Two-stage dynamic control over redox state improves cystolic expression of disulfide containing proeteins in e. coli
AssigneeDuke University
InventorsJennifer Noel Hennigan, Michael Lynch, Romel Menacho Melgar
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Abstract
Methods and microorganism for expression of a protein requiring at least one disulfide bond for proper folding of the protein are described. The biofermentation methods comprise growth of a microorganism that may conditionally expressing the protein in addition to at least one synthetic metabolic valve designed to regulate of at least one enzyme effective for altering the redox characteristics of the cytosolic environment of the genetically modified microorganism. In a product producing step of the method, an oxidative cytosolic environment is conditionally provided.
Claims24
A method of expressing a heterologous protein product in a microorganism, the protein requiring at least one disulfide bond for proper folding of the protein, the method comprising:
- (a) providing a genetically modified microorganism, the genetically modified microorganism comprising:
- means for conditional expression or overexpression of a heterologous protein product;
- a synthetic metabolic valve(s) comprising:
- a gene expression-silencing synthetic metabolic valve characterized by silencing gene expression of one or more genes encoding one or more enzymes;
- or
- an enzymatic degradation synthetic metabolic valve characterized by inducing enzymatic degradation of one or more enzymes, or
- a combination thereof.
- wherein the one or more enzymes of each synthetic metabolic valve(s) are the same or different;
- and
- wherein reducing expression of at least one of the enzymes of a synthetic metabolic valve(s) is effective for altering the redox characteristics of the cytosolic environment of the genetically modified microorganism.
- (b) growing the genetically modified microorganism in a growth media comprising a limiting nutrient,
- wherein the microorganism maintains a reducing cytosolic environment during the growth phase comparable to the reducing cytosolic environment of a microorganism lacking genetic modifications;
- (c) transitioning from a growth phase to a stationary and protein product producing phase, the transition comprising:
- stopping the growth of the microorganism;
- inducing the synthetic metabolic valve(s) resulting in a shift to an oxidative cytosolic environment;
- inducing expression or overexpression of the heterologous protein product, thereby producing the protein product under oxidative conditions so that the necessary disulfide bonds are formed for proper folding of the heterologous protein product in the oxidative environment of the stationary phased genetically modified microorganism produced by induction of the synthetic metabolic valve(s).
A genetically modified microorganism comprising:
- means for conditional expression or overexpression of a heterologous protein product;
- a synthetic metabolic valve(s) comprising:
- a gene expression-silencing synthetic metabolic valve characterized by silencing gene expression of one or more genes encoding one or more enzymes;
- or
- an enzymatic degradation synthetic metabolic valve characterized by inducing enzymatic degradation of one or more enzymes, or
- a combination thereof.
- wherein the one or more enzymes of each synthetic metabolic valve(s) are the same or different;
- and
- wherein reducing expression of at least one of the enzymes of a synthetic metabolic valve(s) is effective for altering the redox characteristics of the cytosolic environment of the genetically modified microorganism,
- wherein the microorganism grows in a media while maintaining a reducing cytosolic environment during the growth phase, the reducing cytosolic environment comparable to the reducing cytosolic environment of a microorganism lacking genetic modifications;
- and
- wherein the synthetic metabolic valve(s) of the genetically modified microorganism, when activated, produce an oxidative cytosolic environment to promote formation of disulfide bonds for proper folding of the heterologous protein product.
Dependencies are inferred from the claim text.