Bacteria engineered to treat diseases associated with hyperammonemia
Genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating and treating disorders associated with hyperammonemia are disclosed.
1. A genetically engineered bacterium comprising an arginine regulon,
wherein the bacterium comprises a gene encoding an arginine feedback resistant N-acetylglutamate synthetase (ArgA fbr ), wherein the ArgA fbr has reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions and wherein expression of the gene encoding ArgA fbr is controlled by a promoter that is induced by low-oxygen or anaerobic conditions; and
wherein the bacterium has been genetically engineered to lack a functional ArgR.
2. The bacterium of claim 1 , wherein each copy of a functional argR gene normally present in a corresponding wild-type bacterium has been deleted.
3. The bacterium of claim 1 , wherein each copy of a functional argG gene normally present in a corresponding wild-type bacterium has been independently deleted or rendered inactive by one or more nucleotide deletions, insertions or substitutions.
4. The bacterium of claim 3 , wherein each copy of the functional argG gene normally present in a corresponding wild-type bacterium has been deleted.
5. The bacterium of claim 1 , wherein under low-oxygen or anaerobic conditions, the transcription of each gene that is present in an operon comprising a functional ARG box and which encodes an arginine biosynthesis enzyme is increased as compared to a corresponding gene in a wild-type bacterium under the same conditions.
6. The bacterium of claim 1 , wherein the promoter that is induced under low-oxygen or anaerobic conditions is an FNR promoter.
7. The bacterium of claim 1 , wherein the arginine feedback resistant N-acetylglutamate synthetase gene has a DNA sequence selected from:
a) SEQ ID NO:28, and
b) a DNA sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as encoded by SEQ ID NO:28.
8. The bacterium of claim 1 , wherein the bacterium is a non-pathogenic bacterium.
9. The bacterium of claim 1 , wherein the bacterium is a probiotic bacterium.
10. The bacterium of claim 1 , wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus , and Lactococcus.
11. The bacterium of claim 1 , wherein the bacterium is Escherichia coli strain Nissle.
12. The bacterium of claim 1 , wherein the gene encoding the arginine feedback resistant N-acetylglutamate synthetase (ArgA fbr ) is present on a plasmid in the bacterium and operably linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions.
13. The bacterium of claim 1 , wherein the gene encoding the arginine feedback resistant N-acetylglutamate synthetase (ArgA fbr ) is present in the bacterial chromosome and is operably linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.
14. The bacterium of claim 1 , wherein the bacterium is an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut.
15. The bacterium of claim 14 , wherein the mammalian gut is a human gut.
16. A pharmaceutically acceptable composition comprising a genetically engineered bacterium comprising an arginine regulon, wherein the bacterium comprises a gene encoding an arginine feedback resistant N-acetylglutamate synthetase (ArgA fbr ), wherein the ArgA fbr has reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding ArgA fbr is controlled by a promoter that is induced by low-oxygen or anaerobic conditions; and wherein the bacterium has been genetically engineered to lack a functional ArgR; and a pharmaceutically acceptable carrier.
17. The bacterium of claim 14 , wherein the bacterium is a thyA or dapB auxotroph.
18. The bacterium of claim 1 , wherein the bacterium comprises an antibiotic resistance gene.