WO2025199288A1 - Programmable payload overproduction and delivery - Google Patents

Abstract

Prophage-based platforms for producing and releasing cargo of interest from microbial cell factories are provided. The prophages are engineered to exhibit a high rate of spontaneous induction and to eliminate non-essential genes without disrupting phage-mediated lysis and lysis timing. Methods of using the platform for sustained delivery and release from long term cultures in an in vitro setting (e.g., industrial protein production) or an in vivo setting (e.g., therapeutic cargo delivery) are also provided.

Description

TITLE: PROGRAMMABLE PAYLOAD OVERPRODUCTION AND DELIVERY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional application U.S. Serial No. 63/567,501, filed March 20, 2024, which is hereby incorporated herein by reference in its entirety.

SEQUENCE LISTING XML

[0002] The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on March 14, 2025, is named P14679WOOO.xml and is 241,431 bytes in size.

TECHNICAL FIELD

[0003] The present disclosure relates to compositions and methods for the production and delivery of polypeptides, complexes of polypeptides, and bacteriophage particles.

BACKGROUND

[0004] Current protein delivery practices mostly rely on mobile plasmids to express the protein, and the bacterial secretion to release the protein, where hyper-production and efficient release of protein are limited by the necessity of finding stable plasmid expression vectors, optimizing the bacterial secretion system, the high metabolic burden posed by protein secretion on the host cells.

SUMMARY

[0005] Prophage-based platforms for producing and releasing desired proteins or bacteriophage particles from microbial cell factories are provided. Such delivery systems are designed by engineering the payloads into the activatable prophages, which are dormant phages residing in the bacterial chromosome. The delivery approach is based on expression of a desired payload from a refactored prophage element that has been engineered to exhibit a high rate of spontaneous induction. Such designs capitalize on the ability of phages to express large amounts of protein (>10⁵ copies per cell) during their lytic cycles, coupled with the intrinsic ability of phages to lyse their host cells, to produce and release large amounts of proteins from rapidly growing microbial cell factories. The overproduction and release of the payloads can either be constant during bacterial cell growth due to engineering of the spontaneous prophage induction rate, or programmable to be triggered by specific signals regulating the prophage repressors in a given bacterial cell factory. The payload delivery systems of the disclosure address multiple bottlenecks of current protein overexpression and delivery practices. Payload expression by prophage induction allows for release of payloads into the medium via cell lysis, which can release fully folded cytoplasmic proteins, proteins first directed to the periplasm for optimum folding, protein complexes, non-proteinaceous payloads derived from inserted gene elements, or functional native or heterologous bacteriophage particles. The summation of these prophage modifications and resulting technology is referred to herein as PIPER: Prophage Induction for Payload Over-Expression and Release.

[0006] Bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a payload of interest are provided. In certain embodiments, the payload comprises a polypeptide, a complex of polypeptides, or a native or heterologous bacteriophage. Cell cultures comprising one or more bacterial cells of the disclosure are also provided.

[0007] Methods for producing a payload of interest are provided. In certain embodiments, the methods comprise culturing one or more bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a payload of interest. In certain embodiments, the methods further comprise recovering the payload of interest.

[0008] Methods for producing a payload of interest in a subject are also provided. In certain embodiments, the methods comprise administering to the subject one or more bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a payload of interest.

[0009] Pharmaceutical compositions comprising one or more bacterial cells of the disclosure and a pharmaceutically acceptable carrier are provided.

[0010] Methods of treating or preventing a disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising one or more bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a therapeutic polypeptide, complexes of polypeptides, and native or heterologous bacteriophage particles are also provided.

[0011] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent based on the detailed description, which shows and describes illustrative embodiments of the disclosure. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE FIGURES

[0012] The following drawings form part of the specification and are included to further demonstrate certain embodiments. In some instances, embodiments can be best understood by referring to the accompanying figures in combination with the detailed description presented herein. The description and accompanying figures may highlight a certain specific example, or a certain embodiment. However, one skilled in the art will understand that portions of the example or embodiment may be used in combination with other examples or embodiments.

[0013] FIG. 1A-C is schematic illustrating the prophage-induction based payload overexpression and release. FIG. 1A shows a prophage element incorporated into the bacterial chromosome is dormant in normally growing cells. The prophage is engineered to carry the protein payload whose expression is driven by a programmable induction switch. FIG. IB shows the prophage element excises when induced and begins expression of its payload protein. Protein expression is enhanced by the replication of the excised element and large amounts of expressed protein accumulate in the cell. FIG. 1C shows the cell lyses explosively at a time determined by the phage element, releasing the accumulated protein into the environment.

