Reivindicaciones
WHAT IS CLAIMED IS: 1. A fusion polypeptide comprising an amino acid sequence of a bactcriocin flanked at both the N- and C-termini by a split intein that circularizes the bacteriocin. 2. The fusion polypeptide of claim 1, wherein the bacteriocin is a natively circular bacteriocin. 3. The fusion polypeptide of claim 1 or 2, wherein the amino acid sequence of the bacteriocin is circularly permuted compared to a native amino acid sequence of the bacteriocin. 4. The fusion polypeptide of anyone of claims 1-3, wherein the first residue of the amino acid sequence of the bacteriocin is a serine or a cysteine that is present in the native amino acid sequence of the bacteriocin. 5. The fusion polypeptide of anyone of claims 1-3, wherein the first residue of the amino acid sequence of the bacteriocin is a non-native serine or a non-native cysteine. 6. The fusion polypeptide of claim 5, wherein the non-native serine or the non- native cysteine substitutes a native amino acid residue in the amino acid sequence of the bacteriocin. 7. The fusion polypeptide of claim 5, wherein the length of the amino acid sequence of the bacteriocin is increased by one residue due to the non-native serine or the non- native cysteine compared to the length of the native amino acid sequence of the bacteriocin. 8. The fusion polypeptide of any one of claims 5-7, wherein the native amino acid sequence of the bacteriocin does not comprise a serine or cysteine. 9. The fusion polypeptide of any one of the preceding claims, wherein the split intein is based on an intein from one of the following: Npu DnaE, See VMA, Ssp DnaE. 10. The fusion polypeptide of any one of the preceding claims, wherein the split intein is a conditional split intein. 11. The fusion polypeptide of claim 10, wherein the conditional split intein is pH- or temperature-sensitive. 12. The fusion polypeptide of any one of the preceding claims, wherein the split intein comprises a second amino acid sequence of a C-terminal intein fragment (Ic) at least 80% identical to the Ic shown in Table B, and a third amino acid sequence of a N-terminal intein fragment (IN) at least 80% identical to the split intein IN shown in Table B. 13. The fusion polypeptide of any one of the preceding claims, wherein the bactcriocin is selected from any one of the bactcriocins listed in Table A. 14. The fusion polypeptide of any one of the preceding claims, wherein the amino acid sequence of the bacteriocin is at least 80% identical to any one of the sequences listed in Table A. 15. The fusion polypeptide of any one of the preceding claims, wherein the amino acid sequence of the bacteriocin is selected from any one of the sequences listed in Table A. 16. The fusion polypeptide of any one of claims 1-14, wherein the bacteriocin is an engineered bacteriocin. 17. The fusion polypeptide of any one of the preceding claims, wherein one or more amino acids of the polypeptide in the amino acid sequence is a non-natural amino acid. 18. The fusion polypeptide of any one of the preceding claims, further comprising a degradation tag. 19. The fusion polypeptide of claim 18, wherein the degradation tag is at the C- terminus of the fusion polypeptide. 20. The fusion polypeptide of any one of the preceding claims, wherein the split intein comprises a C-terminal intein fragment (“Ic”) fused N-terminal to the amino acid sequence of the bacteriocin and a N-terminal intein fragment (“IN”) fused C-terminal to the amino acid sequence of the bacteriocin, wherein the polypeptide further comprises a degradation tag C-terminal to the IN. 21. The fusion polypeptide of any one of claims 18-20, wherein the degradation tag comprises a sequence at least 80% identical to AANDENYALAA (SEQ ID NO: 873). 22. The fusion polypeptide of any one of the preceding claims, further comprising a signal peptide and/or a leader sequence. 23. A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of any one of the preceding claims. 24. The nucleic acid of claim 23, wherein the nucleotide sequence is operably linked to a promoter sequence. 25. The nucleic acid of claim 23 or 24, wherein the nucleic acid comprises DNA. 26. The nucleic acid of claim 23, wherein the nucleic acid comprises RNA. 27. A genetic vector comprising the nucleic acid of any one of claims 23-26. 28. A genetically engineered microbial cell comprising the nucleic acid of any one of claims 23-26, or the genetic vector of claim 27. 29. The microbial cell of claim 28, wherein the microbial cell is resistant to the bacteriocin. 30. The microbial cell of claim 28 or 29, wherein the microbial cell comprises a second nucleic acid encoding an immunity modulator that confers resistant to the bacteriocin. 31. The microbial cell of claim 30, wherein expression of the immunity modulator from the second nucleic acid is regulatable. 32. The microbial cell of any one of claims 28-31, wherein the microbial cell is a bacteria, fungi, or algae. 33. A composition comprising the fusion polypeptide of any one of claims 1-22. 34. A composition comprising a circular bacteriocin and a split intein. 35. A method of making a circular bacteriocin, comprising contacting the nucleic acid of any one of claims 22-26, or the genetic vector of claim 27 with an in vitro expression system under conditions sufficient to produce a circular bacteriocin. 36. A method of making a circular bacteriocin, comprising culturing the microbial cell of any one of claims 28-31 under conditions sufficient to produce a circular bacteriocin. 37. The method of claim 35 or 36, further comprising purifying the circular bacteriocin. 38. The method of any one of claims 35-37, further comprising purifying the fusion polypeptide. 