Methods for engineering outer membrane vesicle production and cargo packaging in pseudomonas putida
AssigneeAlliance for Sustainable Energy, LLC
InventorsRebecca Ann WILKES, Allison Jean ZIMONT WERNER, Tarryn E. MILLER, Gregg Tyler BECKHAM
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Abstract
Disclosed herein are methods, compositions and systems useful for genetically engineering subcellular compartments such as OMVs for synthetic biology applications. In an embodiment, genetically engineered bacteria use OMVs to secrete compounds or proteins of interest extracellularly where the compounds or proteins of interest can be isolated from the growth media.
Claims20
A genetically modified Pseudomonas sp. comprising at least one deletion of an endogenous gene, wherein:
- the one or more deletion results in an increase in the production of outer membrane vesicles (OMVs) relative to the wild-type Pseudomonas sp.
A genetically modified Pseudomonas sp. comprising at least one deletion of an endogenous gene, wherein:
- the one or more deletion results in an increase in the production of outer membrane vesicles (OMVs) relative to the wild-type Pseudomonas sp.;
- and
- wherein the genetically modified Pseudomonas sp. further comprises at least one exogenous gene encoding an enzyme;
- and
- wherein the expressed enzyme encoded by the at least one exogenous gene encoding an enzyme is connected to an outer membrane protein that is incorporated into the membrane of an outer membrane vesicle;
- and
- wherein the enzyme is connected to the outer membrane protein through a linker.
A system for the production and isolation of a compound of interest comprising:
- a genetically modified Pseudomonas sp. comprising at least one deletion of an endogenous gene, wherein:
- the one or more deletion results in an increase in the production of outer membrane vesicles (OMVs) relative to the wild-type Pseudomonas sp.;
- and
- wherein the genetically modified Pseudomonas sp. further comprises at least one exogenous gene encoding an enzyme;
- and
- wherein the expressed enzyme encoded by the at least one exogenous gene encoding an enzyme is connected to an outer membrane protein that is incorporated into the membrane of an outer membrane vesicle;
- and
- wherein the expressed enzyme is connected to the outer membrane protein through a linker;
- and
- wherein the expressed enzyme encoded by the at least one exogenous gene is contacted with a substrate;
- and
- wherein a product of a reaction catalyzed by the expressed enzyme encoded by the at least one exogenous gene is isolated;
- and
- wherein the product of the reaction catalyzed by the expressed enzyme is the compound of interest.
Dependencies are inferred from the claim text.