[0014] FIG. 2A-B is an illustration of example applications of the proposed technology in human medicine. Colonizing bacteria, such as engineered probiotic strains taken orally, release protein cargo continuously into human intestine to treat disorders such as inflammatory diseases, metabolic enzyme deficiency, or to deliver vaccine antigens and antimicrobials. FIG. 2A shows possible types of protein payloads that could be delivered by colonizing bacteria using the proposed technology. FIG. 2B shows constitutive in situ delivery of therapeutic proteins in intestine through spontaneous prophage induction by colonizing bacteria.

[0015] FIG. 3 shows release of a large proteinaceous enzyme via a prophage-based platform in E. coli. Lambda prophage was engineered to harbor a lacZ gene encoding a large enzyme P- Galactosidase, a glycoside hydrolase enzyme also known as lactase or P-gal. Specifically, the lacZ gene was fused to the endolysin gene in the Lambda prophage genome (cI857 R::lacZ). The induction of the prophage was achieved thermally by shifting the temperature from 30 °C (left plate) to 37 °C (right plate). The production and release of the enzyme surrounding the producing bacterial colonies is visualized as diffused blue color, which is due to enzymatic degradation of X-Gal (5-bromo-4-chloro-3-indolyl-P-d-galactoside) incorporated in the agar plates.

[0016] FIG. 4A-B shows the achievement of high level of spontaneous phage induction by modifying the phage lysogeny control system. Lambda prophage was engineered to harbor a lacZ gene encoding a large enzyme P-Galactosidase, a glycoside hydrolase enzyme also known as lactase or P-gal. Specifically, the lacZ gene was fused to the endolysin gene in the Lambda prophage genome (cI857 R::lacZ). Mutation was introduced into the lambda repressor protein CI (PIPER prophage) and the rates of prophage induction were compared to the parental prophage (WT prophage). FIG. 4A shows normalized amount of E. coli cells harboring parental prophage or prophage mutant (PIPER Prophage) were incubated at 30°C in wells of a 96-well plate. The lambda prophage carrying the mutation (PIPER Prophage) was spontaneously induced at a significantly higher rate (Row C) than the lambda parental prophage (WT prophage) without the mutation (Row B). The increased spontaneous induction rate results in an increased release of the P-galactosidase enzyme carried on the prophage from the E. coli cells, as evidenced by the development of a deeper blue color due to the degradation of the substrate X-Gal (5-bromo-4- chloro-3-indolyl-P-d-galactoside). E. coli cells carrying no prophage was included as a control (Row A). FIG. 4B shows E. coli cells harboring parental prophage or prophage mutant (PIPER Prophage) were incubated at 30°C. The PIPER prophage displays increased payload expression (blue signal), while the parental prophage (WT prophage) with the same payload remains colorless.

[0017] FIG. 5A-C shows quantification of the protein payload released by prophage compared to the plasmid-based expression system and the native bacterial expression. Lambda prophage was engineered to harbor a lacZ gene encoding a large enzyme P-Galactosidase, a glycoside hydrolase enzyme also known as lactase or P-gal. Specifically, the lacZ gene was fused to the endolysin gene in the Lambda prophage genome (cI857 R::lacZ). Mutation was introduced into the lambda repressor protein CI (PIPER prophage). FIG. 5A shows release of protein payload by diffusion of blue color into plate after prolonged incubation on an agar plate, while payload expressed from a traditional plasmid remains internal to the bacterial colony. When quantified, PIPER prophage releases enzyme payload at a much greater level than both parental WT prophage (FIG. 5B) and a native bacterial expression (FIG. 5C).

[0018] FIG. 6A-C is an illustration of the development of miniPIPER from a hypothetical prophage genome and portability as a mobile genetic element. FIG. 6A shows the fully intact PIPER prophage containing all genes necessary to release proteinaceous cargo and generate infectious phage particles. FIG. 6B shows development of miniPIPER by removal of genes nonessential to prophage induction for payload over-expression and release. Proteins such as phage structural proteins and non-essential host takeover proteins will be removed; however, proteins related to phage DNA replication, transcription, translation, regulation, excision, integration, and lysis will be retained. FIG. 6C demonstrates two modes of PIPER transfer as genetic element when desired: full size PIPER can generate infectious heterologous phage particles capable of infecting hosts different from the PIPER-harboring host. Reduction of PIPER size to miniPIPER will increase its portability as a mobile genetic element where in which it can be placed on a plasmid or transposon and transferred to other bacterial genera where specific host traits are desired.

