Granted Patent B2

US 12,545,886 · App. 18/512,389 · Granted Feb 10, 2026

Inventors: Dean Falb (Sherborn, MA); Adam B. Fisher (Cambridge, MA); Vincent M. Isabella (Medford, MA); Jonathan W. Kotula (Berkeley, CA); David Lubkowicz (Somerville, MA); Paul F. Miller (Salem, CT); Yves Millet (Newton, MA); Sarah Elizabeth Rowe (Durham, NC)

Assignee: Synlogic Operating Company, Inc.

C12N1/20A61K9/0053A61K35/74A61K35/741C07K14/245C12N15/63C12N2510/00C12N2795/00021C12N2800/202C12Q1/527

Abstract

Claims (57)

1 . An E. coli Nissle bacterium, wherein the bacterium comprises

(i) one or more gene(s) encoding a phenylalanine ammonia lyase (PAL) enzyme comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 3, 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 genes 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, or the deleted phage genes consist of SEQ ID NO: 130.

5 . The bacterium of claim 1 , wherein the bacterium further comprises an antibiotic resistance gene.

6 . The bacterium of claim 1 , further comprising one or more gene(s) encoding a phenylalanine transporter.

7 . The bacterium of claim 6 , wherein the phenylalanine transporter is PheP.

8 . The bacterium of claim 6 , further comprising one or more gene(s) encoding L-amino acid deaminase (LAAD).

9 . The bacterium of claim 8 , 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.

10 . The bacterium of claim 9 , 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.

11 . The bacterium of claim 9 , 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.

12 . The bacterium of claim 9 , 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.

13 . The bacterium of claim 9 , wherein:

the one or more of the genes encoding PAL are operably linked to an FNR-responsive promoter;

one or more additional genes encoding PAL comprise an amino acid sequence having at least 90% 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 FNR-responsive promoter; and

the one or more genes encoding LAAD are operably linked to an arabinose-inducible promoter.

14 . The bacterium of claim 9 , wherein the bacterium comprises:

three copies of the gene encoding PAL each independently comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 3;

two additional copies of the gene encoding PAL each independently comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 3;

two copies of the gene encoding the phenylalanine transporter, wherein the two copies of the gene encoding the phenylalanine transporter are non-native and derived from Escherichia coli;

one copy of the gene encoding LAAD, wherein the one copy of the gene encoding LAAD is derived from Proteus mirabilis ; and

a mutation in one or more of ThyA or DapA to generate an auxotrophy.

15 . The bacterium of claim 14 , wherein:

the promoter operably linked to the three copies of the gene encoding PAL is inducible under anaerobic or low-oxygen conditions;

the promoter operably linked to the two additional copies of the gene encoding PAL 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.

16 . The bacterium of claim 15 , wherein:

the promoter to which the three copies of the gene encoding PAL are operably linked to is an FNR-responsive promoter; and

the promoter to which the two copies of the non-native gene encoding the phenylalanine transporter derived from Escherichia coli are operably linked to is an FNR-responsive promoter.

17 . The bacterium of claim 9 , wherein the bacterium comprises:

four copies of the gene encoding PAL;

one copy of the gene encoding PheP phenylalanine transporter, wherein the one copy of the gene encoding PheP phenylalanine transporter is non-native and derived from Escherichia coli;

one copy of the gene encoding LAAD, wherein the one copy of the gene encoding LAAD is derived from Proteus mirabilis ; and

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 four copies of the gene encoding PAL is an IPTG-inducible promoter;

the promoter operably linked to the one copy of the non-native 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.

19 . A pharmaceutically acceptable composition comprising the bacterium of claim 1 , and a pharmaceutically acceptable carrier.

20 . The composition of claim 19 formulated for oral administration.

Assignments (1)

Continuity (8)

References Cited (331)