Bacteriocin polypeptides, nucleic acids encoding them and methods of use thereof

Abstract

Provided herein are fusion polypeptides comprising an amino acid sequence of a bacteriocin, the amino acid sequence of the bacteriocin being a cleavage intein both on both the N-terminus and on both the C-terminus that cyclizes the bacteriocin. Also provided are nucleic acids and genetic vectors encoding the fusion polypeptides, as well as microbial cells genetically engineered with the nucleic acids or genetic vectors. Also provided are methods of making the cyclic bacteriocin, methods of screening using a library of nucleic acids or genetic vectors encoding the fusion polypeptides, and methods of controlling growth of organisms using the cyclic bacteriocin made by the methods provided herein.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 365,584, filed May 31, 2022. The entire contents of this related application are incorporated herein by reference.

[0003] References to sequence listings

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named SeqListSyng.012wo.xml created on May 25, 2023, which is 1,024,021 bytes in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety.

[0005] background

[0006] field

[0007] The present disclosure generally relates to antimicrobial peptides, such as bacteriocins.

[0008] Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria. The application of bacteriocins has traditionally focused on food preservation, mainly due to the widespread occurrence of these peptides in lactic acid bacteria and the regulatory approval of nisin as a food preservative. Bacteriocins are also considered for use as antimicrobial agents in human and animal health and non-food industrial applications.

[0009] Overview

[0010] Circular bacteriocin is a class of antimicrobial peptides produced by Gram-positive bacteria, which undergo head-to-tail connection after production. Compared with their linear counterparts, cyclic bacteriocins are usually very stable to temperature and pH changes and more resistant to proteolytic enzymes, and are considered to be a group of promising antimicrobial peptides for industrial applications. Although many operons encoding new cyclic bacteriocins have been found in the genome of the database, a limited number of cyclic bacteriocins have been produced and fully characterized. The activity of several proteins mediates the production and cyclization of these bacteriocins, and the genes encoding these proteins are expressed by natural bacteriocin production bacteria, or can be expressed in heterologous hosts. This article provides a method for carrying out bacteriocin cyclization by using the split-intein circular ligation of peptides and proteins (split-intein circular ligation of peptides and proteins, SICCLOPPS) system. In some embodiments, the method of the present disclosure provides for rapid and effective selection of in vitro (by cell-free protein system) and in vivo (by Escherichia coli (Escherichia coli)) production and correct cyclization characterization and / or novel cyclic bacteriocins. In some embodiments, the present disclosure provides intein-based synthetic biology tools for the production and characterization of new cyclic bacteriocins, biosynthesis of variants, and / or production of these peptides in other hosts.

[0011] Provided herein is a fusion polypeptide comprising an amino acid sequence of a bacteriocin, wherein the amino acid sequence of the bacteriocin is a fractured intein that cyclizes the bacteriocin on both sides of the N-terminus and the C-terminus. Optionally, the bacteriocin is a natural cyclic bacteriocin. In some embodiments, the amino acid sequence of the bacteriocin is arranged in a ring shape compared to the native amino acid sequence of the bacteriocin. In some embodiments, the first residue of the amino acid sequence of the bacteriocin is a serine or cysteine ​​present in the native amino acid sequence of the bacteriocin.

[0012] In some embodiments, the first residue of the amino acid sequence of the bacteriocin is a non-natural serine or a non-natural cysteine. Optionally, the non-natural serine or the non-natural cysteine ​​replaces the natural amino acid residue in the amino acid sequence of the bacteriocin. Optionally, compared with the length of the natural amino acid sequence of the bacteriocin, the length of the amino acid sequence of the bacteriocin increases by one residue due to the non-natural serine or the non-natural cysteine. In some embodiments, the natural amino acid sequence of the bacteriocin does not contain serine or cysteine.

[0013] In some embodiments, the split intein is based on an intein from one of the following: Npu DnaE, ​​Sce VMA, Ssp DnaE. In some embodiments, the split intein is a conditional split intein. In some embodiments, the conditional split intein is pH sensitive or temperature sensitive. In some embodiments, the split intein comprises a C-terminal intein fragment (I C ) At least 80% identical C The second amino acid sequence of the intein and the N-terminal intein fragment of the split intein shown in Table B (I N ) At least 80% identical N The third amino acid sequence of

[0014] In some embodiments, the bacteriocin is selected from any of the bacteriocins listed in Table A. In some embodiments, the amino acid sequence of the bacteriocin is at least 80% identical to any of the sequences listed in Table A. In some embodiments, the amino acid sequence of the bacteriocin is selected from any of the sequences listed in Table A.

[0015] In some embodiments, the bacteriocin is an engineered bacteriocin.In some embodiments, one or more amino acids in the amino acid sequence of the polypeptide are non-natural amino acids.

[0016] In some embodiments, the fusion polypeptide further comprises a degradation tag. Optionally, the degradation tag is at the C-terminus of the fusion polypeptide. In some embodiments, the split intein comprises a C-terminal intein fragment ("I C ”) and an N-terminal intein fragment fused to the C-terminus of the amino acid sequence of the bacteriocin (“I N ”), wherein the polypeptide further comprises I N In some embodiments, the degradation tag comprises a sequence at least 80% identical to AANDENYALAA (SEQ ID NO: 873).

[0017] In some embodiments, the fusion polypeptide further comprises a signal peptide and / or a leader sequence.

[0018] Also provided herein is a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide as described in any of the preceding claims. Optionally, the nucleotide sequence is operably linked to a promoter sequence. In some embodiments, the nucleic acid comprises DNA. Optionally, the nucleic acid comprises RNA. Genetic vectors comprising the nucleic acid of the present disclosure are also provided.

[0019] Also provided are genetically engineered microbial cells comprising nucleic acids of the present disclosure or genetic vectors of the present disclosure. Optionally, the microbial cells are resistant to bacteriocins. In some embodiments, the microbial cells comprise a second nucleic acid encoding an immunomodulator that confers resistance to bacteriocins. Optionally, the expression of the immunomodulator from the second nucleic acid is adjustable. In some embodiments, the microbial cells are bacteria, fungi, or algae.

[0020] Also provided herein are compositions comprising the fusion polypeptides of the disclosure.Provided herein are compositions comprising a cyclic bacteriocin and a split intein.

[0021] Also provided herein is a method for preparing a cyclic bacteriocin, comprising contacting a nucleic acid of the present disclosure or a genetic vector of the present disclosure with an in vitro expression system under conditions sufficient to produce the cyclic bacteriocin. Also provided is a method for preparing a cyclic bacteriocin, comprising culturing a microbial cell of the present disclosure under conditions sufficient to produce the cyclic bacteriocin.

[0022] In some embodiments, the method further comprises purifying the cyclic bacteriocin. In some embodiments, the method further comprises purifying the fusion polypeptide. In some embodiments, the cleavage intein is a conditional cleavage intein that cyclizes the bacteriocin under permissive conditions rather than under non-permissive conditions, and wherein the method further comprises exposing the fusion polypeptide to permissive conditions after exposure to non-permissive conditions to induce cyclization of the bacteriocin. In some embodiments, the method further comprises changing pH or temperature to induce cyclization of the bacteriocin, wherein the cleavage intein is pH sensitive or temperature sensitive, respectively. In some embodiments, the method further comprises allowing the cleavage intein to degrade after producing the cyclic bacteriocin.

[0023] Also provided is a library comprising a plurality of genetic vectors, each genetic vector comprising a nucleic acid of the present disclosure, wherein at least two of the plurality of genetic vectors comprise nucleotide sequences encoding different bacteriocins. Optionally, the nucleotide sequence encodes bacteriocins from different microbial species. Optionally, the nucleotide sequence comprises different sequence variants of a parent bacteriocin. Optionally, the parent bacteriocin is a natural cyclic bacteriocin, and the sequence variant comprises a first variant that eliminates the natural cyclization of the parent bacteriocin.

[0024] The present invention also provides a method for screening, which comprises: providing a library of the present disclosure; expressing a plurality of polypeptides encoded by one or more genetic vectors in the library; generating a plurality of cyclic bacteriocins from a plurality of expressed polypeptides; and determining the desired activity of the plurality of cyclic bacteriocins. Optionally, the desired activity comprises antimicrobial activity.

[0025] Also provided is a method for controlling microbial growth, comprising contacting a composition comprising a microorganism and / or a composition that helps support microbial growth with a microbial cell of the present disclosure under conditions sufficient to produce a cyclic bacteriocin, thereby controlling the growth of the microorganism. Also provided is a method for controlling microbial growth, comprising contacting a composition comprising a microorganism and / or a composition that helps support microbial growth with a cyclic bacteriocin prepared by the method of the present disclosure, thereby controlling the growth of the microorganism. Provided herein is a method for controlling microbial growth, comprising contacting a composition comprising a microorganism and / or a composition that helps support microbial growth with a fusion polypeptide of the present disclosure, thereby controlling the growth of the microorganism.

[0026] In some embodiments, the microorganism is a bacterium. In some embodiments, the composition is a culture medium, a feedstock, or a microbial group. In some embodiments, the split intein is a conditional split intein that cyclizes the bacteriocin under permissive conditions but not under non-permissive conditions, and wherein the method further comprises providing permissive conditions to the composition, thereby inducing cyclization of the bacteriocin. In some embodiments, the method comprises changing the pH or temperature of the composition to induce cyclization of the bacteriocin, wherein the split intein is pH-sensitive or temperature-sensitive, respectively.

[0027] Also provided is a method for designing a nucleic acid encoding a polypeptide precursor of a bacteriocin, comprising: identifying a natural amino acid sequence of a candidate bacteriocin, wherein the natural amino acid sequence does not contain serine or cysteine ​​at the N-terminus; providing a second amino acid sequence having serine or cysteine ​​at its N-terminus by at least one of the following methods: cyclically arranging the natural amino acid sequence; or introducing serine or cysteine ​​into the natural amino acid sequence; providing a nucleotide sequence encoding a polypeptide comprising a second amino acid sequence, wherein both the N-terminus and the C-terminus are split inteins configured to cyclize the bacteriocin; and expressing the polypeptide encoded by the nucleotide sequence. Optionally, based on the genome sequence of the microorganism encoding the candidate bacteriocin in the genome of the microorganism, the candidate bacteriocin is predicted to be a cyclic bacteriocin. Optionally, the method comprises: identifying multiple natural amino acid sequences of multiple different candidate bacteriocins; for each of the multiple natural amino acid sequences: providing a second amino acid sequence; and providing a nucleotide sequence encoding a polypeptide comprising the second amino acid sequence, wherein both the N-terminus and the C-terminus are split inteins configured to cyclize the bacteriocin, thereby generating a library of nucleic acids representing each of the multiple natural amino acid sequences.

[0028] In some embodiments, the polypeptide further comprises a degradation tag. In some embodiments, the polypeptide further comprises a signal peptide and / or a leader sequence. In some embodiments, the polypeptide is expressed in vitro. In some embodiments, the polypeptide is expressed by a genetically engineered microbial cell, which is configured to express a polypeptide encoded by a nucleotide sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram showing a polypeptide of a bacteriocin flanked by split inteins that are spliced ​​to produce a circular bacteriocin according to some non-limiting embodiments of the present disclosure.

[0031] Figure 2A is a schematic diagram showing cyclic bacteriocins according to some non-limiting embodiments of the present disclosure.

[0032] Figure 2B is a schematic diagram showing the structure of nucleic acids encoding bacteriocins with or without a functional split intein according to some non-limiting embodiments of the present disclosure.

[0033] Figure 2C Depicted are amino acid sequences of split intein-flanked bacteriocins according to some non-limiting embodiments of the present disclosure.

[0034] Figure 2D is a schematic diagram showing a polypeptide of a bacteriocin flanked by split inteins that are spliced ​​to produce a circular bacteriocin according to some non-limiting embodiments of the present disclosure.

[0035] Figure 3A are images showing the antimicrobial activity of cyclic bacteriocins produced by bacteria genetically engineered with nucleic acids encoding bacteriocins flanked by split inteins according to some non-limiting embodiments of the present disclosure.

[0036] Figure 3B are images showing the antimicrobial activity of cyclic bacteriocins produced by bacteria genetically engineered with nucleic acids encoding bacteriocins flanked by split inteins according to some non-limiting embodiments of the present disclosure.

[0037] Figure 4A is a schematic diagram showing the in vitro and in vivo production of cyclic bacteriocins according to some non-limiting embodiments of the present disclosure, followed by the evaluation of the antimicrobial activity of the cyclic bacteriocins and mass spectrometry analysis.

[0038] Figure 4B is a collection of mass spectra of mass spectrometric analysis of purified cyclic bacteriocins produced by genetically engineered bacteria according to some non-limiting embodiments of the present disclosure.

[0039] Figure 5 is a block diagram showing screening methods according to some non-limiting embodiments of the present disclosure.

[0040] Details

[0041] Bacteriocins can be divided into two main groups: class I bacteriocins that undergo post-translational modification and class II or unmodified bacteriocins. Bacteriocins such as lantibiotics, thiopeptides, lassopeptides or sactibiotics belong to class I, and pediocin-like bacteriocins, dipeptide bacteriocins and linear non-pediocin-like single peptide bacteriocins belong to class II. Some bacteriocins undergo enzymatic modification during biosynthesis, where an amide bond is formed between the N-terminal and C-terminal amino acids, thereby obtaining a head-to-tail structure or a cyclic structure. Without being bound by theory, it is believed that their cyclic structure contributes to their higher stability to heat stress, pH changes and degradation by many proteolytic enzymes compared to the linear counterparts of these bacteriocins. Therefore, cyclic bacteriocins may have a variety of industrial applications.

[0042] The biosynthesis of circular bacteriocins involves the action of different proteins encoded by genes that are usually clustered together. The gene organization in head-to-tail circular bacteriocin clusters is very conserved and may include a minimum of 5 to 7 genes encoding the bacteriocin precursor peptide, an immunity protein, a membrane DUF95 protein (presumably involved in circularization), and one or more other proteins [9]

[10] .

[0043] A typical biosynthetic gene cluster for head-to-tail cyclized bacteriocins consists of genes encoding a bacteriocin precursor peptide, a transporter, a SpoIIM (phase II sporulation protein M) membrane protein (formerly known as DUF95), an immunity protein, and one or more unknown hydrophobic proteins. The inactive precursor peptide has an N-terminal leader sequence and a C-terminal core peptide. During maturation, the leader peptide is cleaved and a peptide bond is formed between the new N-terminal amino acid and the C-terminal residue, resulting in an active head-to-tail cyclized bacteriocin.

[0044] Advances in sequencing and bioinformatics have exponentially accelerated the discovery of novel cyclic bacteriocins. Through the identification of putative cyclic bacteriocin clusters in microbial genomes, many potential novel cyclic bacteriocins have been identified in a wide range of Gram-positive strains.

[0045] Novel bacteriocins can be experimentally confirmed by the production and purification of antimicrobial peptides in the supernatant of natural strains or heterologous hosts carrying all the genes required for the biosynthesis of mature bacteriocins. This process can be laborious, expensive and time-consuming, and in most cases requires natural bacteriocin-producing bacteria. Alternatively, cell-free protein synthesis methods can be used to produce bacteriocins. In vitro production can allow the properties of bacteriocins to be tested, including industrially relevant properties that may be more difficult to test by other methods (e.g., by fermentation) (see Gabant and Borrero 2019). In vitro production is also compatible with high-throughput methods for screening collections of bacteriocin genes or collections of variants thereof. Suitable options for in vitro production include PARAGEN 1.0 as described by Gabant and Borrero (2019), which demonstrates the synthetic production of a collection of 164 different class II bacteriocins (referred to as PARAGEN 1.0) using cell-free protein synthesis methods.

