Characterizing and utilizing oxygen-dependent promoters for efficient dynamic metabolic engineering

Promoters adjust cellular gene expression in response to internal or external signals and are key elements for implementing dynamic metabolic engineering concepts in fermentation processes. One useful signal is the dis-solved oxygen content of the culture medium, since production phases often proceed in anaerobic conditions. Although several oxygen-dependent promoters have been described, a comprehensive and comparative study is missing. The goal of this work is to systematically test and characterize 15 promoter candidates that have been previously reported to be induced upon oxygen depletion in Escherichia coli. For this purpose, we developed a microtiter plate-level screening using an algal oxygen-independent flavin-based fluorescent protein and addi-tionally employed flow cytometry analysis for verification. Various expression levels and dynamic ranges could be observed, and six promoters (nar-strong, nar-medium, nar-weak, nirB-m, yfiD-m, and fnrF8) appear partic-ularly suited for dynamic metabolic engineering applications. We demonstrate applicability of these candidates for dynamic induction of enforced ATP wasting, a metabolic engineering approach to increase productivity of microbial strains that requires a narrow level of ATPase expression for optimal function. The selected candidates exhibited sufficient tightness under aerobic conditions while, under complete anaerobiosis, driving expression of the cytosolic F1-subunit of the ATPase from E. coli to levels that resulted in unprecedented specific glucose uptake rates. We finally utilized the nirB-m promoter to demonstrate the optimization of a two-stage lactate production process by dynamically enforcing ATP wasting, which is automatically turned on in the anaerobic (growth -arrested) production phase to boost the volumetric productivity. Our results are valuable for implementing metabolic control and bioprocess design concepts that use oxygen as signal for regulation and induction.

Automated summary

This study systematically tested and characterized 15 promoter candidates that are induced upon oxygen depletion in Escherichia coli. Six of these promoters are particularly suited for dynamic metabolic engineering applications. The study also demonstrated the optimization of a two-stage lactate production process using these promoters.