Bacteria for the treatment of disorders
Modified probiotics, pharmaceutical compositions thereof, and methods of modulating and treating disorders are disclosed.
1. An E. coli Nissle bacterium, wherein the bacterium comprises
(i) one or more gene(s) encoding a non-native phenylalanine metabolizing enzyme (PME), and
(ii) one or more non-native deletions in one or more phage genes in E. coli Nissle Phage 3, wherein the one or more phage queens are selected from the group consisting of ECOLIN_09965, ECOLIN_09970, ECOLIN_09975, ECOLIN_09980, ECOLIN_09985, ECOLIN_09990, ECOLIN_09995, ECOLIN_10000, ECOLIN_10005, ECOLIN_10010, ECOLIN_10015, ECOLIN_10020, ECOLIN_10025, ECOLIN_10030, ECOLIN_10035, ECOLIN_10040, ECOLIN_10045, ECOLIN_10050, ECOLIN_10055, ECOLIN_10065, ECOLIN_10070, ECOLIN_10075, ECOLIN_10080, ECOLIN_10085, ECOLIN_10090, ECOLIN_10095, ECOLIN_10100, ECOLIN_10105, ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, ECOLIN_10175, ECOLIN_10180, ECOLIN_10185, ECOLIN_10190, ECOLIN_10195, ECOLIN_10200, ECOLIN_10205, ECOLIN_10210, ECOLIN_10220, ECOLIN_10225, ECOLIN_10230, ECOLIN_10235, ECOLIN_10240, ECOLIN_10245, ECOLIN_10250, ECOLIN_10255, ECOLIN_10260, ECOLIN_10265, ECOLIN_10270, ECOLIN_10275, ECOLIN_10280, ECOLIN_10290, ECOLIN_10295, ECOLIN_10300, ECOLIN_10305, ECOLIN_10310, ECOLIN_10315, ECOLIN_10320, ECOLIN_10325, ECOLIN_10330, ECOLIN_10335, ECOLIN_10340, and ECOLIN_10345.
2. The bacterium of claim 1 , wherein the non-native deletions comprise a complete or partial deletion of ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, and ECOLIN_10175.
3. The bacterium of claim 1 , wherein the non-native deletions are: a complete deletion of ECOLIN_10110, ECOLIN_10115, ECOLIN 10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN 10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, and a partial deletion of ECOLIN_10175.
4. The bacterium of claim 3 , wherein the deleted phage genes comprise SEQ ID NO:130.
5. The bacterium of claim 4 , wherein the deleted phage genes consist of SEQ ID NO:130.
6. The bacterium of claim 1 , wherein the bacterium further comprises an antibiotic resistance gene.
7. The bacterium of claim 1 , wherein the PME is phenylalanine ammonia lyase (PAL).
8. The bacterium of claim 7 , further comprising one or more gene(s) encoding a phenylalanine transporter.
9. The bacterium of claim 8 , wherein the phenylalanine transporter is PheP.
10. The bacterium of claim 8 , further comprising one or more gene(s) encoding L-amino acid deaminase (LAAD).
11. The bacterium of claim 10 , wherein:
the one or more genes encoding PAL are operably linked to a promoter;
the one or more genes encoding the phenylalanine transporter are operably linked to a promoter; and
the one or more genes encoding LAAD are operably linked to a promoter.
12. The bacterium of claim 11 , wherein:
the one or more genes encoding PAL are operably linked to an IPTG-inducible promoter; and
the one or more genes encoding LAAD are operably linked to an arabinose-inducible promoter.
13. The bacterium of claim 11 , wherein:
the one or more genes encoding PAL are operably linked to an IPTG-inducible promoter;
the one or more genes encoding the phenylalanine transporter are operably linked to an IPTG-inducible promoter; and
the one or more genes encoding LAAD are operably linked to an arabinose-inducible promoter.
14. The bacterium of claim 11 , wherein:
the one or more genes encoding PAL comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 3 and are operably linked to an IPTG-inducible promoter;
the one or more genes encoding the phenylalanine transporter are operably linked to an IPTG-inducible promoter; and
the one or more genes encoding LAAD are operably linked to an arabinose-inducible promoter.
15. The bacterium of claim 11 , wherein:
the one or more genes encoding PAL are operably linked to an FNR-responsive promoter;
the one or more genes encoding the phenylalanine transporter are operably linked to an FNR-responsive promoter; and
the one or more genes encoding LAAD are operably linked to an arabinose-inducible promoter.
16. The bacterium of claim 11 , wherein:
one or more of the genes encoding PAL are operably linked to an FNR-responsive promoter;
one or more additional genes encoding PAL are operably linked to an IPTG-inducible promoter;
the one or more genes encoding the phenylalanine transporter are operably linked to an FNR-responsive promoter; and
the one or more genes encoding LAAD are operably linked to an arabinose-inducible promoter.
17. The bacterium of claim 11 wherein:
three copies of the gene encoding PAL are derived from Photorhabdus luminescens;
two additional copies of the gene encoding PAL are derived from Photorhabdus luminescens;
two copies of the gene encoding the phenylalanine transporter are derived from Escherichia coli;
one copy of the gene encoding LAAD is derived from Proteus mirabilis ; and
the bacterium further comprises a mutation in one or more of ThyA or DapA to generate an auxotrophy.
18. The bacterium of claim 17 , wherein:
the promoter operably linked to the three copies of the gene encoding PAL derived from Photorhabdus luminescens is inducible under anaerobic or low-oxygen conditions;
the promoter operably linked to the two additional copies of the gene encoding PAL derived from Photorhabdus luminescens is an IPTG-inducible promoter;
the promoter operably linked to the two copies of the non-native gene encoding the phenylalanine transporter derived from Escherichia coli is inducible under anaerobic or low-oxygen conditions;
the promoter operably linked to the one copy of the gene encoding LAAD derived from Proteus mirabilis is an arabinose-inducible promoter; and
the mutation in one or more of ThyA or DapA is a mutation in the DapA gene.
19. The bacterium of claim 18 , wherein:
the promoter to which the three copies of the gene encoding PAL derived from Photorhabdus luminescens are operably linked to is an FNR-responsive promoter; and
the promoter to which the two copies of the gene encoding the phenylalanine transporter derived from Escherichia coli are operably linked to is an FNR-responsive promoter.
20. The bacterium of claim 11 , wherein:
four copies of a gene encoding PAL are derived from Photorhabdus luminescens;
one copy of a gene encoding PheP phenylalanine transporter is derived from Escherichia coli;
one copy of a gene encoding LAAD is derived from Proteus mirabilis ; and
the bacterium further comprising a mutation in one or more of ThyA or DapA to generate an auxotrophy.
21. The bacterium of claim 20 , wherein:
the promoter operably linked to the four copies of the gene encoding PAL derived from Photorhabdus luminescens is an IPTG-inducible promoter;
the promoter operably linked to the one copy of the gene encoding the phenylalanine transporter derived from Escherichia coli is an IPTG-inducible promoter;
the promoter operably linked to the one copy of the gene encoding LAAD derived from Proteus mirabilis is an arabinose-inducible promoter; and
the mutation in one or more of ThyA or DapA is a mutation in the DapA gene.
22. The bacterium of claim 1 , wherein the PME is PAL derived from Anabaena variabilis (PAL1) or Photorhabdus luminescens (PAL3).
23. A pharmaceutically acceptable composition comprising the bacterium of claim 1 , and a pharmaceutically acceptable carrier.
24. The composition of claim 23 formulated for oral administration.