[0046] Split intein (internal protein) can be used to cyclize peptides. In some embodiments, the present invention provides a fast and reliable method for producing cyclic bacteriocins by combining the split intein circular ligation of peptides and proteins (SICLOPPS) method with cell-free protein synthesis. In some embodiments, the fusion of the C-terminal and N-terminal intein fragments of the DnaE split intein of Nostoc punctiforme (Npu) with the mature peptide of the bacteriocin garvicin ML allows the production and cyclization of the peptide without any other protein involved in the cyclization of the peptide in the natural environment. In some embodiments, active garvicin ML is produced in vitro (by cell-free synthesis) and in vivo (by Escherichia coli). The purification and subsequent analysis of garvicin ML have demonstrated the correct cyclization of the peptide, thereby obtaining a peptide with the same molecular weight as the natural peptide. In some embodiments, other cyclic bacteriocins characterized or not yet characterized are produced. In some embodiments, new candidates can be tested, or libraries of cyclic bacteriocins can be produced.

[0047] Provided herein are fusion polypeptides for producing cyclic bacteriocins and nucleic acids encoding them. In general, the fusion polypeptides of the present disclosure comprise the amino acid sequence of a bacteriocin, and the amino acid sequence of the bacteriocin is a broken intein capable of cyclizing the bacteriocin on both sides of the two ends of the amino acid sequence. In some embodiments, the fusion polypeptides and nucleic acids of the present disclosure promote the production of cyclic bacteriocins. The cyclic bacteriocins prepared by the fusion polypeptides of the present disclosure or the nucleic acids encoding them and the genetic vectors can have antimicrobial activity. In some embodiments, the cyclic bacteriocins prepared by the fusion polypeptides of the present disclosure or the nucleic acids encoding the fusion polypeptides disclosed herein and the genetic vectors have significant antimicrobial activity. In some embodiments, the cyclic bacteriocins prepared by the fusion polypeptides of the present disclosure or the nucleic acids encoding the fusion polypeptides disclosed herein and the genetic vectors have antimicrobial activity at least about the same level as the corresponding naturally produced cyclic bacteriocins. Since the cyclization of bacteriocins by split inteins can be achieved without any additional components that may be involved in the natural environment (e.g., other proteins encoded by genes of the bacteriocin cluster in natural microbial organisms), cyclic bacteriocins can be produced or expressed in various heterologous environments, for example, in heterologous organisms without additional proteins, or in vitro without additional components). In some embodiments, the fusion polypeptides and nucleic acids of the present disclosure provide high-throughput expression of known or putative cyclic bacteriocins for screening. In some embodiments, the fusion polypeptides and nucleic acids of the present disclosure provide expression of cyclic bacteriocin variants with mutations that will affect the cyclization of bacteriocins via natural mechanisms, thereby expanding the mutation space for screening variant bacteriocins with desired activities. In some embodiments, the fusion polypeptides and nucleic acids of the present disclosure provide expression of cyclic bacteriocin variants comprising non-natural amino acids, thereby expanding the mutation space for screening variant bacteriocins of interest. In some embodiments, the use of split inteins to cyclize bacteriocins allows for additional levels of control of regulating bacteriocin activity by regulating the cyclization activity of the split intein. In some embodiments, cyclizing the bacteriocin (including cyclizing a native linear bacteriocin) improves the stability of the bacteriocin, for example, by making the bacteriocin more resistant to degradation by heat, pH, or proteases.

[0048] the term

[0049] Unless otherwise noted, terms used herein have their customary and ordinary meanings as understood by those skilled in the art in light of this disclosure.

[0050] As used herein, "bacteriocin" and variants of this root term have their conventional and common meanings as understood by those skilled in the art in view of the present disclosure. It refers to a polypeptide that is secreted by a host cell and is capable of neutralizing at least one microbial organism different from the single host cell that produces the polypeptide, including cells associated with host cell clones and other microbial cells. Unless otherwise indicated, "bacteriocin" refers to natural cyclic bacteriocins and natural linear bacteriocins. "Cyclic bacteriocin" means a bacteriocin that is cyclized when expressed by the natural host from which the bacteriocin is derived, or a bacteriocin that is predicted to be cyclized based on the sequence of the bacterial genome, or a bacteriocin that has been designed or engineered to be active when cyclized. "Linear bacteriocin" means a bacteriocin that maintains linearity (and no cyclization) when expressed by the natural host from which the bacteriocin is derived, or a bacteriocin that is predicted to be linear based on the genome background, or a bacteriocin that has been designed or engineered to be active when in linear form. "Bacteriocin" also includes a cell-free or chemically synthesized form of this polypeptide, such as an engineered bacteriocin according to some embodiments of this invention. Host cells can exert cytotoxic or growth inhibitory effects on one or more other microbial organisms by secreting bacteriocins.

[0051] "Circularized" and "cyclized" are used interchangeably and have their conventional and ordinary meanings as understood by those skilled in the art in view of the present disclosure, and are used to refer to a polypeptide that has undergone head-to-tail cyclization or cyclization of the peptide backbone to form an amide bond between the N-terminal amino group and the C-terminal carboxyl group of the polypeptide. "Linear" as used herein has its conventional and ordinary meaning as understood by those skilled in the art in view of the present disclosure, and refers to a polypeptide with a free (non-bonded) amino group at the N-terminus and / or a free (non-bonded) carboxyl group at the C-terminus.

[0052] As used herein, "cyclic arrangement" means modifying a linear sequence of elements by moving the positions of the elements while maintaining the positions of each element relative to each other, wherein the elements moved past the first or last position in the linear sequence wrap around to the opposite end of the sequence. For example, a cyclic arrangement of the sequence "ABCDE" can produce any one of "BCDEA", "CDEAB", "DEABC", and "EABCD".

[0053] As used herein, the term "operably linked" has its conventional and ordinary meaning as understood by those skilled in the art in view of the present disclosure, and refers to the connection of nucleic acid elements in a functional relationship. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid. For example, if a transcriptional regulatory sequence affects the transcription of a coding sequence, it is operably linked to a coding sequence. "Operably linked" means that the DNA sequences being linked are generally contiguous, and when necessary to connect two protein coding regions, are contiguous and in reading frame.

[0054] The terms "protein" or "polypeptide" have their ordinary and common meaning as understood by those skilled in the art in view of this disclosure and are used interchangeably and refer to molecules composed of chains of amino acids without reference to a particular mode of action, size, three-dimensional structure or origin.

[0055] The term "gene" has its conventional and ordinary meaning as understood by those skilled in the art in view of the present disclosure, and means a DNA fragment comprising a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell, which is operably linked to a suitable regulatory region (e.g., a promoter). A gene will typically comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end), such as comprising a polyadenylation site and / or a transcription termination site.

[0056] In the amino acid sequences described herein, amino acids or "residues" are represented by three-letter or single-letter symbols. These three-letter symbols and the corresponding single-letter symbols are well known to those skilled in the art and have the following meanings: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine. The residue can be any proteinogenic amino acid, but can also be any non-proteinogenic amino acid, such as D-amino acids and modified amino acids formed by post-translational modifications, and can also be any non-natural amino acid. As used herein, "natural" and "non-natural" each have their ordinary and conventional meanings as understood by those of ordinary skill in the art in view of the present disclosure. A "natural" amino acid refers to an amino acid that occurs naturally in nature. A "non-natural" amino acid refers to an amino acid that is not genetically encoded, regardless of whether it occurs in nature.Unnatural amino acids that may be present in the peptide mimetics described herein include: b-amino acids; p-acyl-L-phenylalanine; N-acetyl lysine; O-4-allyl-L-tyrosine; 2-aminoadipic acid; 3-aminoadipic acid; β-alanine; 4-tert-butylhydrogen 2-azidosuccinate; β-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid; 2,4-diaminobutyric acid; 6-aminohexanoic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; p-aminophenylalanine; 2,3-diaminobutyric acid; 2,3-diaminopropionic acid; 2,2'-diaminopinnelic acid acid; p-amino-L-phenylalanine; p-azido-L-phenylalanine; D-allylglycine; p-benzoyl-L-phenylalanine; 3-benzothienylalanine; p-bromophenylalanine; tert-butylalanine; tert-butylglycine; 4-chlorophenylalanine; cyclohexylalanine; cysteine; D-citrulline; thio-L-citrulline; desmosine; ε-aminocaproic acid; N-ethylglycine; N-ethylasparagine; 2-fluorophenylalanine; 3-fluorophenylalanine; 4-fluorophenylalanine; homoarginine; homocysteine; homoserine; hydroxylysine; allohydroxylysine; 3-(3-methyl-4-nitrobenzyl)-L-histidine methyl ester; isodesmosine; allo-isoleucine; isopropyl-L-phenylalanine; 3-methyl-phenylalanine; N-methylglycine; N-methylisoleucine; 6-N-methyllysine; O-methyl-L-tyrosine; N-methylvaline; methionine sulfoxide; 2-naphthylalanine; L-3-(2-naphthylalanine); isoserine; 3-phenylserine; norvaline; norleucine; 5,5,5-trifluoro-DL-leucine; ornithine; 3-chloro-tyrosine; N5-carbamoyl-ornithine; penicillamine; phenylglycine; piperidinic acid acid); pyridylalanine; 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid; β-2-thienylalanine; γ-carboxy-DL-glutamic acid; 4-fluoro-DL-glutamic acid; D-thyroxine; allo-threonine; 5-hydroxy-tryptophan; 5-methoxy-tryptophan; 5-fluoro-tryptophan; 3-fluoro-valine. In some embodiments, the natural amino acids of the fusion polypeptides of the present disclosure are substituted with corresponding non-natural amino acids. As used herein, "corresponding non-natural amino acids" refers to non-natural amino acids that are derivatives of the referenced natural amino acids. For example, a natural amino acid can be substituted with a corresponding β-amino acid, which has an amino group bonded to the β-carbon instead of the α-carbon.

[0057] The terms "homology", "sequence identity" and the like have their conventional and ordinary meanings as understood by those skilled in the art in view of the present disclosure, and are used interchangeably herein. Sequence identity is described herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (nucleic acid) sequences, as determined by comparing the sequences. In one embodiment, sequence identity is calculated based on the full length of two given sequences (including those identified by SEQ ID NO) or a portion thereof. A portion thereof means at least 50%, 60%, 70%, 80%, 90% or 100% of the two SEQ ID NOs. "Identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as determined by the match between a series of such sequences. “Identity” can be easily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004.

[0058] " sequence identity " can be determined by using overall or local alignment algorithm to compare two peptides or two nucleotide sequences according to the length of two sequences.In embodiments, use overall alignment algorithm (for example Needleman-Wunsch) to compare sequences of similar length, and use local alignment algorithm (for example Smith-Waterman) to compare sequences of substantially different lengths.When sequence (when for example using the program EMBOSS needle or EMBOSS water of default parameters to carry out optimal comparison) shares at least a certain minimum percentage sequence identity (as described below), sequence can be referred to as "substantially identical".

[0059] When two sequences have similar length, global comparison is suitably used to determine sequence identity.In embodiments, when sequence has substantially different total lengths, local comparison can be used, such as those using Smith-Waterman algorithm.EMBOSS needle uses Needleman-Wunsch global comparison algorithm to compare two sequences over their entire length (total length), maximizing the number of matching and minimizing the number of spaces.EMBOSS water uses Smith-Waterman local comparison algorithm.Usually, using EMBOSS needle and EMBOSS water default parameters, wherein gap open penalty=10 (nucleotide sequence) / 10 (protein) and gap extension penalty=0.5 (nucleotide sequence) / 0.5 (protein).For nucleotide sequence, the default score matrix used is DNAfull, and for protein, the default score matrix is ​​BLOSUM62 (Henikoff & Henikoff, 1992, PNAS 89,915-919).

[0060] The percent identity can be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of some embodiments of the present disclosure can be further used as a "query sequence" to search public databases, for example to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the BLASTx program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. In order to obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., BLASTx and BLASTn) can be used.See the home page of the National Center for Biotechnology Information, which can be accessed on the World Wide Web at ncbi.nlm.nih.gov / .

[0061] As used herein, "conservative" amino acid substitutions have their conventional and common meanings as understood by those skilled in the art in view of the present disclosure, and refer to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains are serine and threonine; a group of amino acids with amide-containing side chains are asparagine and glutamine; a group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains are lysine, arginine, and histidine; a group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. Suitable conservative amino acid substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue has been inserted in its place. In some embodiments, the amino acid changes are conservative. For each of the naturally occurring amino acids, suitable conservative substitutions include: Ala to ser; Arg to lys; Asn to gln or his; Asp to glu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asn or gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and Val to ile or leu.

[0062] As used herein, "microbial organism", "microorganism", "microbial cell" or "microbial host" and variations of these root terms (e.g., plural, etc.) have their ordinary and plain meanings as understood by those skilled in the art in view of the present disclosure, including any naturally occurring species or synthetic or fully synthetic prokaryotic or eukaryotic unicellular organisms. Thus, this expression may refer to cells of any of the three domains of bacteria, archaea and eukaryotes.

[0063] "Comprise" and its variations are used herein in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, "consisting of" may be replaced by "consisting essentially of", which means that the features described herein may include additional features other than the specifically identified features, which additional features do not change the unique characteristics of the features described.

[0064] Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the element be present. Thus, the indefinite article "a" or "an" generally means "at least one".

[0065] As used herein, the use of "at least" a particular value means the particular value or more. For example, "at least 2" should be understood to be the same as "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ... etc.

[0066] The word "about" or "approximately" when used in conjunction with a numerical value (e.g., about 10) means that the value may be 10% greater or less than a given value (e.g., 10) or that value. As used herein, the term "and / or" indicates that one or more of the described situations may occur alone or in combination with at least one of the described situations, or even with all of the described situations.

[0067] Unless stated otherwise, each of the embodiments identified herein can be combined together.

[0068] All patent applications, patents, and printed publications cited herein are incorporated herein by reference in their entirety (except for any definitions, subject matter disclaimers, or disavowals) at least for the subject matter cited, except to the extent the incorporated material is inconsistent with the express disclosure herein, in which case the language of the present disclosure controls.

[0069] Those skilled in the art will recognize many features similar or equivalent to those described herein that can be used in the practice of the subject matter of the present application.In fact, the present disclosure is in no way limited to the features described.

