- 82
- ISSN:
- 1096-7184
- Page Range / eLocation ID:
- 262-273
- Subject(s) / Keyword(s):
- Iterative metabolic pathways Combinatorial Biosynthesis Orthogonal control of gene expression RBOX TriO
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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