Compositions and methods for nucleic acid expression and protein secretion in bacteroides

Abstract

Provided are nucleic acids that include a promoter, where the promoter is operable in a Bacteroides cell and is operably linked to a heterologous nucleotide sequence of interest. Also provided are nucleic acids that include a promoter (operable in a prokaryotic cell such as a Bacteroides cell) operably linked to a sequence encoding a synthetic ribosomal binding site (RBS). Also provided are fusion proteins (and nucleic acids encoding them) in which a secreted Bacteroides polypeptide is fused to a heterologous polypeptide of interest. Also provided are prokaryotic cells (e.g., E. coli, a Bacteroides cell, and the like) that include one more nucleic acids such as those described above. Also provided are methods of expression in a prokaryotic cell, methods of detectably labeling a Bacteroides cell in an animal's gut, and methods of delivering a protein to an individual's gut.

What is claimed is: 
     
         1 . A nucleic acid, comprising:
 (a) a promoter operable in a prokaryotic cell, wherein the promoter comprises a nucleotide sequence comprising one or more of the following:
 (i) 80% or more sequence identity of defined nucleotides of the nucleotide sequence: GTTAA (n) 4-7  GTTAA (n) 34-38  TA (n) 2  TTTG, 
 (ii) 80% or more sequence identity with a sequence set forth in any of SEQ ID NOs: 388 and 407, 
 (iii) a nucleotide sequence comprising GTTAA (n) 4-7  GTTAA, 
 (iv) a nucleotide sequence comprising GTTAA (n) 44-50  TA, 
 (v) a nucleotide sequence comprising GTTAA (n) 48-54  TTTG, 
 (vi) a nucleotide sequence comprising GTTAA (n) 36-38  TA, 
 (vii) a nucleotide sequence comprising GTTAA (n) 40-42  TTTG, 
 (viii) a nucleotide sequence comprising GTTAA (n) 3-7  GTTAA (n) 36-38  TA, 
 (ix) a nucleotide sequence comprising GTTAA (n) 3-7  GTTAA (n) 40-42  TTTG, 
 (x) a nucleotide sequence comprising GTTAA (n) 44-50  TA (n) 2  TTTG, 
 (xi) a nucleotide sequence comprising GTTAA (n) 36-38  TA (n) 2  TTTG, 
 (xii) a nucleotide sequence comprising GTTAA (n) 0-20  GTTAA (n) 10-60  TA (n) 0-10  TTTG, 
 (xiii) a nucleotide sequence comprising TTAA (n) 0-10  TTAA (n) 30-50  TA (n) 2  TTTG, 
 (xiv) a nucleotide sequence comprising GTTAA (n) 4-7  GTTAA (n) 36-39  TA (n) 2  TTTGC, 
 (xv) a nucleotide sequence comprising GTTAA (n) 4-7  GTTAA (n) 36-39  TA (n) 2  TTTG, 
 (xvi) a nucleotide sequence comprising GTTAA (n) 4-7  GTTAA (n) 34-38  TA (n) 2  TTTG, 
 (xvii) a nucleotide sequence comprising GTTAA (n) 4-7  GTTAA (n) 36-38  TA (n) 2  TTTG, 
 (xviii) a nucleotide sequence comprising GTTAA (n) 3-7  GTTAA (n) 36-38 TA (n) 2  TTTG, 
 (xix) a nucleotide sequence comprising GTTAA (n) 4-7  GTTAA (n) 12-16  TTG (n) 18-22  TA (n) 2  TTTGC, 
 (xx) a nucleotide sequence comprising GTTAA (n) 3-7  GTTAA (n) 12-16  TTG (n) 18-22  TA (n) 2  TTTG, 
 (xxi) a nucleotide sequence comprising GTTAA (n) 4-8  GTTAA (n) 12-16  TTG (n) 18-22  TA (n) 2  TTTG, and 
 (xxii) a nucleotide sequence comprising GTTAA (n) 4-7  GTTAA (n) 12-16  TTG (n) 18-22  TA (n) 2  TTTG, 
   wherein each n is independently selected from A, C, G, and T; and   (b) a nucleotide sequence of interest that is operably linked to the promoter, wherein the nucleotide sequence of interest and the promoter are not found operably linked in nature.   
     
     
         2 . The nucleic acid of  claim 1 , wherein the prokaryotic cell is a  Bacteroides  cell. 
     