[0070] Peptides

[0071] As described above, a fusion polypeptide for producing a cyclic bacteriocin is provided. Figure 1, provides a schematic diagram of a fusion polypeptide of the present disclosure. The fusion polypeptide may include an amino acid sequence 110 of a bacteriocin, which in some embodiments may be a mature sequence of a bacteriocin. The mature sequence typically includes the full sequence of a bacteriocin without a natural signal peptide, a leader sequence, or other additional N-terminal or C-terminal regulatory sequences (e.g., involved in processing and / or secretion). In some embodiments, the amino acid sequence is arranged in a circular shape compared to the natural mature sequence of the bacteriocin. The amino acid sequence 110 may be flanked by split inteins 121, 122, which are arranged such that the split inteins cyclize the bacteriocin by cyclization of the peptide backbone. The amino acid sequence may be flanked at the N-terminus by a C-terminal intein fragment ("I C ”) 121, and the C-terminus may be flanked by an N-terminal intein fragment fused to the last amino acid residue 114 of the amino acid sequence 110 of the bacteriocin (“I N ”) 122. Thus, when the amino acid sequence 110 of the bacteriocin is flanked by split inteins at both the N-terminus and the C-terminus, the split intein mediates the formation of a peptide bond between the first amino acid residue 112 and the last amino acid residue 114 of the amino acid sequence 110 of the bacteriocin to produce a cyclized bacteriocin 115. After cyclization, the intein 125 can be cleaved from the cyclized bacteriocin. In some embodiments, the N-terminal amino acid residue 112 of the bacteriocin is directly cleaved from the cyclized bacteriocin. C In some embodiments, the amino acid sequence 110 of the bacteriocin is modified (e.g., by cyclization) from the native sequence (e.g., the native mature sequence) so that the first amino acid residue in the sequence is serine or cysteine, as provided herein. In some embodiments, after cyclization, the intein 125 is removed via a C-terminal degradation tag.

[0072] In some embodiments, the fusion polypeptide comprises the amino acid sequence of any suitable bacteriocin. In some embodiments, the amino acid sequence is the amino acid sequence of a cyclic bacteriocin (e.g., a bacteriocin known as a cyclic bacteriocin produced in a natural environment, a bacteriocin predicted to be a cyclic bacteriocin based on a genomic background, a bacteriocin designed or engineered to be functional in a cyclic form, etc.). In some embodiments, the bacteriocin has antimicrobial activity only when cyclized. In some embodiments, the bacteriocin has significant antimicrobial activity only when cyclized. In some embodiments, the bacteriocin has antimicrobial activity when in a linear form. In some embodiments, the bacteriocin has antimicrobial activity when cyclized and when in a linear form. In some embodiments, compared with when in a linear form, the bacteriocin has greater antimicrobial activity when cyclized. In some embodiments, the bacteriocin has an antimicrobial activity of greater than at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 150%, or at least about 200% or more, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 10%-200%, 10%-100%, 50%-200%, 50%-100%, 70%-200%, or 50%-150%) when cyclized compared to when in linear form.

[0073] The fusion polypeptide may include any suitable amino acid sequence of a bacteriocin or a variant thereof (e.g., a variant of its cyclic arrangement as described herein). In some embodiments, the amino acid sequence is a naturally occurring bacteriocin or is derived from a naturally occurring bacteriocin. In some embodiments, the amino acid sequence is a mature sequence of a bacteriocin or a variant thereof (e.g., a variant of its cyclic arrangement as described herein). In some embodiments, the amino acid sequence is an amino acid sequence of a bacteriocin without a natural signal peptide sequence. In some embodiments, the amino acid sequence is an amino acid sequence of a bacteriocin without any signal peptide sequence. In some embodiments, the amino acid sequence does not contain any sequence required for processing bacteriocins (e.g., intracellular processing, cyclization) in a natural environment. In some embodiments, the amino acid sequence of a bacteriocin is modified from a natural sequence (e.g., a natural mature sequence) to promote cyclization by breaking an intein. In some embodiments, the amino acid sequence of a bacteriocin comprises an amino acid as the first amino acid residue, which is preferably used for cyclization by a broken intein. In some embodiments, the amino acid preferably used for cyclization by a broken intein depends on the type of broken intein in the fusion polypeptide. In some embodiments, the amino acid preferably used for cyclization by a broken intein is cysteine ​​or serine. In some embodiments, the amino acid sequence of the bacteriocin comprises cysteine ​​or serine as the first amino acid residue. In some embodiments, as disclosed herein, the native amino acid sequence of the bacteriocin is arranged in a ring such that the cysteine ​​or serine present in the native amino acid sequence is the first amino acid residue of the amino acid sequence of the bacteriocin of the fusion polypeptide.

[0074] In some embodiments, the amino acid sequence of the bacteriocin in the fusion polypeptide is arranged in a circular pattern compared to the natural amino acid sequence of the bacteriocin (e.g., the natural mature sequence). As used herein, "circular arrangement" means to modify the linear sequence of elements by moving the position of the elements while maintaining the position of each element relative to each other, wherein the element moved through the first or last position in the linear sequence is looped back to the opposite end of the sequence. For example, the circular arrangement of the sequence "ABCDE" can produce any one of "BCDEA", "CDEAB", "DEABC" and "EABCD". In some embodiments, the amino acid residue that is not the N-terminal residue in the natural amino acid sequence of the bacteriocin (e.g., the natural mature sequence) is the first amino acid residue of the circular arrangement of the amino acid sequence of the bacteriocin in the fusion polypeptide. In some embodiments, the first amino acid residue of the circular arrangement of the amino acid sequence of the bacteriocin in the fusion polypeptide is the amino acid of the first amino acid that is preferably used for cyclization by the broken intein. In some embodiments, the preferred amino acid is cysteine ​​or serine. In some embodiments where the native amino acid sequence of the bacteriocin includes cysteine ​​or serine, the amino acid sequence of the bacteriocin is arranged in a ring compared to the native amino acid sequence, such that the native cysteine ​​or serine is the first amino acid residue of the amino acid sequence of the bacteriocin. In some embodiments where the native amino acid sequence of the bacteriocin includes cysteine ​​or serine, the amino acid sequence of the bacteriocin is arranged in a ring compared to the native amino acid sequence, such that the native cysteine ​​or serine is the first amino acid residue of the amino acid sequence of the bacteriocin and is directly adjacent to the amino acid residue of the bacteriocin. C Fusion. As used herein in the context of a fusion polypeptide, "the amino acid sequence of a bacteriocin" is intended to include the sequence of a circular arrangement of the bacteriocin relative to its native sequence.

[0075] In some embodiments, the first amino acid residue of the amino acid sequence of the bacteriocin in the fusion polypeptide is a non-natural amino acid residue. As used herein, "non-natural" has its common and conventional meaning as understood by those of ordinary skill in the art in view of the present disclosure, and represents an amino acid that is not present in a natural amino acid sequence or a cyclically arranged sequence thereof. In some embodiments, the first amino acid residue of the amino acid sequence of the bacteriocin of the fusion polypeptide is a non-natural amino acid residue, which is a preferred amino acid for cyclization by a broken intein. In some embodiments, the natural amino acid sequence of the bacteriocin is modified by adding a non-natural amino acid residue to the natural sequence, or by replacing a natural amino acid residue with a non-natural amino acid residue. In some embodiments, the natural amino acid sequence of the bacteriocin is modified by adding an amino acid preferred by a broken intein to the N-terminus of the natural sequence, or by replacing the first amino acid residue of the natural sequence with an amino acid preferred by a broken intein, to provide an amino acid sequence in a fusion polypeptide. In some embodiments, the natural amino acid sequence of the bacteriocin does not contain an amino acid preferred by a broken intein. In some embodiments, the length of the amino acid sequence of the bacteriocin is increased by one residue due to the addition of a non-natural amino acid compared to the length of the natural amino acid sequence of the bacteriocin.

[0076] In some embodiments, the cysteine ​​or serine as the first amino acid residue of the amino acid sequence of the bacteriocin is a cysteine ​​or serine that is not present in the natural amino acid sequence of the bacteriocin (or its cyclically arranged sequence). In some embodiments, the natural amino acid sequence of the bacteriocin is modified by adding a cysteine ​​or serine residue to the natural sequence, or by replacing a natural amino acid residue that is not cysteine ​​or serine with a cysteine ​​or serine. In some embodiments, the natural amino acid sequence of the bacteriocin is modified by adding an N-terminal cysteine ​​or serine to the natural sequence, or by replacing the first amino acid residue of the natural sequence with a cysteine ​​or serine to provide an amino acid sequence in a fusion polypeptide. In some embodiments, the natural amino acid sequence of the bacteriocin can be modified by inserting a cysteine ​​or serine into the natural sequence, or by replacing an amino acid of the natural sequence (except the first N-terminal residue) with a cysteine ​​or serine to provide an amino acid sequence in a fusion polypeptide, and arranging the modified sequence (as disclosed herein) in a cyclic manner so that the non-natural cysteine ​​or serine is the first amino acid residue of the amino acid sequence of the bacteriocin in the fusion polypeptide. In some embodiments, the natural amino acid sequence of the bacteriocin does not include serine or cysteine. In some embodiments, the length of the amino acid sequence of the bacteriocin is increased by one residue due to a non-natural serine or non-natural cysteine ​​compared to the length of the natural amino acid sequence of the bacteriocin.

[0077] The amino acid sequence of the bacteriocin can have any suitable length. In some embodiments, the amino acid sequence of the bacteriocin in the fusion polypeptide is 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-175, 175-200, 200-300. In some embodiments, the present invention relates to a polypeptide having a length of 20-300, 20-400, 40-600, 600-800, 800-1000 amino acids in length, or longer, or a length within the range defined by any two of the preceding values, such as 20-1000, 20-800, 20-600, 100-800, 20-150, 80-150, 40-130, 100-150, or 20-80 amino acids in length.

[0078] Suitable amino acid sequences of cyclic bacteriocins include, but are not limited to, any of the sequences shown in Table A. In some embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage in a range defined by any two of the foregoing values ​​(e.g., 70-100%, 70-90%, 75-95%, 80-90%, 90-98%) identical to any of the sequences in Table A. In some embodiments, the fusion polypeptide comprises an amino acid sequence of any of the sequences shown in Table A, with up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions thereto. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of the sequences shown in Table A, having up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions thereto. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of the sequences shown in Table A, having up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid additions and / or deletions thereto. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of the sequences shown in Table A, having up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and / or deletions thereto. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of the sequences shown in Table A, having up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid conservative substitutions, additions and / or deletions thereto. In some embodiments, amino acid substitution or addition includes substitution or addition of non-natural amino acids with non-natural amino acids. In some embodiments, amino acid substitution or addition includes substitution or addition of only natural amino acids with natural amino acids.

[0079] In Table A, each bacteriocin is represented by two amino acid sequences (except for bacteriocin F9 from Staphylococcus felis, which is represented by three sequences), wherein for each bacteriocin entry, the native mature sequence is shown at the top, and a modified form of the native mature sequence is shown at the bottom, each modified form having serine as the first amino acid residue of the bacteriocin by cyclic permutation of the native sequence and / or insertion or substitution of serine of the native sequence. For example, for Amylocyclicin (Alc) from Bacillus amyloliquefaciens, the native mature sequence has the following sequence:

[0080] LASTLGISTAAAKKAIDIIDAASTIASIISLIGIVTGAGAISYAIVAT AKTMIKKYGKKYAAAW(SEQID NO:751),

[0081] And the circular arrangement form of the natural mature sequence has the following sequence:

[0082] STAAAKKAIDIIDAASTIASIISLIGIVTGAGAISYAIVATAKTMIKKYGKKYAAAWLASTLGI (SEQ ID NO: 752).

[0083] In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of the modified sequences of Table A (the bottom line of each bacteriocin entry). In some embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or about 100% or a percentage in a range defined by any two of the foregoing values ​​(e.g., 70-100%, 80-100%, 90-95%, 85-95% or 95-99%) identical to any one of the modified sequences of Table A (the bottom line of each bacteriocin entry). In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of the modified sequences of Table A (the bottom line of each bacteriocin entry) with up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and / or deletions thereto. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of the modified sequences of Table A (the lower row of each bacteriocin entry), with up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions, additions and / or deletions thereto. In some embodiments, the amino acid substitutions or additions comprise substitutions or additions of non-natural amino acids with non-natural amino acids. In some embodiments, the amino acid substitutions or additions comprise substitutions or additions of only natural amino acids with only natural amino acids.

[0084] Table A

[0085]

[0086]

[0087]

[0088]

[0089] Other non-limiting examples of bacteriocins (e.g., linear bacteriocins) suitable for the fusion polypeptide of the present application are shown in the even-numbered sequences of SEQ ID NO:4-450 and the odd-numbered sequences of SEQ ID NO:699-737. A detailed description of suitable bacteriocins can be found in, for example, U.S. Patent No. 9,333,227 and International Publication No. WO2019 / 046577, each of which is hereby incorporated by reference in its entirety. Some examples of suitable bacteriocins and bacteriocin classes are taught in Table 1.1 and Table 1.2 of U.S. Patent No. 9,333,227 and International Publication No. WO2019 / 046577. The amino acid sequence of the bacteriocin in the fusion polypeptide may comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or about 100% or a percentage within a range defined by any two of the aforementioned values ​​(e.g., 70-100%, 70-90%, 75-95%, 80-90%, 90-98%) identical to any amino acid sequence of the bacteriocin disclosed herein (including those described above). Any amino acid sequence of the bacteriocin disclosed herein (including those described above) may be modified by any suitable selection disclosed herein so that serine or cysteine ​​(e.g., native serine or cysteine) is the first amino acid of the bacteriocin sequence. In some embodiments, any amino acid sequence of the bacteriocin disclosed herein (including those described above) may be arranged in a ring to place serine or cysteine ​​as the first amino acid of the bacteriocin sequence in the fusion polypeptide. In some embodiments, serine or cysteine ​​may be added to or may be substituted for a native amino acid in any of the amino acid sequences of the bacteriocins disclosed herein, including those described above, and optionally may be further cyclically arranged to place the non-native serine or cysteine ​​as the first amino acid of the bacteriocin sequence in the fusion polypeptide.

[0090] In some embodiments, the bacteriocin in the fusion polypeptide of the present application is an engineered bacteriocin, for example, a polypeptide engineered to have antimicrobial activity when cyclized. In some embodiments, the fusion polypeptide comprises a non-natural amino acid in the amino acid sequence of the bacteriocin and / or the cleavage intein. In some embodiments, the fusion polypeptide comprises 1, 2, 3, 4, 5 or more non-natural amino acids in the amino acid sequence of the bacteriocin and / or the cleavage intein. In some embodiments, 1, 2, 3, 4, 5 or more amino acids in the amino acid sequence of the bacteriocin and / or the cleavage intein of the fusion polypeptide are replaced by corresponding non-natural amino acids.

[0091] The split intein of the fusion polypeptide of the present application can be any suitable intein that can mediate the cyclization of the bacteriocin. In some embodiments, the split intein comprises C-terminal and N-terminal intein fragments (respectively I C and I N ). In some embodiments, I C The amino acid sequence of the bacteriocin is fused to the N-terminus, and I N Fusion to the C-terminus of the amino acid sequence of the bacteriocin. In some embodiments, the split intein is a constitutively active split intein (e.g., a split intein capable of cyclizing the bacteriocin under conditions where the fusion polypeptide is expressed from a nucleic acid encoding it). In some embodiments, the split intein is a conditional split intein, for example, a split intein that cyclizes the bacteriocin under permissive conditions and does not cyclize the bacteriocin under non-permissive conditions. In some embodiments, the split intein cyclizes the bacteriocin under permissive conditions and does not substantially cyclize the bacteriocin under non-permissive conditions. In some embodiments, the split intein preferentially or specifically cyclizes the bacteriocin under permissive conditions. In some embodiments, the split intein cyclizes the bacteriocin under permissive conditions at a faster rate than under non-permissive conditions. In some embodiments, the split intein cyclizes the bacteriocin to a greater extent under permissive conditions than under non-permissive conditions. In some embodiments, the conditional split intein is sensitive to pH, temperature, light stimulation, and / or small molecule ligands. As used herein, "sensitive" has its ordinary and plain meaning as understood by those skilled in the art in view of the present disclosure and with reference to the environmental conditions of the conditionally split intein, meaning that the cyclization activity of the split intein (e.g., its rate and / or extent) is affected by the environmental conditions to which the split intein is exposed.