[0019] FIG. 7 shows growth of the E. coli hosts harboring either the parental lambda prophage or PIPER prophage as measured via direct plating and quantification of CFU/ml. Mutations to the CI repressor in the PIPER leads to an increased rate of prophage induction compared to the parental prophage. This increased rate of spontaneous PIPER prophage induction leads to a mild reduction population growth, but it still maintains a positive growth trajectory and a stable bacterial population. Each error bar is constructed using one standard error from the mean. [0020] FIG. 8A-B is an illustration of the strategies for constructing “miniPIPER”. FIG. 8A shows a full-length genome map of phage lambda (48 kb). FIG. 8B shows a theoretical reduction of the parental prophage to the smallest minimum required components for the miniPIPER genome (lOkb). Structural genes are dispensable to eliminate phage particles as well auxiliary genes such as abortive infection systems, or non-essential genes of unknown function. Required genes loci include DNA replication, integration/excision, lysis/lysogeny switch, and host lysis.

[0021] FIG. 9A-B shows a proof-of-concept miniPIPER. Through sequential deletion of major structural genes, a miniPIPER was generated from the parental lambda prophage (c/857). FIG. 9A shows a genome map of WT lambda (top) (NCBI accession: NC_001416.1; SEQ ID NO: 1), and its alignment to miniPIPER (bottom). Deleted structural locus indicated by redline, aligning parts of the genome connected by grey shaded region. Dispensable components to the PIPER system and beneficial components to the PIPER system are indicated. FIG. 9B shows lysis curves of the parental lambda prophage and miniPIPER prophage monitored via OD. Arrows indicate initiation of culture lysis following induction for parental prophage (~30min) and beginning of culture lysis for miniPIPER (~45min). Despite large deletions, lysis timing and thermal induction were largely unaffected, indicating the miniPIPER systems were still suitable for cargo release.

[0022] FIG. 10A-B shows Lactobacillus phage A2 contains a similar master lysis/lysogeny to that of lambda. FIG. 10A shows a comparison of genetic switches in lambda and A2. Box indicates “master” switch consisting of cl and cro. This conservation of lysis/lysogeny switch synteny implies similar modifications to the Ci-repressor could convert phage A2 to the PIPER system. FIG. 10B shows an alignment of lambda and A2 CI proteins (SEQ ID NO: 151 and SEQ ID NO: 152). Boxes indicate acidic residues that interact with RNA polymerase in phage lambda, and the equivalent residues in phage A2. The conservation of acidic residues critical to maintenance of lysogeny imply that the same or similar mutations utilized to destabilize the lysogen of lambda could be employed to convert phage A2 to the PIPER delivery system.

[0023] FIG. 11A-D shows lysis curves of position 49, 50, 51 and 52 mutant series. Mutations to the lambda holin are labeled on the graph. FIG 11A shows position 49 series; FIG 11B shows position 50 series; FIG 11C shows position 51 series; and FIG 11D shows position 52 series. Mutations to the TMD2 of the lambda S holin modulate the time to lysis following an induction amount. In some cases, this is a shortened lysis time, while other mutations confer a delayed lysis phenotype. These mutations provide the PIPER system with a large dynamic range of cargo production per lysis event and the dosage levels which can be achieved for therapeutic or industrial purposes.

[0024] FIG. 12 shows accumulation of phage particles (cargo) over time in a lambda lysogen that does not undergo lysis due to a mutation in the lambda holin gene. E. coli MC4100 (X CI857 Sam7) was thermally induced (42 °C for 15 min) and the level of phage particles inside bacterial cells was quantified every 15 min after induction. Continued accumulation of phage particles overtime well beyond that of WT lambda (to levels >600 virions per cells) demonstrates that lysis timing can utilized to modulate level of cargo made per PIPER induction event.

DETAILED DESCRIPTION

[0025] Bacteria have been used to overproduce medically or industrially important proteins for decades. While bacteria are versatile platforms for protein production, the release of the accumulated proteins into the medium has relied on either mechanical disruption of the cell wall or bacterial secretion. Both of these routes have limitations: mechanical disruption is only practicable in controlled fermentations, and secretion is dependent on protein compatibility with a bacterial secretion system, which limits the type and size of proteins that can be secreted. Exploiting bacterial secretory pathways requires additional protein engineering, is not compatible with many protein types (e.g., folded or multimeric proteins) and places a significant burden on the cell. Engineering bacteria to secrete recombinant protein is not trivial and it involves screening the mutagenized signal peptide libraries, native and/or heterologous signal peptides. To ease the cellular burden on the production host cells, some experimental approaches have been proposed including developing orthogonal ribosomes and feedback controllers to seek control of cellular resource allocation and lower burden. However, controlling cellular response to exogenous expression is still a big challenge. In addition, the expression level of the desired proteins often relies on the stability of the expression vector maintained in the host, which is not always guaranteed over long periods of time.