     
         3 . A nucleic acid, comprising:
 (a) a promoter operable in a  Bacteroides  cell, and   (b) a nucleotide sequence of interest that is operably linked to the promoter, wherein the nucleotide sequence of interest and the promoter are not found operably linked in nature,   wherein the promoter provides one or more of the following when the nucleic acid is expressed in the  Bacteroides  cell:   (i) an increase in mRNA production of at least 30% relative to a native  Bacteroides  promoter,   (ii) an increase in fluorescence of at least 2000% relative to autofluorescence, wherein the nucleotide sequence of interest encodes super-folding GFP, or   (iii) a cytoplasmic protein concentration of at least 1.5 μM, wherein the nucleotide sequence of interest encodes the protein.   
     
     
         4 . The nucleic acid of  claim 3 , wherein the native  Bacteroides  promoter is a native  Bacteroides  rRNA promoter. 
     
     
         5 . The nucleic acid of  claim 3 , wherein the increase in mRNA production is at least 50%. 
     
     
         6 . The nucleic acid of  claim 3 , wherein the increase in fluorescence is at least 5000%. 
     
     
         7 . The nucleic acid of  claim 3 , wherein the increase in fluorescence is at least 8000%. 
     
     
         8 . The nucleic acid of  claim 3 , wherein the cytoplasmic protein concentration is at least 2 μM. 
     
     
         9 . The nucleic acid of  claim 3 , wherein the cytoplasmic protein concentration is at least 5 μM. 
     
     
         10 . The nucleic acid of  claim 3 , wherein the cytoplasmic protein concentration is at least 10 μM. 
     
     
         11 . The nucleic acid of  claim 3 , wherein the protein is luciferase. 
     
     
         12 . The nucleic acid of  claim 1  or  3 , wherein the promoter is a phage promoter or a functional fragment thereof. 
     
     
         13 . The nucleic acid of  claim 12 , wherein the phage is ϕB124-14. 
     
     
         14 . The nucleic acid of  claim 1  or  3 , wherein the promoter is a non-naturally occurring promoter. 
     
     
         15 . The nucleic acid of  claim 1  or  3 , wherein the promoter comprises a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence: GTTAA (n) 3-7  GTTAA (n) 36-38 TA (n) 2  TTTG (SEQ ID NO: 400). 
     
     
         16 . The nucleic acid of  claim 1  or  3 , wherein the promoter comprises the nucleotide sequence set forth in any of SEQ ID NOs: 381-388. 
     
     
         17 . The nucleic acid of  claim 1  or  3 , wherein the nucleotide sequence of interest comprises a transgene sequence that encodes a protein. 
     
     
         18 . The nucleic acid of  claim 17 , wherein the protein encoded by the transgene sequence comprises a reporter protein, a selectable marker protein, a metabolic enzyme, or a therapeutic protein. 
     
     
         19 . The nucleic acid of  claim 17 , wherein the protein encoded by the transgene sequence is a fusion protein comprising a cleavable linker and a secreted  Bacteroides  polypeptide fused to a heterologous polypeptide of interest, wherein the cleavable linker is positioned between the secreted  Bacteroides  polypeptide and the polypeptide of interest. 
     
     
         20 . The nucleic acid of  claim 1  or  3 , wherein the nucleotide sequence of interest comprises a transgene sequence that encodes a non-coding RNA. 
     
     
         21 . A prokaryotic cell comprising the nucleic acid of any of  claims 1 - 20 . 
     
     
         22 . The prokaryotic cell of  claim 21 , wherein the nucleic acid is integrated into a chromosome of the prokaryotic cell. 
     
     
         23 . The prokaryotic cell of any of  claims 21 - 22 , wherein the prokaryotic cell is a  Bacteroides  cell. 
     
     
         24 . The prokaryotic cell of any of  claims 21 - 22 , wherein the prokaryotic cell is not a  Bacteroides  cell. 
     
     
         25 . The prokaryotic cell of  claim 24 , wherein the prokaryotic cell is an  E. coli  cell. 
     
     
         26 . A method of expressing a nucleic acid in a prokaryotic cell, the method comprising:
 introducing the nucleic acid of any of  claims 1 - 20  into the prokaryotic cell.   
     
     
         27 . The method of  claim 26 , wherein the prokaryotic cell is a  Bacteroides  cell. 
     
     
         28 . The method of  claim 27 , wherein the  Bacteroides  cell is a cell of a species selected from:  B. fragilis  (Bf),  B. distasonis  (Bd),  B. thetaiotaomicron  (Bt),  B. vulgatus  (Bv),  B. ovatus  (Bo),  B. eggerrthii  (Be),  B. merdae  (Bm),  B. stercoris  (Bs),  B. uniformis  (Bu), and  B. caccae  (Bc). 
     