[0092] In some embodiments, the split intein is configured to preferentially or specifically cyclize the bacteriocin at a permissive pH (or pH range). In some embodiments, the split intein is configured to preferentially or specifically cyclize the bacteriocin at a permissive temperature (or temperature range). In some embodiments, the split intein is pH sensitive. In some embodiments, the split intein cyclizes the bacteriocin at a pH or pH range below a threshold pH or above a threshold pH. In some embodiments, the threshold pH is less than 3.0, or is about 3.0, 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.5, or about 10.0, or is a pH value within a range defined by any two of the foregoing values, e.g., pH 3-5, pH 4-7, pH 4-6, pH 5-6, pH In some embodiments, the pH range is limited by any two of the following pH values: 3.0, 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.5, 10.0. In some embodiments, the split intein is temperature sensitive. In some embodiments, the split intein cyclizes the bacteriocin at a temperature or temperature range below or above a threshold temperature. In some embodiments, the threshold temperature is less than 15°C, or is about 15°C, 16.0°C, 17.0°C, 18.0°C, 19.0°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 45°C, 50°C, 55°C, or about 60°C, or is a temperature within a range defined by any two of the foregoing values, e.g., 15-20°C, 15-18°C, 30-40°C, 40-50°C, 25-35°C.In some embodiments, the temperature range is defined by any two of the following temperatures: 15°C, 16.0°C, 17.0°C, 18.0°C, 19.0°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 45°C, 50°C, 55°C, 60°C. In some embodiments, the split intein is configured to preferentially or specifically cyclize the bacteriocin in the presence of a small molecule ligand. In some embodiments, the split intein is configured to preferentially or specifically cyclize the bacteriocin by light stimulation. Non-limiting examples of suitable conditional inteins are disclosed in DiVentura et al., (Biological Chemistry, vol. 400, no. 4, 2019, pp. 467-475), which is incorporated herein by reference in its entirety.

[0093] In some embodiments, the split intein is based on an intein from one of the following: Npu DnaE, ​​Sce VMA, Ssp DnaE. In some embodiments, the split intein is a natural split intein (e.g., found as a split intein in the genome of the host microorganism). In some embodiments, the split intein is not a split intein in its natural background, but is engineered to be a split intein. In some embodiments, the split intein is a constitutively active split intein derived from any one of Npu DnaE, ​​SceVMA, Ssp DnaE (e.g., a split intein capable of cyclizing bacteriocin under conditions where the fusion polypeptide is expressed from a nucleic acid encoding it). In some embodiments, the split intein is a conditional split intein derived from any one of Npu DnaE, ​​Sce VMA, Ssp DnaE. In some embodiments, the split intein is derived from the Nostoc punctata (Npu) DnaE split intein. In some embodiments, the split intein comprises C-terminal and N-terminal intein fragments from the Npu DnaE split intein. C and I N Contains the corresponding amino acid sequence shown in Table B. In some embodiments, I C and I N Comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage within a range defined by any two of the foregoing values ​​(e.g., 70-100%, 70-90%, 75-95%, 80-90%, 90-98%) identical to the corresponding sequence shown in Table B.

[0094] Table B

[0095]

[0096] In some embodiments, the fusion polypeptide comprises one or more additional functional sequences in addition to the bacteriocin flanked by split inteins. In some embodiments, the fusion polypeptide comprises a degradation tag, e.g., configured to degrade the intein after the bacteriocin is cyclized and the intein is cleaved from the cyclized bacteriocin. In some embodiments, the fusion polypeptide comprises a fragment of the N-terminal intein fragment I. N In some embodiments, the split intein comprises a C-terminal intein fragment ("I C ”) and an N-terminal intein fragment fused to the C-terminus of the amino acid sequence of the bacteriocin (“I N ”), wherein the polypeptide further comprises I N The degradation tag at the C-terminus. The degradation tag can be any suitable peptide that can induce the degradation of the cleaved intein after the bacteriocin is cyclized and the intein is cleaved from the cyclized bacteriocin. In some embodiments, the degradation tag is an SsrA sequence. In some embodiments, the degradation tag comprises the sequence: AANDENYALAA (SEQ ID NO: 873). In some embodiments, the degradation tag comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or about 100% identical to AANDENYALAA (SEQ ID NO: 873) or a percentage within a range defined by any two of the foregoing values ​​(e.g., 70-100%, 70-90%, 75-95%, 80-90%, 90-98%). In some embodiments, the degradation tag comprises a sequence that differs from AANDENYALAA (SEQ ID NO: 873) by at most 4, 3, 2, or 1 amino acids (e.g., substitutions, additions, and / or deletions). In some embodiments, the degradation tag comprises a sequence that differs from AANDENYALAA (SEQ ID NO: 873) by at most 4, 3, 2, or 1 conservative amino acid substitutions, additions, and / or deletions. In some embodiments, the amino acid substitutions or additions comprise substitutions or additions of non-natural amino acids with non-natural amino acids. In some embodiments, the amino acid substitutions or additions comprise substitutions of only natural amino acids with only natural amino acids or additions of only natural amino acids.

[0097] In some embodiments, the fusion polypeptide comprises one or more affinity tags. In some embodiments, the affinity tag is associated with a break intein, which can promote the purification of the fusion protein, but can be separated from the bacteriocin after cyclization. In some embodiments, the affinity tag is associated with the amino acid sequence of the bacteriocin and can be incorporated into the cyclic bacteriocin. The affinity tag can be used to purify the cyclic bacteriocin after cyclization. Optionally, one or more cleavage sites can be located between the affinity tag and the rest of the fusion polypeptide to facilitate affinity purification and remove the affinity tag. The affinity tag can be used for purification, for example, by contacting a molecule that binds to an affinity tag fixed on a solid phase (e.g., beads). An exemplary affinity tag suitable for the fusion polypeptide of the present disclosure can include, consist of, or consist of: His-tag, glutathione-S-transferase (GST) tag, FLAG tag, strep tag, maltose binding protein (MBP), chitin binding protein (CBP), myc tag, HA tag, NE tag, and V5 tag, variants in any of these tags, or any combination of two or more of these tags. In some embodiments, the affinity tag is chitin binding protein (CBP).In some embodiments, chitin resin is used to purify fusion polypeptides or cyclized bacteriocins with a CBP affinity tag.

[0098] In some embodiments, the fusion polypeptide comprises a signal peptide and / or a leader sequence. In some embodiments, the signal peptide or leader sequence is configured to promote secretion of the fusion polypeptide from a genetically engineered microbial cell expressing the fusion polypeptide, as disclosed herein. Any suitable signal peptide and / or leader sequence that can promote secretion of the fusion polypeptide or cyclic bacteriocin from a genetically engineered microbial cell can be used. In some embodiments, the fusion polypeptide also comprises post-translational or co-translational modifications, for example, glycosylation, acetylation, methylation, PEGylation, SUMOylation, ubiquitination or any of these two or more.

[0099] Compositions comprising the fusion polypeptides of the present disclosure are also provided. In some embodiments, the composition comprises a physiologically compatible carrier, such as water or a buffer solution. In some embodiments, the fusion polypeptide is lyophilized in the composition. Compositions comprising a cyclic bacteriocin and a split intein are also provided. The cyclic bacteriocin can be any cyclic bacteriocin produced by the fusion polypeptide, including those described herein. In some embodiments, the split intein comprises a C-terminal intein fragment (I C ) and the N-terminal intein fragment (I N ). In some embodiments, I C and I N The split intein can be any suitable split intein provided herein. In some embodiments, the split intein further comprises a degradation tag.

[0100] Nucleic acids, vectors, genetically engineered microbial cells

[0101] Also provided herein are nucleic acids comprising nucleotide sequences encoding fusion polypeptides as described herein. In some embodiments, nucleic acids (e.g., DNA or RNA) are included in regulatory elements driving fusion polypeptide expression under suitable conditions. In some embodiments, nucleic acids include DNA. In some embodiments, nucleic acids (e.g., DNA) are included in regulatory elements (e.g., promoters) driving nucleic acid transcription under suitable conditions (e.g., in vivo expression or in vitro transcription). In some embodiments, the nucleotide sequence is operably linked to a promoter sequence, e.g., in a DNA vector, as disclosed herein. Any suitable promoter sequence can be used to drive the transcription of nucleic acids. In some embodiments, the promoter sequence is a promoter sequence suitable for driving nucleic acid transcription in vitro (e.g., in an in vitro transcription solution). In some embodiments, the promoter sequence is a promoter sequence suitable for expressing fusion polypeptides from nucleic acids in vivo (e.g., in microbial cells). In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a conditional active promoter, e.g., depending on the presence or absence of environmental conditions, compounds, gene products, cell cycle stages, etc.

[0102] Non-limiting example nucleic acids encoding some of these bacteriocins (e.g., linear bacteriocins) are shown in the odd-numbered sequences of SEQ ID NO: 5-451 and the even-numbered sequences of SEQ ID NO: 700-738. Detailed descriptions of suitable bacteriocins and some polynucleotide sequences encoding bacteriocins, including methods and compositions for using bacteriocins to control the growth of microbial cells, can be found in, for example, U.S. Patent No. 9,333,227 and International Publication No. WO2019 / 046577, each of which is hereby incorporated by reference in its entirety.

[0103] In some embodiments, nucleic acid is included in the regulatory element that drives fusion polypeptide translation from nucleic acid under suitable conditions (for example, in vivo expression or in vitro translation).In some embodiments, nucleic acid is RNA.Generally, the translation initiation of a particular transcript is regulated by the 5' end of the specific sequence of the 5' end of the coding sequence of the transcript or the 5' end of the 5' end of the coding sequence of the transcript.For example, the coding sequence can start from the start codon configured to be paired with the initiator tRNA.Although naturally occurring translation systems usually use Met (AUG) as the initiator codon, it will be easily understood that the initiator tRNA can be engineered to combine any desired triplet, therefore, in certain embodiments, the triplet except AUG can also be used as the initiator codon. In addition, sequences near the start codon can promote ribosome assembly, such as the Kozak sequence ((gcc)gccRccAUGG, SEQ ID NO: 542, wherein R represents "A" or "G") or the internal ribosome entry site (IRES) in a typical eukaryotic translation system, or the Shine-Delgarno sequence (GGAGGU, SEQ ID NO: 543) in a typical prokaryotic translation system. Thus, in some embodiments, a transcript comprising an "encoding" nucleotide sequence of the present disclosure comprises an appropriate start codon and a translation initiation sequence. In some embodiments, for example, if two or more "encoding" nucleotide sequences are positioned in cis on a transcript, each nucleotide sequence comprises an appropriate start codon and a translation initiation sequence. In some embodiments, for example, if two or more "encoding" nucleotide sequences are positioned in cis on a transcript, the two sequences are under the control of a single translation initiation sequence, and either a single polypeptide capable of functioning with two polypeptides encoded in cis is provided, or a method for separating two polypeptides encoded in cis (e.g., 2A sequences, etc.) is provided. In some embodiments, the translation initiator tRNA is regulatable so as to regulate the initiation of translation of the bacteriocin from the nucleic acid.

[0104] Genetic vectors comprising nucleic acids of the present disclosure are also provided. Any suitable genetic vector can be used to contain nucleic acids having nucleotide sequences encoding fusion polypeptides as described herein. In some embodiments, the genetic vector is an expression vector. Suitable genetic vectors include, but are not limited to, plasmids, viruses (including bacteriophages), and transposable elements.

[0105] In some embodiments, the genetic vector may include one or more additional nucleotide sequences encoding the target gene product. In some embodiments, the genetic vector may include additional nucleotide sequences encoding gene products, and the gene products confer resistance to the cyclized bacteriocin of the microbial cells expressing the cyclized bacteriocin from the genetic vector. In some embodiments, the gene product conferring resistance to the cyclized bacteriocin is an immunomodulator. Any suitable immunomodulator can be encoded by the additional nucleotide sequence in the genetic vector. Suitable immunomodulators are provided in, for example, but not limited to, U.S. Patent No. 9,333,227. In some embodiments, the genetic vector is configured to express the target gene product under suitable conditions. In some embodiments, the promoter in the genetic vector drives transcription from the nucleotide sequence encoding the bacteriocin and the additional nucleotide sequence encoding the gene product, and the gene product confer resistance to the cyclized bacteriocin of the microbial cells expressing the cyclized bacteriocin from the genetic vector. In some embodiments, the expression of the nucleotide sequence encoding the bacteriocin and the additional nucleotide sequence encoding the gene product conferring resistance to the cyclized bacteriocin is under the control of different promoters. In some embodiments, one or both promoters controlling the expression of the nucleotide sequence encoding the bacteriocin and the additional nucleotide sequence encoding the gene product conferring resistance to the cyclized bacteriocin are conditional promoters. In some embodiments, expression from the conditional promoter operably linked to the nucleotide sequence encoding the bacteriocin is regulated by different conditions than expression from the conditional promoter operably linked to the additional nucleotide sequence encoding the gene product conferring resistance to the cyclized bacteriocin.

[0106] Also provided is a genetically engineered microbial cell comprising a nucleic acid of the present disclosure or a genetic vector provided herein. Microbial cells can be genetically engineered by any suitable selection. In some embodiments, the genetic vector of the present disclosure is used to transform microbial cells. In some embodiments, nucleic acid is stably integrated into the chromosome, or can be a self-replicating unit (for example, as a plasmid, an extrachromosomal array, an episome, a minichromosome, etc.) independent of the chromosome. In some embodiments, plasmid conjugation can be used for the desired plasmid to be imported into a recipient microbial cell from a "donor" microbial cell.

[0107] Any suitable microbial cell can be genetically engineered to contain nucleic acid or genetic vectors of the present disclosure. In some embodiments, the microbial cell is a cell that does not naturally produce a bacteriocin encoded by nucleic acid or genetic vector. In some embodiments, the microbial cell is a cell that does not endogenously encode a bacteriocin encoded by nucleic acid or genetic vector in its genome. In some embodiments, the microbial cell is resistant to bacteriocin. In some embodiments, the microbial cell expresses a gene product (e.g., an immunomodulator) that imparts resistance to bacteriocin. In some embodiments, the microbial cell is genetically engineered to express a gene product (e.g., an immunomodulator) that imparts resistance to bacteriocin. In some embodiments, the expression of the immunomodulator from the second nucleic acid is adjustable. In some embodiments, the expression of the immunomodulator from the second nucleic acid is controlled by a conditional promoter.

[0108] The exemplary microbial cells that can be used according to the embodiments of this paper include but are not limited to bacteria, yeast, filamentous fungi and algae, such as photosynthetic microalgae. In addition, the microbial genome that is completely synthetic can be synthesized and transplanted into a single microbial cell to produce a synthetic microorganism that can continuously replicate itself (see Gibson et al. (2010), "Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome," Science 329: 52-56, which is incorporated herein by reference). Therefore, in some embodiments, microbial cells are completely synthetic. The expected combination of genetic elements comprising the elements of regulatory gene expression and the elements of the coding gene product (such as immunomodulators, toxins, antidotes and industrially useful molecules (also referred to as target products)) can be assembled into partially or completely synthetic microbial cells on the chassis (chassis) desired. Descriptions of genetically engineered microbial organisms for industrial applications can also be found in Wright, et al. (2013) "Building-in biosafety for synthetic biology" Microbiology 159: 1221-1235, which is incorporated herein by reference.