[0026] The engineered prophage-induction strategy of the disclosure overcomes the common bottleneck of the current practices by eliminating the necessity of finding stable plasmid vectors for expressing the gene in situ, and offers efficient release of proteins independent of the bacterial secretion system.

[0027] So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the disclosure pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosure without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present disclosure, the following terminology will be used in accordance with the definitions set out below.

[0028] It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the content clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicate otherwise. The word “or” means any one member of a particular list and also includes any combination of members of that list. Further, all units, prefixes, and symbols may be denoted in its SI accepted form. [0029] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various embodiments of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, P , and 4³/4. This applies regardless of the breadth of the range.

[0030] The term “gene” is used broadly to refer to any segment of nucleic acid molecule that encodes a protein or that can be transcribed into a functional RNA. Genes may include sequences that are transcribed but are not part of a final, mature, and/or functional RNA transcript, and genes that encode proteins may further comprise sequences that are transcribed but not translated, for example, 5' untranslated regions, 3' untranslated regions, introns, etc. Further, genes may optionally further comprise regulatory sequences required for their expression, and such sequences may be, for example, sequences that are not transcribed or translated. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0031] The term “heterologous” when used in reference to a polynucleotide, a gene, or a nucleic acid molecule refers to a polynucleotide, gene, or a nucleic acid molecule that is not derived from the host species. For example, “heterologous gene” or “heterologous nucleic acid sequence” as used herein, refers to a gene or nucleic acid sequence from a different species than the species of the host organism it is introduced into. When referring to a gene regulatory sequence or to an auxiliary nucleic acid sequence used for manipulating expression of a gene sequence (e.g. a 5' untranslated region, 3' untranslated region, poly A addition sequence, etc.) or to a nucleic acid sequence encoding a protein domain or protein localization sequence, “heterologous” means that the regulatory or auxiliary sequence or sequence encoding a protein domain or localization sequence is from a different source than the gene with which the regulatory or auxiliary nucleic acid sequence or nucleic acid sequence encoding a protein domain or localization sequence is juxtaposed in a genome. Thus, a promoter operably linked to a gene to which it is not operably linked to in its natural state (for example, in the genome of a non-genetically engineered organism) is referred to herein as a “heterologous promoter,” even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked. [0032] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. Nucleic acid molecules can have any three-dimensional structure. A nucleic acid molecule can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). Non-limiting examples of nucleic acid molecules include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, tracrRNAs, crRNAs, guide RNAs, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes and nucleic acid primers. A nucleic acid molecule may contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a polynucleotide molecule.

[0033] As used herein, the term “percent sequence identity” or “% sequence identity” refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference (“query”) sequence (or its complementary strand) as compared to a test (“subject”) sequence (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the Sequence Analysis software package of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.), MEGAlign (DNAStar Inc., Madison, Wis.), and MUSCLE (Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput” Nucleic Acids Research 32(5): 1792-7 (2004)) for instance with default parameters. The BLAST program set to the default parameters, available from the National Center for Biotechnology Information (NCBI), can also be used to obtain an optimal alignment of protein or nucleic acid sequences and to calculate the percentage of sequence identity. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in the portion of the reference sequence segment being aligned, that is, the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more sequences may be to a full-length sequence or a portion thereof, or to a longer sequence.

[0034] The terms “polypeptide”, “peptide”, and “protein” are used interchangeably and refer to a polypeptide chain which may or may not be modified by addition of non-amino acid groups. The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms “protein”, “peptide” and “polypeptide” as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the disclosure as described herein.

[0035] As used herein, the terms “subject”, “individual” or “patient” are interchangeable and refer to an animal, preferably to a mammal, even more preferably to a human, including adult, child, newborns and human at the prenatal stage. The term “subject” can also refer to nonhuman animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep, donkeys, rabbits, ferrets, rats, mice, guinea pigs and non-human primates, among others, that are in need of treatment.

Bacteriophages

[0036] Bacteriophages (or phages) are viruses that infect bacteria. Following phage infection (lytic cycle), the phage takes over host genetic replication machinery and rapidly synthesizes new phage particles, which are then released from the host cell by destroying the host cell envelope, leading to the bulk release of the new phage particles and other intracellular components. Some phages, designated as temperate or lysogenic, can become stable components of the host genome by entering a stable and dormant prophage state. Prophages may remain dormant within the host (which is now called a lysogen) for many generations. When reactivated in a process called induction, the phage resumes its lytic pathway and produces progeny phage that are released by host lysis.