39. The method of any one of claims 35-38, wherein the split intein is a conditional split intein that circularizes the bacteriocin under a permissive condition but not under a non- permissive condition, and wherein the method further comprises exposing the fusion polypeptide to the permissive condition, following exposure to the non-permissive condition, to induce circularization of the bacteriocin. 40. The method of any one of claims 35-39, further comprising modifying the pH or temperature to induce circularization of the bacteriocin, wherein the split intein is pH- or temperature- sensitive, respectively. 41. The method of any one of claims 35-40, further comprising allowing the split intein to be degraded after the circular bacteriocin is produced. 42. A library comprising a plurality of genetic vectors, each genetic vector comprising the nucleic acid of any one of claims 23-26, wherein at least two of the plurality of genetic vectors comprise nucleotide sequences encoding different bacteriocins. 43. The library of claim 41, wherein the nucleotide sequences encode bacteriocins from different microbial species. 44. The library of claim 41, wherein the nucleotide sequences comprise different sequence variants of a parent bacteriocin. 45. The library of claim 44, wherein the parent bacteriocin is a natively circular bacteriocin, and the sequence variants comprise a first variant that abrogates natural circularization of the parent bacteriocin. 46. A method of screening, comprising: providing the library of any one of claims 42-45 ; expressing a plurality of polypeptides encoded by one of more genetic vectors of the library; generating a plurality of circular bacteriocins from the plurality of expressed polypeptides; and assaying the plurality of circular bacteriocins for a desired activity. 47. The method of claim 46, wherein the desired activity comprises antimicrobial activity. 48. A method of controlling the growth of a microorganism, comprising contacting a composition comprising and/or conducive to supporting the growth of a microorganism with the microbial cell of any one of claims 28-32 under conditions sufficient to produce a circular bacteriocin, to thereby control the growth of the microorganism. 49. A method of controlling the growth of a microorganism, comprising contacting a composition comprising and/or conducive to supporting the growth of a microorganism with a circular bacteriocin made by the method of any one of claims 35-38, to thereby control the growth of the microorganism. 50. A method of controlling the growth of a microorganism, comprising contacting a composition comprising and/or conducive to supporting the growth a microorganism with the fusion polypeptide of any one of claims 1-22, to thereby control the growth of the microorganism. 51. The method of any one of claims 48-50, wherein the microorganism is a bacteria. 52. The method of any one of claims 48-51, wherein the composition is a culture medium, feedstock, or a microbiome. 53. The method of any one of claims 48-52, wherein the split intein is a conditional split intein that circularizes the bacteriocin under a permissive condition but not under a non- permissive condition, and wherein the method further comprises providing the permissive condition to the composition to thereby induce circularization of the bacteriocin. 54. The method of any one of claims 48-53, wherein the method comprises modifying the pH or temperature of the composition to induce circularization of the bacteriocin, wherein the split intein is pH- or temperature- sensitive, respectively. 55. A method of designing a nucleic acid encoding a polypeptide precursor of a bacteriocin, comprising: identifying a native amino acid sequence of a candidate bacteriocin, wherein the native amino acid sequence does not comprise a serine or cysteine at the N- terminus; providing a second amino acid sequence having a serine or cysteine at the N- terminus thereof by at least one of: circularly permuting the native amino acid sequence; or introducing a serine or cysteine to the native amino acid sequence; providing a nucleotide sequence encoding a polypeptide comprising the second amino acid sequence flanked at both the N- and C -termini by a split intein configured to circularize the bacteriocin; and expressing the polypeptide encoded by the nucleotide sequence. 56. The method of claim 55, wherein the candidate bacteriocin is predicted to be a circular bacteriocin based on a genomic sequence of a microorganism that encodes the candidate bacteriocin in its genome. 57. The method of claim 55 or 56, comprising: identifying a plurality of native amino acid sequences of a plurality of different candidate bacteriocins; for each of the plurality of native amino acid sequences: providing the second amino acid sequence; and providing the nucleotide sequence encoding a polypeptide comprising the second amino acid sequence flanked at both the N- and C-termini by a split intein configured to circularize the bacteriocin, thereby generating a library of nucleic acids representing each of the plurality of native amino acid sequences. 58. The method of any one of claims 55-57, wherein the polypeptide further comprises a degradation tag. 59. The method of any one of claims 55-58, wherein the polypeptide further comprises a signal peptide and/or leader sequence. 60. The method of any one of claims 55-59, wherein the polypeptide is expressed in vitro. 61. The method of any one of claims 55-59, wherein the polypeptide is expressed from a genetically engineered microbial cell configured to express the polypeptide encoded by the nucleotide sequence. 62. The fusion protein, nucleic acid, vector, library, microbial cell, or method of any one of the preceding claims, wherein the polypeptide comprises an affinity tag, optionally wherein the affinity tag is chitin binding protein (CBP).
