Bacteria engineered to treat disorders in which oxalate is detrimental
The present invention provides recombinant bacterial cells comprising at least one heterologous gene encoding at least one oxalate catabolism enzyme. In another aspect, the recombinant bacterial cells further comprise at least one heterologous gene encoding an importer of oxalate. The invention further provides pharmaceutical compositions comprising the recombinant bacteria, and methods for treating disorders in which oxalate is detrimental, such as hyperoxaluria, using the pharmaceutical compositions of the invention.
1. A bacterium comprising gene sequence(s) encoding one or more oxalate catabolism enzyme(s) operably linked to a directly or indirectly first inducible promoter that is not associated with the oxalate catabolism enzyme gene in nature, wherein the gene sequence(s) encoding the one or more oxalate catabolism enzyme(s) are present in a cassette and encode:
(1) ScAAE3, Oxc, and Frc; or
(2) ScAAE3, Oxc, Frc, and YfdE; or
(3) Oxc, Frc, and YfdE.
2. The bacterium of claim 1 , wherein the bacterium further comprises gene sequence(s) encoding one or more transporter(s) of oxalate operably linked to a second promoter that is not associated with the oxalate transporter gene in nature.
3. The bacterium of claim 2 , wherein the bacterium further comprises gene sequence(s) encoding one or more exporter(s) of formate operably linked to a third promoter that is not associated with the formate exporter gene in nature.
4. The bacterium of claim 3 , wherein the first inducible promoter operably linked to the gene sequence(s) encoding the oxalate catabolism enzyme is directly or indirectly induced by exogenous environmental conditions found in the mammalian gut; wherein the second promoter operably linked to the gene sequence(s) encoding the transporter of oxalate is directly or indirectly induced by exogenous environmental conditions found in the mammalian gut; and/or wherein the third promoter operably linked to the gene sequence(s) encoding the exporter of formate is directly or indirectly induced by exogenous environmental conditions found in the mammalian gut.
5. The bacterium of claim 4 , wherein the first inducible promoter operably linked to the gene sequence(s) encoding the oxalate catabolism enzyme is directly or indirectly induced under low-oxygen or anaerobic conditions; wherein the second promoter operably linked to the gene sequence(s) encoding the oxalate transporter is directly or indirectly induced under low-oxygen or anaerobic conditions; and/or wherein the third promoter operably linked to the gene sequence(s) encoding the formate exporter is directly or indirectly induced under low-oxygen or anaerobic conditions.
6. The bacterium of claim 5 , wherein the first inducible promoter operably linked to the gene sequence(s) encoding the oxalate catabolism enzyme is selected from the group consisting of an FNR-responsive promoter, an ANR-responsive promoter, and a DNR-responsive promoter; wherein the second promoter operably linked to the gene sequence(s) encoding the oxalate transporter is selected from the group consisting of an FNR-responsive promoter, an ANR-responsive promoter, and a DNR-responsive promoter; and/or wherein the third promoter operably linked to the gene sequence(s) encoding the formate exporter is selected from the group consisting of an FNR-responsive promoter, an ANR-responsive promoter, and a DNR-responsive promoter.
7. The bacterium of claim 1 , wherein the one or more oxalate catabolism enzyme(s) convert oxalate to formate; convert oxalate to oxalyl-CoA; or convert oxalyl-CoA to formate.
8. The bacterium of claim 1 , wherein the gene sequence(s) encoding the one or more oxalate catabolism enzyme(s) are present in a cassette and encode ScAAE3, Oxc, and Frc.
9. The bacterium of claim 1 , wherein the gene sequence(s) encoding the one or more oxalate catabolism enzyme(s) are present in a cassette and encode ScAAE3, Oxc, Frc, and YfdE.
10. The bacterium of claim 1 , wherein the gene sequence(s) encoding the one or more oxalate catabolism enzyme(s) are present in a cassette and encode Oxc, Frc, and YfdE.
11. The bacterium of claim 1 , wherein the bacterium is a probiotic bacterial cell.
12. The bacterium of claim 11 , wherein the bacterium is a member of a genus selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus and Lactococcus.
13. The bacterium of claim 1 , wherein the engineered bacterial cell is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut.
14. The bacterium of claim 13 , wherein the engineered bacterial cell is an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway.
15. The bacterium of claim 1 , wherein the engineered bacterial cell is further engineered to harbor a gene encoding a substance that is toxic to the bacterium, wherein the gene is under the control of a promoter that is directly or indirectly induced by an environmental condition not naturally present in the mammalian gut.
16. A pharmaceutical composition comprising the bacterium of claim 1 , and a pharmaceutically acceptable carrier.
17. A method for reducing the levels of oxalate in a subject, the method comprising administering a pharmaceutical composition of claim 16 to the subject.
18. A method for treating a disease or disorder in which oxalate is detrimental in a subject, the method comprising administering a pharmaceutical composition of claim 16 .
19. The method of claim 18 , wherein the disorder in which oxalate is detrimental is a hyperoxaluria.
20. The bacterium of claim 1 , wherein the bacterium further comprises a knockout of a clbA gene.