     
         29 . The method of  claim 26 , wherein the prokaryotic cell is an  E. coli  cell. 
     
     
         30 . The method of any of  claims 26 - 29 , wherein the nucleotide sequence of interest is a transgene encoding a fusion protein comprising a cleavable linker and a secreted  Bacteroides  polypeptide fused to a heterologous polypeptide of interest, wherein the cleavable linker is positioned between the secreted  Bacteroides  polypeptide and the polypeptide of interest. 
     
     
         31 . A fusion protein comprising: a secreted  Bacteroides  polypeptide fused to a heterologous polypeptide of interest. 
     
     
         32 . The fusion protein of  claim 31 , wherein the secreted  Bacteroides  polypeptide comprises an amino acid sequence that has 80% or more sequence identity with an amino acid sequence set forth in any of SEQ ID NOs: 458-484. 
     
     
         33 . The fusion protein of any of  claims 31 - 32 , comprising a cleavable linker positioned between the secreted  Bacteroides  polypeptide and the polypeptide of interest. 
     
     
         34 . The fusion protein of  claim 33 , wherein the cleavable linker is cleavable by one or more gut proteases. 
     
     
         35 . The fusion protein of  claim 34 , wherein the cleavable linker is cleavable by one or more gut proteases selected from: a trypsin, a chymotrypsin, and an elastase. 
     
     
         36 . The fusion protein of  claim 34 , wherein the cleavable linker is set forth in any of SEQ ID NOs: 420-453. 
     
     
         37 . The fusion protein of any of  claims 31 - 36 , wherein the polypeptide of interest is an anti-inflammatory peptide. 
     
     
         38 . The fusion protein of  claim 37 , wherein the anti-inflammatory peptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 411-417. 
     
     
         39 . A nucleic acid encoding the fusion protein of any of  claims 31 - 38 . 
     
     
         40 . The nucleic acid of  claim 39 , wherein the nucleic acid is a plasmid. 
     
     
         41 . The nucleic acid of  claim 40 , wherein the plasmid comprises an origin of replication that functions in prokaryotic cells other than  Bacteroides  cells, but does not function in  Bacteroides  cells. 
     
     
         42 . An outer membrane vesicle, comprising the fusion protein of any of  claims 31 - 38 . 
     
     
         43 . A method of delivering a polypeptide, comprising:
 recombinantly expressing the fusion protein of any of  claims 31 - 38  in a prokaryotic cell;   and delivering the fusion protein or the polypeptide of interest outside of the prokaryotic cell.   
     
     
         44 . The method of  claim 43 , further comprising delivering the fusion protein or the polypeptide of interest to a gut. 
     
     
         45 . The method of  claim 43 , further comprising packaging the fusion protein or the polypeptide of interest into an outer membrane vesicle. 
     
     
         46 . The method of  claim 45 , further comprising fusing the outer membrane vesicle with a cell membrane of a second cell. 
     
     
         47 . The method of  claim 43 , further comprising delivering the fusion protein or the polypeptide of interest to a second cell. 
     
     
         48 . The method of  claim 47 , wherein the second cell is a eukaryotic cell. 
     
     
         49 . The method of  claim 47 , wherein the second cell is a mammalian cell. 
     
     
         50 . A method of delivering a protein to an individual's gut, the method comprising: introducing, into an individual's gut, a  Bacteroides  cell comprising the nucleic acid of any one of  claims 1 - 20  and  39 - 41 . 
     
     
         51 . The method of  claim 50 , wherein the individual has a disease impacted by gut microbiota. 
     
     
         52 . The method of  claim 50 , wherein the individual has a disease selected from: obesity, diabetes, heart disease, central nervous system diseases, rheumatoid arthritis, metabolic disorders, and cancer. 
     
     
         53 . The method of  claim 50 , wherein the individual has gut inflammation. 
     
     
         54 . The method of  claim 50 , wherein the individual has colitis. 
     
     
         55 . The method of any of  claims 50 - 54 , wherein the  Bacteroides  cell is a cell of a species selected from:  B. fragilis  (Bf),  B. distasonis  (Bd),  B. thetaiotaomicron  (Bt),  B. vulgatus  (Bv),  B. ovatus  (Bo),  B. eggerrthii  (Be),  B. merdae  (Bm),  B. stercoris  (Bs),  B. uniformis  (Bu), and  B. caccae  (Bc).