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 bacterium comprising 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 the bacterium has been genetically engineered to lack a functional ArgR, and
wherein the bacterium comprises one or more genes encoding a biosynthetic pathway for producing butyrate.
2. The bacterium of claim 1 , wherein the gene encoding ArgA fbr and the one or more genes encoding the biosynthetic pathway for producing butyrate are operably linked to a promoter that is induced by low-oxygen or anaerobic conditions.
3. The bacterium of claim 1 , wherein the promoter that is induced by low-oxygen or anaerobic conditions is selected from an FNR, ANR, and DNR promoter.
4. The bacterium of claim 3 ,wherein the FNR promoter is selected from nirB1, nirB2, nirB3, ydZ, fnrS1, and fnrS2.
5. The bacterium of claim 1 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate is selected from bcd2, etB3, etfA3, thiA1, hbd, crt2, pbt, buk, ter, and tesB genes.
6. The bacterium of claim 5 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate comprise bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk genes.
7. The bacterium of claim 5 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate comprise ter, thiA1, hbd, crt2, pbt, and buk genes.
8. The bacterium of claim 5 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate comprise ter, thiA1, hbd, crt2, and tesB genes.
9. The bacterium of claim 2 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate is selected from bcd2, etB3, etfA3, thiA1, hbd, crt2, pbt, buk, ter, and tesB genes.
10. The bacterium of claim 9 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate comprise ter, thiA1, hbd, crt2, and tesB genes.
11. The bacterium of claim 1 , wherein the ArgA fbr gene comprises a polynucleotide sequence selected from:
a) SEQ ID NO: 30,
b) a polynucleotide sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as encoded by SEQ ID NO: 30,
c) a polynucleotide sequence having at least 95% homology to the polynucleotide sequence of a) or b),
d) a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 31, and
e) a polynucleotide sequence that encodes a polypeptide having at least 95% homology with the polypeptide of SEQ ID NO: 31.
12. The bacterium of claim 2 , wherein the ArgA fbr gene comprises a polynucleotide sequence selected from:
a) SEQ ID NO: 30,
b) a polynucleotide sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as encoded by SEQ ID NO: 30,
c) a polynucleotide sequence having at least 95% homology to the polynucleotide sequence of a) or b),
d) a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 31, and
e) a polynucleotide sequence that encodes a polypeptide having at least 95% homology with the polypeptide of SEQ ID NO: 31.
13. The bacterium of claim 6 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate comprise a polynucleotide sequence selected from:
a.) nucleotides 135-8012 of SEQ ID NO: 81;
b.) a polynucleotide sequence that, but for the redundancy of the genetic code, encodes the same polypeptides as encoded by nucleotides 135-8012 of SEQ ID NO: 81;
c.) a polynucleotide sequence having at least 95% homology to nucleotides 135-8012 of SEQ ID NO: 81; and
d.) a polynucleotide sequence having at least 95% homology to the polynucleotide sequence that, but for the redundancy of the genetic code, encodes the same polypeptides as nucleotides 135-8012 of SEQ ID NO: 81.
14. The bacterium of claim 7 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate comprise a polynucleotide sequence selected from:
a.) nucleotides 181-6270 of SEQ ID NO: 104;
b.) a polynucleotide sequence that, but for the redundancy of the genetic code, encodes the same polypeptides as encoded by nucleotides 181-6270 of SEQ ID NO: 104;
c.) a polynucleotide sequence having at least 95% homology to nucleotides 181-6270 of SEQ ID NO: 104; and
d.) a polynucleotide sequence having at least 95% homology to the polynucleotide sequence that, but for the redundancy of the genetic code, encodes the same polypeptides as nucleotides 181-6270 of SEQ ID NO: 104.
15. The bacterium of claim 8 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate comprise a polynucleotide sequence selected from:
a.) nucleotides 773-5719 of SEQ ID NO: 88;
b.) a polynucleotide sequence that, but for the redundancy of the genetic code, encodes the same polypeptides as encoded by nucleotides 773-5719 of SEQ ID NO: 88;
c.) a polynucleotide having at least 95% homology to nucleotides 773-5719 of SEQ ID NO: 88; and
d.) a polynucleotide having at least 95% homology to the polynucleotide that, but for the redundancy of the genetic code, encodes the same polypeptides as nucleotides 773-5719 of SEQ ID NO: 88.
16. The bacterium of claim 11 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate comprise ter, thiA1, hbd, crt2, and tesB genes.
17. The bacterium of claim 12 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate comprise ter, thiA1, hbd, crt2, and tesB genes.
18. 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.
19. The bacterium of claim 9 , wherein the bacterium is an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut.
20. The bacterium of claim 17 , wherein the bacterium is an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut.
21. The bacterium of claim 18 , wherein the bacterium is a thyA or dapB auxotroph.
22. The bacterium of claim 20 , wherein the bacterium is a thyA or dapB auxotroph.
23. The bacterium of claim 19 , wherein the bacterium comprises an antibiotic resistance gene.
24. The bacterium of claim 1 , wherein the gene encoding the ArgA fbr is present on a plasmid in the bacterium.
25. The bacterium of claim 1 , wherein the gene encoding the ArgA fbr is present in the bacterial chromosome.
26. The bacterium of claim 1 , wherein the gene encoding the ArgA fbr is present in the bacterial chromosome.
27. The bacterium of claim 1 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate is present on a plasmid in the bacterium.
28. The bacterium of claim 1 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate is present in the bacterial chromosome.
29. The bacterium of claim 25 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate is present in the bacterial chromosome.
30. The bacterium of claim 26 , wherein the one or more genes encoding the biosynthetic pathway for producing butyrate is present in the bacterial chromosome.
31. The bacterium of claim 20 , wherein the gene encoding the ArgA fbr and the one or more genes encoding the biosynthetic pathway for producing butyrate are present in the bacterial chromosome.
32. The bacterium of claim 1 , wherein the bacterium is a probiotic bacterium.
33. The bacterium of claim 1 , wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus , and Lactococcus.
34. The bacterium of claim 33 , wherein the bacterium is Escherichia coli strain Nissle.
35. The bacterium of claim 31 , wherein the bacterium is Escherichia coli strain Nissle.
36. A pharmaceutical composition comprising the bacterium of claim 1 and a pharmaceutically acceptable carrier.
37. A pharmaceutical composition comprising the bacterium of claim 1 and a pharmaceutically acceptable carrier.
38. A pharmaceutical composition comprising the bacterium of claim 35 and a pharmaceutically acceptable carrier.