[0109] According to the embodiments herein, a variety of bacterial species and strains can be used, and genetically modified variants or synthetic bacteria based on the "chassis" of known species can be provided. Exemplary bacteria that can be used according to the embodiments herein (including those with industrially applicable characteristics) include, but are not limited to, Bacillus species (e.g., Bacillus coagulans, Bacillus subtilis, Bacillus spp. ... subtilis and Bacilluslicheniformis), Paenibacillus species, Streptomyces species, Micrococcus species, Corynebacterium species, Acetobacter species, Cyanobacteria species, Salmonella species, Rhodococcus species, Pseudomonas species, Lactobacillus species, Enterococcus species, Alcaligenes species, Klebsiella species, Paenibacillus species, Arthrobacter species, Corynebacterium species, Brevibacterium species, Thermus aquaticus, Pseudomonas stutzeri, Clostridium thermocellus, and Escherichia coli.

[0110] According to the embodiments herein, a variety of yeast species and strains can be used, and genetically modified variants or synthetic yeasts based on the "chassis" of known species can be provided. Exemplary yeasts with industrially applicable features that can be used according to the embodiments herein include, but are not limited to, species of Saccharomyces (e.g., Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces boulardii), species of Candida (e.g., Candida utilis, Candida krusei), species of Schizosaccharomyces (e.g., Schizosaccharomyces pombe, Schizosaccharomyces japonicus), species of Pichia or Hansenula (e.g., Pichia pastoris), species of Pichia spp. pastoris or Hansenula polymorpha) species and Brettanomyces species (e.g., Brettanomyces claussenii).

[0111] According to the embodiments of this paper, multiple algae species and bacterial strains can be used, and genetically modified variants or synthetic algae based on " chassis " of known species can be produced. In some embodiments, algae comprises photosynthetic microalgae, is basically made up of photosynthetic microalgae or is made up of photosynthetic microalgae. The exemplary algae species that can be used for biofuel and can use according to some embodiments of this paper include Brown grape algae (Botryococcus braunii), Chlorella (Chlorella) species, Dunaliella tertiolecta (Dunaliella tertiolecta), Gracilaria (Gracilaria) species, Pleurochrysis carterae (Pleurochrysis carterae) and Sargassum (Sargassum) species. In addition, many algae can be used for food products, fertilizer products, waste neutralization, environmental restoration and carbohydrate manufacturing (for example, biofuel).

[0112] According to the embodiments herein, a variety of filamentous fungi species and strains can be used, and genetically modified variants or synthetic filamentous fungi based on the "chassis" of known species can be provided. Exemplary filamentous fungi (including those with industrially applicable features) that can be used according to the embodiments herein include but are not limited to Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Coccidioidomyces. chliobolus), Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex ), Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocaffimastix, Neurospora, Paecilomyces, Peniciffium, Phanerochaete, Piromyces, Poitra sia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium,Volvariella or Xylaria. In some embodiments, filamentous fungal species include, but are not limited to, Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola), Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum), Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenaturn, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0113] Also provided is a library comprising nucleic acid or genetic vectors of the present disclosure. In some embodiments, the library can be used to screen for cyclic bacteriocins that have not yet characterized antimicrobial activity, or for screening cyclic bacteriocins with desired antimicrobial activity from different strains. In some embodiments, the library can be used to screen for different variants of cyclic bacteriocins with desired or altered activity. In some embodiments, at least two genetic vectors in the library comprise nucleotide sequences encoding different bacteriocins. The bacteriocins encoded by the genetic vectors of the library can be different in any suitable manner. In some embodiments, the library is a mutation library comprising sequence variants of bacteriocins with one or more mutations compared to the parent sequence. In some embodiments, the mutation in the sequence variant can include random mutations. In some embodiments, the mutation in the sequence variant can include targeted mutations. In some embodiments, the library can include sequence variants that will eliminate or cancel bacteriocin cyclization in a natural environment. In some embodiments, the parent bacteriocin is a natural cyclic bacteriocin, and the sequence variant includes a first variant that cancels the natural cyclization of the parent bacteriocin.

[0114] In some embodiments, the library comprises bacteriocins from different strains or species of microbial organisms (e.g., bacteria). In some embodiments, the library comprises different previously uncharacterized bacteriocins, e.g., bacteriocins predicted based on individual sequences or bacteriocins for which antimicrobial activity has not yet been observed. In some embodiments, the library comprises different bacteriocins known to have antimicrobial activity.

[0115] The library can contain any suitable number of variants. In some embodiments, the library contains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, 400, 500, 600, 700, 800, 900, 10 3 10 4 10 5 10 6 10 7 10 8 10 9 or more variations, or multiple variations within the range defined by any two of the aforementioned values.

[0116] method

[0117] Also provided is a method for preparing a cyclic bacteriocin (which may be referred to as a production method for convenience). In some embodiments, the method includes contacting a nucleic acid having a nucleotide sequence encoding a fusion polypeptide as described herein with an in vitro expression system under conditions sufficient to produce the cyclic bacteriocin. In some embodiments, the in vitro expression system is a cell-free transcription / translation solution. In some embodiments, an in vitro expression system is used to allow the expression of cyclic bacteriocin from nucleic acids encoding cyclic bacteriocins, wherein the nucleic acid cannot be expressed in vivo, for example, the nucleic acid cannot be expressed from microbial cells engineered with nucleic acid genes. Without being bound by theory, in some cases, the toxicity of the nucleic acid or the gene product encoded therein can prevent the gene product from being expressed from the nucleic acid.

[0118] Any suitable cell-free expression system can be used for in vitro transcription and / or translation of nucleic acid.In some embodiments, the in vitro expression system comprises a cell extract, is composed of a cell extract, or is substantially composed of a cell extract.In some embodiments, the in vitro expression system comprises RNA polymerase, ribosomes, tRNA (and corresponding amino acids), an energy source, and an enzyme cofactor.In some embodiments, the in vitro expression system may also include an enzyme for co-translation or post-translational modification, and / or a cellular component mediating protein folding, such as a heat shock protein.For example, in embodiments where nucleic acid comprises an RNA transcript encoding a fusion polypeptide, it is expected that an in vitro expression system comprising a translation solution, being substantially composed of a translation solution, or being composed of a translation solution is sufficient (because it will be understood that RNA is already a transcript).For example, in embodiments where nucleic acid comprises a DNA encoding a fusion polypeptide, it is expected that the in vitro expression system comprises a transcription solution (for transcribing DNA into RNA) and a translation solution (for translating RNA into polypeptides).In some embodiments, transcription and translation solutions are together in a single solution (for example, the components of the transcription solution and the translation solution are uniformly distributed in the same volume). In some embodiments, the transcription and translation solutions are in separate solutions, such as in vesicles suspended in a single solution, and / or in separate solutions applied sequentially, and / or in separate compartments. In some embodiments, the components of the in vitro transcription / translation solution are lyophilized and configured to be reconstituted into the in vitro transcription / translation solution upon addition of water. In some embodiments, the in vitro transcription / translation solution is reconstituted by adding water to the lyophilized components.

[0119] The translation solution can be used for translating the nucleic acid provided herein. Suitable translation solution can include a reagent for in vitro translation (for convenience, it can be referred to as "translation reagent" in this article), is basically composed of a reagent for in vitro translation or is composed of a reagent for in vitro translation, and can therefore be configured for in vitro translation of transcripts such as RNA. Some embodiments include a transcription solution containing a reagent for transcription (for convenience, it can be referred to as "transcription reagent" herein), and is therefore configured to be used for in vitro transcription and translation, such as to transcribe and translate the nucleic acid encoding fusion polypeptide provided herein. It is expected that in vitro transcription and translation in a single solution (for example, a transcription solution that additionally includes a translation solution as described herein) can promote the effective in vitro production of the fusion polypeptide of some embodiments. Therefore, according to some embodiments described herein, the in vitro expression system includes an in vitro transcription reagent and / or an in vitro translation reagent.

[0120] According to some embodiments described herein, the translation solution comprises, consists essentially of, or consists of one or more translation reagents or in vitro translation reagents. Examples of translation reagents include, but are not limited to, ribosomes, buffers, amino acids, tRNA (which may be combined with amino acids), lysates or extracts such as E. coli lysates or E. coli extracts, and cofactors or metal ions such as Mg. 2+ Or any combination of two or more of the listed items. According to some embodiments described herein, the translation solution also includes a transcription reagent and is therefore configured to be used for in vitro transcription and translation. As described herein, the transcription solution that additionally includes a translation reagent contemplates a single solution that is suitable for in vitro transcription and translation. Therefore, the transcription solution that additionally includes a translation reagent includes a single transcription / translation solution. It should be understood that some components (e.g., ribosomes) of the transcription and / or translation solution may not be liquids and may potentially be separated from the transcription and / or translation solution, for example, by filtration and / or centrifugation.

[0121] In some embodiments, the translation solution comprises a post-translational modification enzyme. Examples of post-translational modification enzymes include, but are not limited to, cleavage enzymes, kinases, phosphatases, glycosyltransferases, or a mixture of any two of the listed items.

[0122] The transcription solution of some embodiments described herein (and which may be included in a translation solution as described herein) may comprise, consist essentially of, or consist of one or more transcription reagents. Examples of transcription reagents include RNA polymerase, buffer, nucleic acid mixture (e.g., NTP, including ATP, GTP, CTP and UTP), cofactors or metal ions such as Mg. 2+, transcription inducers (e.g., transcription factors, IPTG or lactose), polyadenylation enzymes, capping enzymes, lysates or extracts, such as bacterial lysates or extracts, such as E. coli lysates or E. coli extracts, SP6 polymerase, T3 polymerase, T7 RNA polymerase, or a mixture of two or more of any of the listed items. The transcription solution can be used to transcribe a template, such as a candidate nucleic acid described herein. The translation solution of some embodiments includes one or more transcription reagents in combination with one or more translation reagents.

[0123] In vitro expression systems can be provided in any suitable volume. According to some embodiments described herein, the in vitro expression system is provided in a volume of 1 μl-1000 μl, 1 μl-50 μl, 1 μl-500 μl, 1 μl-900 μl, 50 μl-100 μl, 50 μl-500 μl, 50 μl-1000 μl, 100 μl-200 μl, 100 μl-500 μl, 100 μl-1000 μl, 200 μl-500 μl, 200 μl-1000 μl, 500 μl-900 μl, 500 μl-1000 μl, 1 ml-2 ml, 3 ml-5 ml, 5 ml-10 ml, 10 ml-20 ml, 20 ml-50 ml, 50 ml-100 ml or more.

[0124] According to some embodiments described herein, the in vitro transcription / translation solution is lyophilized. In some embodiments, the in vitro transcription / translation solution is configured to be reconstituted in a solution, such as water.

[0125] Contacting can be performed for any suitable amount of time. In some embodiments, contacting is performed for at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 1.5 hours, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, at least about 2 days, at least about 3 days or more or for a duration within the range defined by any two of the aforementioned time periods, e.g., 10-60 minutes, 1 hour-24 hours, 1-12 hours, 24-48 hours, 1-3 days.

[0126] In any production method, in some embodiments, the method includes culturing a microbial cell genetically engineered with a nucleic acid or genetic vector encoding a fusion polypeptide as described herein under conditions sufficient to produce the cyclic bacteriocin. In some embodiments, the method includes culturing a second microbial cell in combination with a microbial cell genetically engineered with a nucleic acid or genetic vector encoding a fusion polypeptide. In some embodiments, the second microbial cell is an industrially useful microbial cell resistant to the cyclic bacteriocin.

[0127] In any production method, in some embodiments, the method includes purifying cyclic bacteriocin. In some embodiments, cyclic bacteriocin is purified from an in vitro expression system. In some embodiments, after culturing a nucleic acid or genetic vector genetically engineered microbial cell encoding a fusion polypeptide as described herein, the cyclic bacteriocin is purified. Any suitable selection can be used to purify the cyclic bacteriocin. In some embodiments, the method includes purifying the fusion polypeptide, for example, using an affinity tag associated therewith. In some embodiments, when the fusion polypeptide or cyclic bacteriocin comprises an affinity tag, the cyclic bacteriocin can be purified by contacting the fusion polypeptide or cyclic bacteriocin with a support (for example, a column, beads, etc.) connected with a binding agent, wherein the binding agent binds the affinity tag, and elutes the bound fusion polypeptide or cyclic bacteriocin. Any suitable affinity tag (such as those disclosed herein) can be used to purify cyclic bacteriocin and / or fusion polypeptide. In some embodiments, the affinity tag is CBP. In some embodiments, the affinity tag is CBP, and purifying cyclic bacteriocin and / or fusion polypeptide comprises using chitin resin.

[0128] Contact or cultivation can be carried out under any suitable conditions for producing cyclic bacteriocins by in vitro expression systems or genetically engineered microbial cells. In some embodiments, contacting nucleic acid with an in vitro expression system includes incubating nucleic acid at a suitable temperature in a transcription and / or translation solution. In some embodiments, contact is carried out at room temperature. In some embodiments, contact is carried out at less than 15°C, or about 15°C, about 18°C, about 20°C, about 22°C, about 25°C, about 27°C, about 30°C, about 34°C, about 36°C, about 38°C or about 40°C or higher, or at a temperature within the range limited by any two of the aforementioned values. In some embodiments, cultivation is carried out at a temperature suitable for the growth of genetically engineered microbial cells. In some embodiments, the culturing is carried out at a temperature of less than 15°C, or about 15°C, about 18°C, about 20°C, about 22°C, about 25°C, about 27°C, about 30°C, about 34°C, about 36°C, about 38°C, or about 40°C or more, or in a range defined by any two of the foregoing values.

[0129] In any production method, in some embodiments, contacting the nucleic acid with an in vitro expression system includes incubating the nucleic acid at a suitable pH in a transcription and / or translation solution. In some embodiments, contacting is less than 3.0, or about 3.0, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 6.7, about 7.0, about 7.2, about 7.5, about 8.0, about 8.5, about 9.0 or about 10.0 or more pH, or at a pH within the range defined by any two of the foregoing values. In some embodiments, culturing is carried out at a pH suitable for growth of genetically engineered microbial cells. In some embodiments, culturing is carried out at a pH of less than 3.0, or about 3.0, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 6.7, about 7.0, about 7.2, about 7.5, about 8.0, about 8.5, about 9.0, or about 10.0 or more, or at a pH within the range defined by any two of the foregoing values.

[0130] In any of the production methods, in some embodiments where the split intein is a conditional intein, the method includes exposing the fusion polypeptide to permissive conditions after exposure to non-permissive conditions to induce cyclization of the bacteriocin. In some embodiments, the method also includes changing the temperature during (or after) contacting or culturing. In some embodiments where the split intein is temperature sensitive, the method includes changing the temperature from a non-permissive temperature to a permissive temperature, or vice versa. In some embodiments, the method also includes changing the pH during (or after) contacting or culturing. In some embodiments where the split intein is pH sensitive, the method includes changing the pH from a pH that does not allow cyclization to a pH that allows cyclization, or vice versa.

