Metabolic pathways of bacterial cells can be genetically modified to overproduce industrial fuels, chemicals, and pharmaceutical products in a cost-effective manner. Dynamic control systems that flexibly regulate metabolic pathway activity—or “flux”— can be used to enhance yield and optimize productivity by switching between “growth mode” and “production mode” by redirecting flux at the glucose-6-phosphate (G6P) node for the production of myo-inositol (MI). They can also be generalized to other applications such as enhancing flux in the pentose phosphate pathway to produce excess NADPH and enhance fatty acid synthesis.
The engineered production of metabolic pathway components in microbial hosts is generally low-yield, in part due to competition with endogenous pathways. As such, the overexpression of synthetic pathway compounds necessitates the coordinated regulation of host enzyme levels. However, metabolic flux balance varies significantly throughout the course of fermentation. Strategies that involve the static knockdown of native genes often result in detrimental effects to the host cells, including reduced growth and poor expression of recombinant proteins. Integrated, dynamic knockdown systems capable of regulating metabolic flux in response to cellular conditions without providing additional strain on the host cells offer a novel advantage in microbial chemical production.
This technology describes a method for the dynamic regulation of E. coli metabolism for the purpose of overexpressing MI and additional industrially relevant compounds. Controlling native enzyme levels is necessary when using engineered microbial systems to produce heterologous compounds. Flux through the glycolytic pathway is endogenously determined in response to intra- and extracellular conditions. The redirection of flux into a pathway for the production of MI from G6P is achieved via controlled degradation of the enzyme phosphofructokinase (Pfk-I). Pfk-I levels control the utilization of G6P; by adding the degradation tag SsrA to the coding sequence of PfkA, Pfk-I protein is degraded rapidly in the presence of adaptor protein SspB and slowly in its absence, thus in turn regulating glycolytic flux and the pool of G6P available for conversion into MI. Endogenous SspB is replaced with an anhydrotetracycline (aTc)-inducible version of the protein, thus generating a switch between growth mode (no aTc; Pfk-I ON) and production mode (aTc added; Pfk-I OFF). Further fine-tuning is achieved with respect to the timing and level of aTc induction. The system can also be optimized for use with different feedstocks, affording further flexibility in the production of MI.
Two-fold increase in MI yield
Dynamic regulation of native gene expression allows for fine-tuned
control over growth and production states
Post-translational control yields a quick phenotypic
Growth on different feedstocks (glucose, xylose, arabinose)