In the field of metabolic engineering, one of the key challenges is to address the imbalance of metabolic fluxes causing bottlenecks and undesired byproduct formation. Recent advances in synthetic biology can play an important role in addressing this challenge, one of which is the development of tools that allow dynamic control of gene expression. Various sensors have been demonstrated to control gene expression in transcription and translation level including temperature, pH, light and inducer molecules. A recent development of independent inducible promoter system is particularly interesting as it can be utilized to control relative gene expression of multiple pathway enzymes in an orthogonal manner. This approach can be useful especially for iterative pathways as it can introduce an additional control over regulating flux toward desired product formation with specific chain length and functional groups. Here, we developed a novel TriO vector containing three operons controlled by three independently inducible promoters, where each operon can easily be replaced in a plug and play manner via Golden Gate Assembly. The orthogonality of gene expression was validated through expression of different fluorescent proteins under each operon. The TriO vector is compatible with commonly used Duet vectors from Novagen, which harbors two IPTG-inducible T7lac promoters. Combining the two vector systems, we demonstrated the implementation of 4 independently inducible promoters into iterative β-oxidation reversal (rBOX) for butyrate production, of which the core and termination enzymes are independently controlled under the 4 promoters. Under the single inducible promoter system, butyrate production from rBOX has been successful only with endogenous thioesterase activity of E. coli because promiscuity of overexpressed thioesterase led to hydrolysis of acetyl-CoA, which is at higher concentration than butyryl-CoA. However, we have demonstrated for the first time that controlled expression of thioesterase can lead to higher selectivity toward butyrate than acetate in a thioesterase-deficient E. coli strain. Moreover, we have implemented Design of Experiment (DoE) analysis to find the optimum of 4x inducer concentrations with reduced number of experiments. The best result under the optimum inducer concentrations improved the butyrate titer by more than an order of magnitude from the same set of enzymes expressed under the single inducible promoter system.

Metabolic flux optimization in the reversal of the β-oxidation cycle via independent gene expression control

ACS Spring 2022

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