[0131] Also provided are screening methods (which for convenience may be referred to as screening methods). Figure 5 , method 500 may include providing a library of nucleic acids or genetic vectors of the present disclosure at frame 510. The method may also include expressing a plurality of polypeptides encoded by one or more genetic vectors of the library at frame 520. The method may also include producing a plurality of cyclic bacteriocins from a plurality of expressed polypeptides at frame 530. In addition, the method may include measuring the desired activity of a plurality of cyclic bacteriocins at frame 540. The desired activity may be any suitable activity of the cyclic bacteriocins. In some embodiments, the desired activity is a change in the activity relative to a reference (e.g., relative to the activity of the parent bacteriocin when screening a mutant library), or relative to a standard level of activity. In some embodiments, the desired activity is to identify an unknown or substantially unknown activity, for example, by screening an uncharacterized and / or predicted bacteriocin library to identify a bacteriocin effective for a microbial species.

[0132] In any screening method, in some embodiments, the desired activity includes antimicrobial activity. In some embodiments, the desired activity includes, for example, an antimicrobial activity increased against one or more microorganisms compared to the parent bacteriocin. In some embodiments, the desired activity includes antimicrobial activity against a particular species or strain of microorganisms. In some embodiments, the desired activity includes resistance to degradation, such as, but not limited to, protease, heat, or pH degradation.

[0133] Also provided are methods for controlling microbial growth (which may be referred to herein as growth control methods). In some embodiments, the method includes contacting a composition (e.g., culture medium, raw material, microbial group, etc.) containing a microorganism (e.g., an undesirable microorganism) with a genetically engineered microbial cell of the present disclosure under conditions sufficient to produce cyclic bacteriocins in the genetically engineered microbial cell to inhibit or slow the growth of the microorganism. In some embodiments, the method includes contacting a composition (e.g., culture medium, raw material, microbial group, etc.) that helps support the growth of a microorganism (e.g., an undesirable microorganism) with a genetically engineered microbial cell of the present disclosure under conditions sufficient to produce cyclic bacteriocins in the genetically engineered microbial cell to prevent the growth of the microorganism in the composition or delay the appearance of the microorganism in the composition.

[0134] In some embodiments, the method includes contacting a composition (e.g., culture medium, raw material, microbiome, etc.) containing a microorganism (e.g., an undesirable microorganism) with a cyclic bacteriocin prepared by a production method disclosed herein to inhibit or slow the growth of the microorganism. In some embodiments, the method includes contacting a composition (e.g., culture medium, raw material, microbiome, etc.) that helps support the growth of a microorganism (e.g., an undesirable microorganism) with a cyclic bacteriocin prepared by a production method disclosed herein to prevent the growth of microorganisms in the composition or delay the appearance of microorganisms in the composition. In some embodiments, the method includes contacting a composition (e.g., culture medium, raw material, microbiome, etc.) containing a microorganism (e.g., an undesirable microorganism) with a fusion polypeptide as disclosed herein to inhibit or slow the growth of the microorganism. In some embodiments, the method includes contacting a composition (e.g., culture medium, raw material, microbiome, etc.) that helps support the growth of a microorganism (e.g., an undesirable microorganism) with a fusion polypeptide as disclosed herein to prevent the growth of microorganisms in the composition or delay the appearance of microorganisms in the composition.

[0135] In any growth control method, in some embodiments, the microorganism targeted by the cyclic bacteriocin is any suitable microorganism, and it is desirable to limit or prevent the growth of the microorganism. In some embodiments, the microorganism is a bacterium. In some embodiments, the microorganism is a pathogenic microorganism.

[0136] In any growth control method, in some embodiments, the composition can be associated with any environment, in which it is desirable to control the growth of microorganisms. In some embodiments, the composition includes but is not limited to culture medium, raw material or microbial group. The microbial group may include any suitable collection of microorganisms associated with the environment. In some embodiments, the microbial group includes the microbial group of animals, human organs, plants, plant roots and / or soil. In some embodiments, the microbial group includes the microbial group of an object, such as skin, intestinal tract, gastrointestinal tract, mammary gland, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or bile duct. In some embodiments, the composition is associated with a commercially relevant environment, such as but not limited to industrial raw materials, or in a fermenter, or in a food, drug or cosmetic manufacturing environment.

[0137] In any of the methods of growth control, in some embodiments where the split intein is a conditional intein, the method comprises exposing the fusion polypeptide to permissive conditions after exposure to non-permissive conditions to induce cyclization of the bacteriocin. In any of the methods of growth control, in some embodiments, the method comprises changing the pH or temperature of the composition to induce cyclization of the bacteriocin, wherein the split intein is pH sensitive or temperature sensitive, respectively, as disclosed herein. In some embodiments, the method comprises changing the temperature of the composition from a non-permissive temperature or pH to a permissive temperature or pH, respectively, to induce cyclization of the bacteriocin.

[0138] Also provided is a method for designing a nucleic acid encoding a polypeptide precursor of a bacteriocin (which may be referred to as a design method for convenience). The method may include identifying a natural amino acid sequence of a candidate bacteriocin, wherein the natural amino acid sequence does not contain serine or cysteine ​​at the N-terminus; providing a second amino acid sequence having serine or cysteine ​​at its N-terminus by at least one of the following methods: arranging the natural amino acid sequence in a ring; or introducing a non-natural serine or cysteine ​​into the natural amino acid sequence; providing a nucleotide sequence encoding a polypeptide comprising a second amino acid sequence, wherein both the N-terminus and the C-terminus are broken inteins configured to cyclize the bacteriocin; and expressing a polypeptide encoded by the nucleotide sequence. In some embodiments, the candidate bacteriocin is, for example, a bacteriocin predicted to be a cyclic bacteriocin based on the sequence or genomic environment of the bacteriocin. The polypeptide comprising a second amino acid sequence of a broken intein configured to cyclize the bacteriocin on both the N-terminus and the C-terminus can be any suitable polypeptide, for example, a fusion polypeptide as disclosed herein. In some embodiments, the candidate bacteriocin is a bacteriocin predicted to be a cyclic bacteriocin based on the genome sequence of the microorganism encoding the candidate bacteriocin in the genome of the microorganism. In some embodiments, introducing a non-natural serine or cysteine ​​into a native amino acid sequence comprises substituting a natural amino acid residue with a serine or cysteine, or adding or inserting a serine or cysteine ​​into a natural amino acid sequence.

[0139] In some embodiments, nucleic acids encoding polypeptide precursors of bacteriocins can be used to generate libraries of candidate bacteriocins for screening. In any design method, in some embodiments, the method includes: identifying multiple natural amino acid sequences of multiple different candidate bacteriocins; for each of the multiple natural amino acid sequences: providing a second amino acid sequence; and providing a nucleotide sequence encoding a polypeptide comprising the second amino acid sequence, wherein the second amino acid sequence is a broken intein configured to cyclize the bacteriocin on both the N-terminus and the C-terminus. In some embodiments, a nucleic acid or genetic vector comprising a nucleotide sequence encoding a polypeptide can be provided in any suitable library, including a library as disclosed herein.

[0140] In some embodiments, the polypeptide further comprises a degradation tag as disclosed herein. In some embodiments, the polypeptide further comprises a signal peptide and / or leader sequence as disclosed herein.

[0141] The split intein can be any suitable split intein as described herein. Expression of a polypeptide encoded by a nucleotide sequence can be performed using any suitable selection. In some embodiments, a polypeptide encoded by a nucleotide sequence is expressed in an in vitro expression system, as provided herein. In some embodiments, a polypeptide encoded by a nucleotide sequence is expressed by a microbial cell genetically engineered with a nucleic acid having the nucleotide sequence.

[0142] Package Products

[0143] A package product for producing cyclic bacteriocins is also provided. In some embodiments, the package product comprises a fusion polypeptide of the present disclosure. In some embodiments, the package product comprises: a lyophilized composition of a fusion polypeptide of the present disclosure; and a liquid (e.g., water or buffer) for reconstructing the lyophilized composition. In some embodiments, the package product comprises a small group of fusion polypeptides with different bacteriocin sequences as disclosed herein. In some embodiments, the package product comprises a nucleic acid or genetic vector encoding a fusion polypeptide as disclosed herein. In some embodiments, the package product comprises a library of nucleic acids or genetic vectors encoding a variety of fusion polypeptides with different bacteriocin sequences as disclosed herein. In some embodiments, the package product comprises: a nucleic acid or genetic vector encoding a fusion polypeptide as disclosed herein; and an in vitro transcription solution (or one or more components thereof) or an in vitro transcription solution (or one or more components thereof) and an in vitro translation solution (or one or more components thereof). In some embodiments, the package product comprises a microbial cell genetically engineered with a nucleic acid or genetic vector encoding a fusion polypeptide as disclosed herein. In some embodiments, the package product comprises an indicator strain of a microorganism, which is known to be sensitive to the cyclic bacteriocin produced by the package product. In some embodiments, the kit further comprises instructions for producing the cyclic bacteriocin from the fusion polypeptide, nucleic acid, genetic vector, or genetically engineered microbial cell.

[0144] Additional Implementations

[0145] Embodiment 1-3 below has proved the cyclization of bacteriocin from fusion polypeptide, and described fusion polypeptide comprises the bacteriocin of fracture intein on both sides.Cyclic bacteriocin becomes the group of promising antimicrobial peptides for industrial application because its higher stability compared with its linear counterpart.Usually, these peptides have stronger resistance to proteolytic enzyme, and can keep its full activity under different pH or temperature.Until now, cyclic bacteriocin remains a very selective group, wherein only 20 candidate materials are found and characterized fully.Benefit from the latest progress of group technology, particularly the latest progress of genome sequencing, recently identified a large number of previously undescribed cyclic bacteriocin clusters of inference in different genomes of gram-positive bacteria from general database, show that these peptides may be more general than previously understood in nature.However, in some cases, when there is no production strain, it is difficult to predict the actual function of these genes / clusters found.

[0146] Synthetic biology tools can be used to quickly and easily produce bacteriocins. Using cell-free protein synthesis (CFPS) technology, a collection of different mature bacteriocins (fully characterized and hypothetical non-characterized candidates) can be produced

[13] . Using only a single gene encoding a mature peptide (without a signal peptide or leader sequence), CFPS allows the production of different active bacteriocins outside the bacterial host in less than 4 hours. Due to the fact that protein production occurs outside the cell, no additional leader sequence / signal peptide cleavage is required, nor is transport from a dedicated protein. Although this technology is suitable for the production of unmodified class II bacteriocins, the production of bacteriocins (class I) that require post-translational modification may not be so efficient without the activity of other dedicated proteins involved in the maturation of peptides in the natural bacterial host. Circular bacteriocins can be included in this last group, where several proteins are known to be involved in maturation (cleavage / cyclization) and secretion to the extracellular space via different dedicated transport systems in the natural bacterial host.

[0147] Examples 1-3 below show the cyclization of bacteriocins using split inteins. Inteins can be used with tags for column purification or protein degradation. Split intein-mediated cyclization of peptides and proteins (SICLOPPS) with different improvements integrated was used to cyclize bacteriocins, such as using an intein from Nostoc punctata (Npu), which is faster and also significantly more tolerant of amino acid diversity in the extein sequence, and also has an Ssra sequence at the C-terminus to reduce the toxic effects of Npu by directing Ssra-tagged proteins to the ClpXP machinery for degradation

[16] .

[0148] SICLOPPS was tested with Garvicin ML, a known cyclic bacteriocin from Lactococcus garvei DCC43, a strain isolated from Mallard Ducks [6]. It has been shown that splicing with the Npu intein is more efficient when Cys or Ser is located at position +1. Ser32 was chosen over the other two serines present in GarML, but other serines could also be chosen. For other bacteriocins produced by CFPS, the recombinant genes were placed under the control of T7 promoter and terminator sequences

[13] . After in vitro production, antimicrobial activity was only observed when the complete SICLOPPS construct was used, but no antimicrobial activity was observed in constructs that did not carry the intein (linear GarML) or in constructs with two point mutations in the intein (preventing splicing). Higher activity was observed when the reaction was left overnight at room temperature. It has been estimated that splicing of Npu occurs within the first 30 to 60 seconds, so the higher activity observed after 24 hours is unlikely to be explained by a higher percentage of splicing over time. Instead, this higher activity may be due to a higher yield of recombinant protein through the in vitro reaction. It has been observed that in some cases, making the in vitro reaction time longer than 2 hours can benefit protein production. These results indicate that SICLOPPS is effective and that GarML has been produced and spontaneously cyclized without any additional auxiliary proteins. To confirm the presence of spliced ​​GarML after CFPS, MS analysis of the reaction was performed and a peak with a mass of 6004 Da was observed, corresponding to the exact mass of the original GarML, confirming the correct splicing of the intein from GarML.

[0149] The correct cyclization of GarML was further confirmed by bringing the SICLOPPS construct into Escherichia coli and producing recombinant GarML in vivo for higher-scale protein production. After protein production and purification, a single FPLC fraction with antimicrobial activity was obtained. MS analysis of this fraction again showed a mass of 6004Da, confirming the results previously obtained with the CFPS method and showing that splicing also occurs in Escherichia coli. In addition, trypsin digestion of the purified peptides and LC-MS / MS analysis of the obtained fragments revealed the presence of 6 peptides covering 100% mature GarML. The presence of one of the peptides of S1 and F60 (TIVNAVSAGMDIATALSLFSGAFTAAGGIMALIK) linked together confirmed the correct cyclization of GarML at the selected position. These results show that the GarML produced with the SICLOPPS system is not only cyclized, but also has exactly the same sequence as the GarML produced by its natural producer, and is likely to adopt the same three-dimensional conformation. This was also observed during the construction of the PARAGEN collection, where different pediocin-like bacteriocins with 2 or 3 disulfide bonds in their native and active final conformation were active after in vitro production and showed a similar activity spectrum to the native bacteriocin. This is the first time that a cyclic bacteriocin has been produced in E. coli. Some options to enhance production and facilitate purification may include using a different host for protein production, adding immunity genes to the construct to prevent the toxic effects of the bacteriocin, fusing a signal peptide to the protein to facilitate extracellular secretion, using a switchable intein for conditional protein splicing, or using a fusion tag to facilitate column purification.

[0150] Finally, the use of SICLOPPS for the cyclization of other cyclic bacteriocins was identified. The list includes some fully characterized candidates, such as enterocin AS-48 or gnaphalactin A, two well-studied bacteriocins with well-documented potential as food biopreservatives or prebiotic compounds in animal health. The other bacteriocins selected in this study correspond to putative cyclic bacteriocins discovered in genome mining studies but not yet confirmed in the laboratory. After CFPS, most of the SICLOPPS were observed to produce bacteriocins showing activity against at least one of the indicators used in the study, thus indicating that, most likely, splicing and cyclization of these bacteriocins are occurring.

[0151] Examples 1-3 demonstrate that the cyclization of bacteriocins using split inteins allows for rapid production and cyclization of bacteriocins, ready to be tested for antimicrobial activity. This is the first time that split inteins have been used for the production and cyclization of bacteriocins, the first time that CFPS has been used to produce them, and the first time that functional cyclic bacteriocins have been produced by E. coli.

