METHODS FOR ENGINEERING OUTER MEMBRANE VESICLE PRODUCTION AND CARGO PACKAGING IN PSEUDOMONAS PUTIDA
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.
1 . 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.
2 . The genetically modified Pseudomonas sp. of claim 1 , wherein the endogenous gene is selected from the group consisting of oprF, and oprI.
3 . The genetically modified Pseudomonas sp. of claim 1 , wherein the Pseudomonas sp. is selected from the group consisting of P. putida, P. fluorescens , and P. stutzeri.
4 . The genetically modified Pseudomonas sp. of claim 3 , wherein the P. putida is P. putida KT2440.
5 . 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.
6 . The genetically modified Pseudomonas sp. of claim 5 , wherein the expressed enzyme encoded by the at least one exogenous gene is tagged with a vesicle nucleating peptide.
7 . The genetically modified Pseudomonas sp. of claim 5 , wherein the expressed enzyme encoded by the at least one exogenous gene is tagged with a vesicle nucleating peptide having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
8 . 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.
9 . The system of claim 8 wherein the expressed enzyme encoded by the at least one exogenous gene is XylE; and the substrate is catechol and the product is 2-hydroxymuconic semialdehyde.
10 . The system of claim 8 wherein the expressed enzyme encoded by the at least one exogenous gene is isolated.
11 . The system of claim 8 wherein the outer membrane protein is encoded by a gene that is endogenous to the genetically modified Pseudomonas sp.
12 . The system of claim 11 wherein the outer membrane protein is selected from the group consisting of OmpA (PP_1122) and EstP.
13 . The system of claim 12 wherein the outer membrane protein is OmpA (PP_1122) and wherein the expressed enzyme encoded by the at least one exogenous gene is on the inside of the outer membrane vesicle.
14 . The system of claim 12 wherein the outer membrane protein is EstP and wherein the expressed enzyme encoded by the at least one exogenous gene is on the outside of the outer membrane vesicle.
15 . The system of claim 8 wherein the outer membrane protein is encoded by a gene that is exogenous to the genetically modified Pseudomonas sp.
16 . The system of claim 15 wherein the outer membrane protein is selected from the group consisting of OmpA from Escherichia coli or INP from Pseudomonas syringae.
17 . The system of claim 16 wherein the outer membrane protein is OmpA from Escherichia coli and wherein the expressed enzyme encoded by the at least one exogenous gene is on the inside of the outer membrane vesicle.
18 . The system of claim 16 wherein the outer membrane protein is INP from Pseudomonas syringae and wherein the expressed enzyme encoded by the at least one exogenous gene is on the outside of the outer membrane vesicle.
19 . The system of claim 8 wherein the expressed enzyme encoded by the at least one exogenous gene is tagged with a vesicle nucleating peptide.
20 . The system of claim 8 wherein the expressed enzyme encoded by the at least one exogenous gene is tagged with a vesicle nucleating peptide having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.