Oxygen-regulated microorganisms
Inventors
Blattner, Frederick R. • Campbell, John Walter • Shi, Nian • Stahl, Buffy
Assignees
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Publication Number
US-8900849-B2
Publication Date
2014-12-02
Expiration Date
Abstract
The present invention relates to novel strains of microorganisms with oxygen-regulated metabolism. The microorganisms have higher growth rates and are more efficient than parental strains. The microorganisms may be used to produce a variety of products of interests, such as recombinant proteins, nucleic acids, such as DNA, amino acids, and chemicals.
Core Innovation
The invention relates to engineered oxygen-regulated metabolism in an E. coli, where metabolic flux from a carbon source is decreased in response to a reduction in oxygen levels. An oxygen-regulated promoter is operatively linked to a sequence that controls carbon-source metabolism, and the disclosed promoters include a cytochrome o oxidase (cyo) promoter and a superoxide dismutase (sodA) promoter, providing oxygen-dependent regulation of metabolic flux.
The oxygen-regulated promoter is operatively linked to a sequence comprising sucrose catabolism (csc) module gene material, including a sequence comprising the cscA gene of the E. coli O157.H7 chromosomally-encoded sucrose catabolism (csc) module. The described E. coli is sucrose catabolizing (Suc+) when the oxygen-regulated promoter is operatively linked to the cscA-containing sequence, coupling oxygen-regulated control to sucrose utilization.
In addition, the disclosed construct includes a sequence comprising the phosphoenol pyruvate-dependent phosphotransferase system (pts) genes ptsH, ptsI and carbohydrate repression resistant (crr), as part of the operatively linked sequence governing oxygen response. The disclosure further describes oxygen-controlled promoter replacement at PTS import/early glycolysis-related regulation (ptsH/ptsI/crr) to provide single-point oxygen control and reduce overflow metabolite formation, including acetate and other organic acids.
Claims Coverage
The independent claim covers an engineered E. coli with oxygen-responsive metabolic flux regulation using an oxygen-regulated promoter linked to defined sucrose catabolism module sequence and to ptsH/ptsI/crr sequences to decrease metabolic flux upon reduced oxygen levels. Two inventive features are identified across the independent claim language.
Oxygen-regulated promoter linked to carbon-source metabolic control
An E. coli comprising an oxygen-regulated promoter selected from the group consisting of a cytochrome o oxidase (cyo) promoter and a superoxide dismutase (sodA) promoter operatively linked to a sequence selected from the group consisting of a sequence comprising the cscA gene of the E. coli O157.H7 chromosomally-encoded sucrose catabolism (csc) module and a sequence comprising the ptsH, ptsI and carbohydrate repression resistant (crr) genes of the E. coli phosphoenol pyruvate-dependent phosphotransferase system (pts) wherein metabolic flux from a carbon source in the E. coli is decreased in response to a reduction in oxygen levels.
Sucrose catabolizing phenotype under oxygen-regulated promoter control
The E. coli is sucrose catabolizing (Suc+) when said oxygen-regulated promoter is operatively linked to said sequence comprising the cscA gene.
Overall, the independent claim structure centers on oxygen-regulated promoters (cyo or sodA) operatively linked to defined sucrose catabolism and PTS-related gene sequences to decrease metabolic flux with reduced oxygen, while maintaining a sucrose catabolizing (Suc+) phenotype when promoter linkage includes the cscA-containing sequence.
Stated Advantages
Reduces overflow metabolite production by decreasing metabolic flux from a carbon source in response to reduced oxygen levels.
Enables sucrose catabolism (Suc+) under oxygen-regulated promoter control while sucrose catabolism is shut down under anaerobic conditions.
Documented Applications
Fermentation use cases involving air/aerobic-to-anaerobic cycling to achieve oxygen-dependent sucrose catabolism shut down and reduced acid/overflow metabolite production.
Culturing microorganisms under nutrient conditions with oxygen concentrations below, above, or at a threshold to alter carbon flow through metabolic pathways that produce overflow metabolites and thereby reduce overflow metabolite production.
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