[0152] Examples 1-3 below demonstrate an effective synthetic biology method for cyclization of many bacteriocins, even in the absence of the original production strain. This method also simplifies the production of cyclic bacteriocins, as only a single gene is required for production and cyclization. This work provides uses of inteins with bacteriocins, including:

[0153] - For production, cyclization and functional testing of other putative circular bacteriocins.

[0154] -For the production and cyclization of acyclic (linear) bacteriocins to enhance stability.

[0155] -For the generation of circular bacteriocin libraries to test and obtain variants with enhanced characteristics.

[0156] - Heterologous production of cyclic bacteriocins in other hosts to expand bacteriocin production.

[0157] References

[0158] [1] M.Younes et al., "Safety of nisin(E 234) as a food additive in the light of new toxicological data and the proposed extension of use," EFSA J., vol.15, no.12, Dec.2017.

[0159] [2] S.Soltani et al., "Bacteriocins as a new generation ofantimicrobials:toxicity aspects and regulations," FEMS Microbiol.Rev., vol.45, no.1, Jan.2021.

[0160] [3] ML Chikindas, R. Weeks, D. Drider, VA Chistyakov, and LMDicks, "Functions and emerging applications of bacteriocins," Curr. Opin. Biotechnol., vol. 49, p. 23, Feb. 2018.

[0161] [4]M.Zimina et al.,“Overview of Global Trends in Classification,Methods of Preparation and Application of Bacteriocins,”Antibiot.2020,Vol.9,Page 553,vol.9,no.9,p.553,Aug.2020.

[0162] [5]P.D.Cotter,R.P.Ross,and C.Hill,“Bacteriocins-a viable alternativeto antibiotics?,”Nat.Rev.Microbiol.,vol.11,no.2,pp.95–105,Feb.2013.

[0163] [6]J.Borrero et al.,“Characterization of garvicin ML,a novel circularbacteriocin produced by Lactococcus garvieae DCC43,isolated from mallardducks(Anas platyrhynchos).,”Appl.Environ.Microbiol.,vol.77,no.1,pp.369–373,Jan.2011.

[0164] [7]P.Alvarez-Sieiro,M.Montalbán-López,D.Mu,and O.P.Kuipers,“Bacteriocins of lactic acid bacteria:extending the family,”Appl.Microbiol.Biotechnol.,vol.100,no.7,pp.2939–2951,Apr.2016.

[0165] [8]J.Borrero et al.,“Plantaricyclin A,a Novel Circular BacteriocinProduced by Lactobacillus plantarum NI326:Purification,Characterization,andHeterologous Production.,”Appl.Environ.Microbiol.,vol.84,no.1,Jan.2018.

[0166] [9]R.H.Perez,T.Zendo,and K.Sonomoto,“Circular and LeaderlessBacteriocins:Biosynthesis,Mode of Action,Applications,and Prospects.,”Front.Microbiol.,vol.9,p.2085,2018.

[0167]

[10] D.Major,L.Flanzbaum,L.Lussier,C.Davies,K.M.P.Caldo,and J.Z.Acedo,“Transporter Protein-Guided Genome Mining for Head-to-Tail CyclizedBacteriocins,”Molecules,vol.26,no.23,Dec.2021.

[0168]

[11] B.Xin et al.,“In Silico Analysis Highlights the Diversity andNovelty of Circular Bacteriocins in Sequenced Microbial Genomes,”mSystems,vol.5,no.3,Jun.2020.

[0169]

[12] B.Vezina,B.H.A.Rehm,and A.T.Smith,“Bioinformatic prospecting andphylogenetic analysis reveals 94 undescribed circular bacteriocins and keymotifs.,”BMC Microbiol.,vol.20,no.1,p.77,Apr.2020.

[0170]

[13] P.Gabant and J.Borrero,“PARAGEN 1.0:A Standardized Synthetic GeneLibrary for Fast Cell-Free Bacteriocin Synthesis.,”Front.Bioeng.Biotechnol.,vol.7,p.213,2019.

[0171]

[14] C.P.Scott,E.Abel-Santos,M.Wall,D.C.Wahnon,and S.J.Benkovic,“Production of cyclic peptides and proteins in vivo,”Proc.Natl.Acad.Sci.U.S.A.,vol.96,no.24,pp.13638–13643,Nov.1999.

[0172]

[15] A.Tavassoli and S.J.Benkovic,“Split-intein mediated circularligation used in the synthesis of cyclic peptide libraries in E.coli.,”Nat.Protoc.,vol.2,no.5,pp.1126–1133,2007.

[0173]

[16] J.E.Townend and A.Tavassoli,“Traceless Production of CyclicPeptide Libraries in E.coli.,”ACS Chem.Biol.,vol.11,no.6,pp.1624–1630,Jun.2016.

[0174]

[17] M. Cheriyan, CS Pedamallu, K. Tori, and F. Perler, "Faster proteins plicing with the Nostoc punctiforme DnaE intein using non-native exteinresidues," J. Biol. Chem., vol. 288, no. 9, pp. 6202–6211, Mar. 2013. Example

[0175] The following materials and methods were used in Examples 1-4.

[0176] Bacterial strains, plasmids and culture conditions

[0177] For GarML production, the microbial strains and plasmids used in this study are listed in Table 1.

[0178] Table 1

[0179]

[0180] a CECT, Colección Tipo de Cultivos.

[0181] b .A. Chopin, MC Chopin, A. Moillo-Batt, and P. Langella, "Two plasmid-determined restriction and modification systems in Streptococcus lactis.," Plasmid, vol.11, no.3, pp.260–263, May 1984.

[0182] exist Figures 2A-2DThe schematic diagram of the design of plasmid is shown in .All amino acid sequences of fusion polypeptide are shown in Table 2.1.When using SICLOPPS method, all amino acid sequences of characterized and uncharacterized cyclic bacteriocin (natural and modified sequences used for use with SICLOPPS (fracture intein ring connection of peptide and protein)) and control are shown in Table 2.2.For plasmid construction, all amino acid sequences are reverse translated and codon optimized (World Wide Web, bioinformatics.org / sms2 / rev_trans.html) for Escherichia coli.Nucleotide sequence is included in the carrier backbone containing T7 promoter region, start codon (ATG), stop codon (TAA) and T7 terminator region.Plasmid synthesis is carried out by Genewiz (New Jersey, USA).

[0183] Table 2.1

[0184]

[0185]

[0186]

[0187]

[0188] List of bacteriocins used in this study. Bacteriocins have been grouped according to the classification made by Vezina et al., 2020. In bold are the names of those fully characterized bacteriocins. The first row of mature amino acid sequences corresponds to the described or assumed linear sequence produced after leader sequence cleavage and before head-to-tail cyclization. The second row corresponds to the amino acid sequences used in this study for cyclization using the SICCLOPPS system. The serine used in position 1 is bold and underlined in the original sequence. Bacteriocin F9 had no serine in its original amino acid sequence. A serine (in bold) was added at the first position.

[0189] Table 2.2

[0190]

[0191] Components of the SICLOPPS system used for this study. Point residue substitutions used to generate non-functional inteins are in bold.

[0192] Cell-free production of bacteriocins

[0193] The recombinant vector was stabilized and amplified in the E. coli DH5α standard strain and used as Templates for cell-free protein synthesis were performed using the In Vitro Protein Synthesis Kit (New England Biolabs). All synthesized bacteriocins were tested for antimicrobial activity against indicator strains on petri dishes. Bacteriocins that showed an inhibition halo against at least one indicator were considered positive.

[0194] Production and purification of Garvicin ML from Escherichia coli BL21-GarvML

[0195] To purify the antimicrobial compounds produced by E. coli BL21-pUC-Npu-GarvML, the culture was grown in 10 ml LB medium (LB-Amp) supplemented with 100 μg / ml ampicillin and grown overnight in a shaking 37°C incubator. 500 ml of LB-Amp was inoculated with the overnight culture to an OD of 600 The culture was grown in a shaking 37°C incubator. When the culture reached OD 600 When the pH was 0.4, IPTG was added to a final concentration of 0.5 mM. The culture was grown for another 3 hours and the cells were pelleted by centrifugation (8,000 rpm; 4°C) for 15 minutes. The cells were resuspended in 20 ml of ice-cold column buffer (20 mM phosphate buffer at pH 6 and 1 M NaCl) and lysed by sonication (45% for 10 seconds, 6 cycles, incubation in ice for 1 minute between each cycle). The insoluble residue was pelleted by centrifugation (8,000 rpm; 4°C) for 15 minutes and the soluble fraction (SF) obtained was filtered through a 0.45 nm filter.

[0196] SF was further subjected to hydrophobic interaction (Octyl Sepharose CL-4B; Merck) chromatography. Ammonium sulfate was first added to SF (10% w / v). The column with 2 ml Octyl Sepharose CL-4B was washed with H2O and equilibrated with 15 ml equilibration buffer (EB; 20 mM phosphate buffer at pH 6 with ammonium sulfate [1% w / v]. SF was then added to the column and the column was washed with 10 ml EB. The bacteriocin was eluted with 10 ml 70% EtOH diluted in 20 mM phosphate buffer at pH 6.

[0197] Finally, the obtained fractions were diluted 5-fold in H2O+TFA (0.1% v / v) and chromatographed on a fast protein liquid chromatography system ( The samples were subjected to reverse phase chromatography (Source 5RPC ST4.6-150; GE Healthcare) in a RP-FPLC. The samples were eluted with a mobile phase consisting of 0.1% (v / v) trifluoroacetic acid (TFA) in a mixture of water (eluent A) and isopropanol (eluent B). (Both reagents were HPLC grade). A gradient program was followed: the sample was initially eluted with 100% A for 5 min, then eluted with a linear gradient of 0-70% B in 50 min, followed by a linear gradient to 100% B in 5 min, and kept at 100% B for 7 min. The flow rate was maintained at 1 ml / min, the absorbance was monitored at 254 nm and the column was maintained at room temperature. The antimicrobial activity of the column eluate and FPLC fractions was determined by spot on agar assays (SPA) using Lactococcus garvieae 5806 as the target organism. The FPLC fractions showing antimicrobial activity were subjected to a second round of FPLC under the same conditions.

[0198] Characterization of Garvivin ML by direct proteomics coupled with mass spectrometry

[0199] The active fractions from the second round of FPLC were concentrated with a Speed-vac and subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a 4800 Proteomics Analyzer using TOF / TOF (AB SCIEX) in positive reflectance mode (Unidad de Proteómica—Universidad Complutense de Madrid, Madrid, Spain).

[0200] After the sample is dried and resuspended in 25 μl 25mM TEAB and buffer S-TRAP to equal parts, it is digested with trypsin in S-Trap microcolumns (PROTIFITM) as recommended by the manufacturer. Soon, the protein is reduced with 10DTT at 56°C for 60min, and then alkylated with 25mM iodoacetamide in the dark for 60min. Then 20% SDS, 1M TEAB and phosphoric acid are added to final concentrations of 10%, 100mM and 1.2% respectively. Next, S-Trap binding buffer is added at a ratio of 6: 1, applied to the column, and digested with 1.5 μg sequencing grade recombinant trypsin (Roche Molecular Biochemicals) in 50mM TEAB under static conditions at 47°C for 90min according to the protocol.

[0201] Finally, the obtained peptides were eluted and dried on a SpeedVac (Savant), reconstituted in 15 μl 2% ACN, 0.1% formic acid, and quantified on a Qubit (ThermoFisher Scientific).The samples were then analyzed by LC-MS / MS.

[0202] Example 1

[0203] This non-limiting example demonstrates the design of a genetic vector encoding a bacteriocin flanked by split inteins and the cell-free production of active bacteriocin thereby.

[0204] Design of expression vector for production of garvicin ML

[0205] Based on the work describing the Split Intein Circular Ligation of Peptides and Proteins (SICLOPPS) system

[16] , C-terminal and N-terminal intein fragments (I and N, respectively) from the Npu DnaE split intein fused to the mature peptide of the bacteriocin Garvicin ML (GarML) were synthesized. C and I N ) genes. Figure 2C , the cleaved intein sequence is underlined with a solid line, and the bacteriocin sequence is underlined with a dashed line. The cyclization of native Garvicin ML occurs after the leader sequence is cleaved and after the head-to-tail ligation between residues Leu1 and Ala60 [6] ( Figure 2A ), but intein chemistry generally requires that the first amino acid of the target peptide be either cysteine ​​or serine. Garvicin ML has no cysteine ​​in its mature sequence but has three serines (Ser19, Ser29, and Ser32). Therefore, the order of residues in Garvicin ML was switched, selecting Ser32 as the first residue in the linear conformation (Ser1 in the new conformation) and Phe31 as the last residue (Phe60 in the new conformation). In addition, a protein degradation tag (SsrA) was included in the C-terminus of the construct ( Figure 2C to overcome the potential toxicity of Npu intein after splicing ( Figure 2B-2D )

[16] . Based on the designed amino acid sequence, a synthetic gene with the codon usage of E. coli was generated and placed under the control of promoter T7 in a pUC expression standard expression vector (called pUC-Npu-GarML). In parallel, a synthetic gene without intein (pUC-GarML) and with intein in I C has N36D substitution and in I N An inactive variant of the Npu DnaE intein with a C1A substitution in pUC-Npu --GarML)

[17] ( Figure 2B ).

[0206] Cell-free production of GarML

[0207] Plasmids pUC-Npu-GarML, pUC-GarML, and pUC-Npu-GarML were used as templates for cell-free protein production of Npu-GarML, GarML, and Npu-GarML, respectively. We then tested the products of the reactions for activity against Lactococcus garneri strains. Neither GarML nor Npu-C1A-GarML was active against the indicator, indicating that linear GarML and GarML with I on both sides of GarML were active against Lactococcus garneri strains. C and I N In contrast, Npu-GarML showed activity against the indicator ( Figure 3A ), and the activity was higher when the product was left overnight at room temperature ( Figure 3B These results indicate that in vitro production of GarML fused to the Npu intein allows circularization of GarML ( Figure 2D ), since the linear form of GarML with or without the Npu intein did not show any activity.

[0208] In some embodiments, designing a nucleic acid encoding a bacteriocin that cyclizes via a split intein involves circularly arranging the amino acid sequence of a naturally mature form of a cyclic bacteriocin such that a serine or cysteine ​​in the native sequence is positioned as the first amino acid, fusing the N-terminal fragment of the split intein to the N-terminus of the circularly arranged bacteriocin sequence, and fusing the C-terminal fragment of the split intein to the C-terminus of the circularly arranged bacteriocin sequence. In some embodiments, a nucleic acid encoding a bacteriocin that is flanked by a split intein that cyclizes the bacteriocin at both the N-terminus and the C-terminus is expressed in vitro in a cell-free expression system to produce a gene product that exhibits the antimicrobial activity of the encoded bacteriocin, wherein the encoded bacteriocin is a naturally occurring cyclic bacteriocin.

[0209] Example 2

[0210] This non-limiting example shows the expression of a circular bacteriocin by genetically engineered bacteria with a vector encoding a bacteriocin flanked by split inteins, and analysis of the expressed bacteriocin to demonstrate head-to-tail cyclization by split inteins. Purification and mass spectrometry (MS) and multiple reaction monitoring (MRM) analysis of garvicin ML from the soluble fraction of recombinant Escherichia coli

[0211] To demonstrate and confirm the correct splicing and circularization of GarML, E. coli BL21 was used as a producer to amplify Npu-GarML production ( Figure 4A ). The soluble fraction of Escherichia coli BL21 pUC-Npu-GarML showed antimicrobial activity against Lactococcus garvei. The active peptide was further purified from the soluble fraction using hydrophobic interaction (HI) and reversed-phase fast high-performance liquid chromatography (FPLC). In two consecutive rounds of purification, the antimicrobial activity was associated with the peak eluted in 39% isopropanol in the FPLC chromatogram. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis revealed that the corresponding fraction had a mass of 6,004,2Da ( Figure 4B ). This correlates with the mass from the native cyclic garvicin ML [6]. This fraction was also subjected to trypsin digestion and analyzed by LC-MRM-MS analysis to analyze the fragment origin. Knowing the mass and amino acid sequence of garvicin ML, the precursor z and fragment m / z (MRM transitions) can be predicted. Each target peptide has a set of accompanying transitions that are then selectively detected in the second stage of MS. All peptides covering 100% of the complete GarML sequence were confirmed by MS / MS. One of the peptides detected and confirmed by MS / MS (TIVNAVSAGMDIATALSFSGAFTAAGGIMALIKK) contained residues S1 and F60 from Npu-GarML linked together, confirming the splicing and head-to-tail cyclization of GarML.

[0212] Example 3

[0213] This non-limiting example demonstrates the cell-free production of known and predicted cyclic bacteriocins using split inteins and demonstrates their antimicrobial activity.

[0214] Plasmid design and cell-free production of characterized and uncharacterized circular bacteriocins

[0215] Following the same protocol as for GarML, different plasmids containing genes encoding recombinant proteins containing Npu I fused to the mature amino acid sequences of different known and putative cyclic bacteriocins were synthesized. C and I N. All of their cyclization points were changed relative to the native peptide, so they all started with Ser at position 1 (Table 2.1). An Ssra tag was also added to the C-terminus. As for GarML, an Npu inactive form was constructed for all peptides. The synthesized plasmid was used as a template for cell-free protein production. The antimicrobial activity of the peptides obtained after cell-free production and overnight incubation at room temperature was tested against the indicator Lactococcus lactis IL1403. None of the proteins containing the inactive intein showed antimicrobial activity. Most of the fractions containing the natural Npu intein fused to the mature sequence of the characterized and hypothesized cyclic bacteriocin showed activity against Lactococcus lactis IL1403, thus showing that the SICLOPPS method can also be used for the cyclization of other cyclic bacteriocins (Table 3).

[0216] Table 3: Antimicrobial activity of different bacteriocins cyclized with inteins

[0217]

[0218]

[0219] In some embodiments, a bacteriocin known or predicted to be a circular bacteriocin is expressed in a cell-free system by designing a nucleic acid encoding the bacteriocin flanked by split inteins, wherein the native amino acid sequence of the bacteriocin is circularly arranged, and / or mutated to introduce a non-native serine such that the serine is at position 1 of the bacteriocin encoded by the nucleic acid.

[0220] Example 4

[0221] This non-limiting example demonstrates screening a library for bacteriocins having a desired activity.

[0222] A nucleic acid encoding the amino acid sequence of the circular bacteriocin enterocin NKR-5-3B is prepared. The amino acid sequence is modified relative to the native sequence of the circular bacteriocin so that serine or cysteine ​​is at the first position of the amino acid sequence, for example, by arranging the native sequence in a circular manner to place the native serine or cysteine ​​at the first position. The nucleic acid is amplified and mutations are introduced, for example, by random mutagenesis or selective point mutations, to produce a collection of variants of nucleic acids encoding circular bacteriocins. These variants are cloned into expression vectors so that each variant of the nucleic acid encoding the circular bacteriocin is flanked by a broken intein configured to cyclize the bacteriocin to produce a library of expression vectors having variant nucleic acids encoding the circular bacteriocins.

[0223] A cell-free expression system is used to express cyclic bacteriocins from a library of expression vectors, and the produced cyclic bacteriocins are tested for antimicrobial activity against one or more bacterial strains of interest to identify those that exhibit the desired activity. For example, in one embodiment, variant nucleic acids encoding enterobactin NKR-5-3B are isolated and sequenced to identify mutations responsible for conferring the desired antimicrobial activity to the cyclic bacteriocin, which variant nucleic acids retain antimicrobial activity against Lactococcus lactis but do not retain antimicrobial activity against Listeria innocua.

[0224] Example 5

[0225] This non-limiting example demonstrates the use of cyclic bacteriocins to control the growth of microbial organisms.

[0226] A polypeptide containing an amino acid sequence of a cyclic bacteriocin (e.g., enterobactin AS-48) flanked by a split intein is produced. The split intein is a conditionally active pH-sensitive split intein and is configured to cyclize the bacteriocin when the pH is below 6.0. The polypeptide is introduced into a culture medium at pH 7.0 where the target microbial organism is grown. The bacteriocin does not cyclize at pH 7.0 and does not exhibit antimicrobial activity. When contaminating microbial species Lactococcus lactis are detected in the culture medium, the pH of the culture medium is reduced to below 6.0, which activates the split intein and causes the bacteriocin to cyclize. Subsequently, the growth of contaminating Lactococcus lactis in the culture medium is inhibited.

[0227] Example 6

[0228] This non-limiting example demonstrates the use of cyclic bacteriocins to control the growth of microbial organisms.

[0229] Microbial cells are genetically engineered with an expression vector encoding a cyclic bacteriocin flanked by a broken intein (e.g., Leucocyclicin Q). The genetically engineered microbial cells are introduced into a culture medium in which the target microbial organism is grown. The microbial cells produce the bacteriocin in a cyclized form and secrete it into the culture medium. The growth of the contaminating microbial species, Lactococcus lactis, is inhibited by the cyclic bacteriocin.

[0230] In at least some embodiments described herein, one or more elements used in the embodiments can be used interchangeably in another embodiment, unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All of these modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0231] With respect to substantially any plural and / or singular terms used herein, those skilled in the art may translate from the plural to the singular and / or from the singular to the plural as appropriate to the context and / or application. For clarity, the different singular / plural permutations may be clearly listed herein.

[0232] Those skilled in the art will understand that, in general, the terms used herein, especially in the appended claims (e.g., the bodies of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be understood as "including but not limited to", the term "having" should be understood as "having at least", the term "includes" should be understood as "including but not limited to", etc.). Those skilled in the art will also understand that if a specific number of introduced claim recitations is intended, such intent is explicitly recited in the claim, and in the absence of such a recitation, such intent is not present. For example, to aid understanding, the appended claims may include the use of the introductory phrases "at least one" and "one or more / one or more" to introduce claim recitations. However, the use of such phrases should not be interpreted as implying that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning at least one" or "one or more"); this is also true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted as meaning at least the recited number (e.g., the basic recitation of "two recitations" without other modifiers means at least two recitations or two or more recitations). In addition, in the case where a phrase similar to "a" or "an" is used, the phrase "a" or "an" should be interpreted as meaning at least one" or "one or more"; In those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally, such construction is intended to mean that a person skilled in the art will understand the convention (e.g., "a system having at least one of A, B, and C" will include but is not limited to systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally, such construction is intended to mean that a person skilled in the art will understand the convention (e.g., "a system having at least one of A, B, or C" will include but is not limited to systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will also understand that almost any transitional word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one, either, or both of these terms.For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0233] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0234] As will be understood by those skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily considered to fully describe and enable the same range to be divided into at least two equal parts, three equal parts, four equal parts, five equal parts, ten equal parts, etc. As a non-limiting example, each range discussed herein can be easily divided into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all languages ​​such as "at most", "at least", "greater than", "less than", etc. include the narrated numbers, and refer to the ranges that can be subsequently divided into sub-ranges as discussed herein. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group with 1-3 products refers to a group with 1, 2 or 3 products. Similarly, a group with 1-5 products refers to a group with 1, 2, 3, 4 or 5 products, etc.

[0235] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the appended claims.

Claims

1. A fusion polypeptide comprising an amino acid sequence of a bacteriocin, wherein both the N-terminus and the C-terminus of the amino acid sequence of the bacteriocin are split inteins that cyclize the bacteriocin.

2. The fusion polypeptide according to claim 1, wherein the bacteriocin is a natural cyclic bacteriocin.

3. The fusion polypeptide according to claim 1 or 2, wherein the amino acid sequence of the bacteriocin is arranged in a circular shape compared to the native amino acid sequence of the bacteriocin.

4. The fusion polypeptide according to any one of claims 1 to 3, wherein the first residue of the amino acid sequence of the bacteriocin is a serine or a cysteine ​​present in the native amino acid sequence of the bacteriocin.

5. The fusion polypeptide according to any one of claims 1 to 3, wherein the first residue of the amino acid sequence of the bacteriocin is a non-natural serine or a non-natural cysteine.

6. The fusion polypeptide according to claim 5, wherein the non-natural serine or the non-natural cysteine ​​replaces a natural amino acid residue in the amino acid sequence of the bacteriocin.

7. The fusion polypeptide according to claim 5, wherein the length of the amino acid sequence of the bacteriocin is increased by one residue due to the non-natural serine or the non-natural cysteine ​​compared to the length of the native amino acid sequence of the bacteriocin.

8. The fusion polypeptide according to any one of claims 5 to 7, wherein the native amino acid sequence of the bacteriocin does not contain serine or cysteine.

9. The fusion polypeptide according to any one of the preceding claims, wherein the split intein is based on an intein from one of the following: Npu DnaE, ​​Sce VMA, Ssp DnaE.

10. The fusion polypeptide according to any one of the preceding claims, wherein the split intein is a conditional split intein. The fusion polypeptide of claim 10 , wherein the conditionally split intein is pH-sensitive or temperature-sensitive.

12. A fusion polypeptide according to any one of the preceding claims, wherein the split intein comprises a C-terminal intein fragment (I C ) At least 80% identical C The second amino acid sequence and the N-terminal intein fragment of the broken intein shown in Table B (I N ) At least 80% identical N The third amino acid sequence of 13. The fusion polypeptide according to any one of the preceding claims, wherein the bacteriocin is selected from any one of the bacteriocins listed in Table A.

14. The fusion polypeptide according to 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 according to 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. A fusion polypeptide according to any one of the preceding claims, wherein one or more amino acids in the amino acid sequence of the polypeptide are unnatural amino acids.

18. The fusion polypeptide according to any one of the preceding claims, further comprising a degradation tag. The fusion polypeptide according to 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 fused to the N-terminus of the amino acid sequence of the bacteriocin ("I C ”) and an N-terminal intein fragment fused to the C-terminus of the amino acid sequence of the bacteriocin (“I N ”), wherein the polypeptide further comprises the I N The C-terminal degradation tag.

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 according to 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 immunomodulator that confers resistance to the bacteriocin.

31. The microbial cell of claim 30, wherein expression of the immunomodulator from said second nucleic acid is regulatable.

32. The microbial cell of any one of claims 28-31, wherein the microbial cell is a bacterium, a fungus or an algae.

33. A composition comprising the fusion polypeptide of any one of claims 1-22.

34. A composition comprising a cyclic bacteriocin and a split intein.

35. A method for preparing a cyclic bacteriocin, comprising contacting the nucleic acid of any one of claims 23 to 26 or the genetic vector of claim 27 with an in vitro expression system under conditions sufficient to produce the cyclic bacteriocin.

36. A method for preparing a cyclic bacteriocin, comprising culturing the microbial cell of any one of claims 28 to 31 under conditions sufficient to produce the cyclic bacteriocin.

37. The method of claim 35 or 36, further comprising purifying the cyclic 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 cyclizes the bacteriocin under permissive conditions but not under non-permissive conditions, and wherein the method further comprises exposing the fusion polypeptide to the permissive conditions after exposure to the non-permissive conditions to induce cyclization of the bacteriocin.

40. The method of any one of claims 35-39, further comprising changing pH or temperature to induce cyclization of the bacteriocin, wherein the split intein is pH-sensitive or temperature-sensitive, respectively.

41. The method of any one of claims 35-40, further comprising allowing the split intein to degrade after producing the cyclic bacteriocin.

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 naturally occurring circular bacteriocin and the sequence variants comprise a first variant that eliminates the naturally occurring circularization of the parent bacteriocin.

46. ​​A method of screening comprising: Providing a library according to any one of claims 42-45; expressing a plurality of polypeptides encoded by one or more genetic vectors in the library; generating multiple cyclic bacteriocins from multiple expressed polypeptides; and The various cyclic bacteriocins were assayed for their desired activities.

47. The method of claim 46, wherein the desired activity comprises antimicrobial activity.

48. A method for controlling the growth of a microorganism, comprising contacting a composition comprising a microorganism and / or aiding in supporting the growth of a microorganism with a microbial cell of any one of claims 28 to 32 under conditions sufficient to produce a cyclic bacteriocin, thereby controlling the growth of the microorganism.

49. A method of controlling the growth of a microorganism comprising contacting a composition comprising a microorganism and / or aiding in supporting the growth of a microorganism with a cyclic bacteriocin prepared by the method of any one of claims 35 to 38, thereby controlling the growth of the microorganism.

50. A method for controlling the growth of a microorganism, comprising contacting a composition comprising a microorganism and / or helping to support the growth of a microorganism with the fusion polypeptide of any one of claims 1-22, thereby controlling the growth of the microorganism.

51. The method of any one of claims 48-50, wherein the microorganism is a bacterium.

52. The method of any one of claims 48-51, wherein the composition is a culture medium, a feedstock, or a microbial composition.

53. The method of any one of claims 48-52, wherein the split intein is a conditional split intein that cyclizes the bacteriocin under permissive conditions but not under non-permissive conditions, and wherein the method further comprises providing the permissive conditions to the composition, thereby inducing cyclization of the bacteriocin.

54. The method of any one of claims 48-53, wherein the method comprises changing the pH or temperature of the composition to induce cyclization of the bacteriocin, wherein the split intein is pH-sensitive or temperature-sensitive, respectively.

55. A method for 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 contain a serine or a cysteine ​​at the N-terminus; The second amino acid sequence having a serine or cysteine ​​at its N-terminus is provided by at least one of the following methods: a circular arrangement of the native amino acid sequence; or Introducing serine or cysteine ​​into the native amino acid sequence; providing a nucleotide sequence encoding a polypeptide comprising the second amino acid, wherein the second amino acid sequence is flanked on both the N-terminus and the C-terminus by a split intein configured to cyclize the bacteriocin; as well as The polypeptide encoded by the nucleotide sequence is expressed.

56. The method of claim 55, wherein the candidate bacteriocin is predicted to be a cyclic bacteriocin based on the genomic sequence of the microorganism encoding the candidate bacteriocin in the genome of the microorganism.

57. The method of claim 55 or 56, comprising: Identification of multiple natural amino acid sequences of a variety of different bacteriocin candidate; For each of the plurality of natural amino acid sequences: providing the second amino acid sequence; and providing a nucleotide sequence encoding a polypeptide comprising the second amino acid sequence, wherein the second amino acid sequence is flanked on both the N-terminus and the C-terminus by split inteins configured to cyclize the bacteriocin, Thereby a library of nucleic acids representing each of the plurality of natural amino acid sequences is generated.

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 a 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 by 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 of the preceding claims, wherein the polypeptide comprises an affinity tag, optionally wherein the affinity tag is chitin binding protein (CBP).

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