[0037] Every prophage has a certain frequency of spontaneous induction. Naturally occurring bacteria already exploit this fundamental aspect of phage biology. For example, strains of shigatoxigenic E. coli express the Shiga toxin from a prophage that spontaneously induces at a rate >10⁵-fold higher than the canonical temperate phage Lambda. Toxin expression can be in excess of 50,000 molecules per cell, and release of the heteromultimeric protein toxin complex is accomplished by cell lysis, in which disruption of the cell wall results in explosive release of the entire cellular contents into the surrounding environment. This “altruistic sacrifice” of a proportion of the bacterial population in each generation results in constant protein production and release in the bacterial culture. The use of phage-mediated lysis as a cargo release mechanism means that proteins or protein complexes of virtually any size may be released into the medium.

[0038] In certain embodiments, the prophage is from the Class Caudoviricetes . In certain embodiments, the prophage is from the Family Inoviridae. Examples of prophages for use in the present disclosure include, without limitation, Lambda, P2, 186, P22, Pasto, P335, phiETA, 77, 80, TP901-1, PBSX, and SP-beta. In certain embodiments, the prophage is Lambda prophage. [0039] TABLE 1 shows a summary of the genes and proteins of Lambda prophage.

[0040] While Table 1 above describes genes and proteins of Lambda prophage specifically, the present disclosure is applicable to a range of prophages. For example, sequences that are homologous, z.e., that share significant sequence identity or similarity, to those provided herein are also part of the present disclosure. In certain embodiments, homologous sequences can be derived from any prophage. In certain embodiments, the prophage of the present disclosure comprises at least one nucleic acid molecule (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, or more nucleic acid molecules) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or a combination thereof. In certain embodiments, the prophage of the present disclosure comprises at least one nucleic acid molecule (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, or more nucleic acid molecules) encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,

SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111,

SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116,

SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121,

SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126,

SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131,

SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136,

SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141,

SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146,

SEQ ID NO: 147, or a combination thereof.

[0041] The prophage of the present disclosure is modified to comprise a heterologous nucleic acid encoding a payload including, but not limited to, polypeptides, complexes of polypeptides, and native or heterologous bacteriophage particles of interest. The polypeptide of interest can be generally any polypeptide and can be, for example, proteins and peptides suitable for pharmaceutical, nutraceutical, or industrial compositions. Non-limiting examples of suitable polypeptides include therapeutic polypeptides, prophylactic polypeptides, diagnostic polypeptides, nutraceutical polypeptides, industrial enzymes, and reporter polypeptides.

[0042] The term “therapeutic polypeptide,” as used herein denotes a bioactive polypeptide that has therapeutic utility. The term encompasses any polypeptide that can be administered to a patient to produce a beneficial therapeutic or diagnostic effect though binding to and/or altering the function of a biological target molecule in the patient. The target molecule can be an endogenous target molecule encoded by the patient's genome (e.g., an enzyme, receptor, growth factor, peptide hormone, cytokine encoded by the patient's genome) or an exogenous target molecule encoded by the genome of a pathogen (e.g., an enzyme encoded by the genome of a virus, bacterium, fungus, nematode or other pathogen). Illustrative categories of therapeutic peptides suitable for practicing the compositions and methods of the present disclosure are hormones, monoclonal antibodies, vaccines, enzymes, cytokines, toxins, and the like. The term therapeutic polypeptide includes functional fragments of therapeutic polypeptides.

[0043] The term “nutraceutical polypeptide” as used herein refers to any polypeptide which may prevent, ameliorate or otherwise confer benefits against an undesirable condition, and used for its associated health benefits, to maintain the healthy condition of the consumer. The term “nutraceutical” as used herein denotes a usefulness in both the nutritional and pharmaceutical field of application. Thus, the nutraceutical polypeptides and compositions of the present disclosure can find use as supplement to food and beverages, and as pharmaceutical formulations not associated with food, suitable for consumption by an individual and usually sold in medicinal forms which may be solid formulations such as caplets, tablet, capsules, soft gel capsules, gel caps and the like, or liquid formulations, such as solutions or suspensions. [0044] A “reporter polypeptide”, as used herein, is a polypeptide that is detectable or has an activity that produces a detectable product. A reporter polypeptide can include a visual marker or enzyme that produces a detectable signal. Non-limiting examples of reporter polypeptides includes cat, lacZ, uidA, xylE, an alkaline phosphatase gene, an a-amylase gene, an a- galactosidase gene, a P -glucuronidase gene, a P-lactamase gene, a horseradish peroxidase gene, a luciferin/luciferase gene, an R-locus gene, a tyrosinase gene, or a gene encoding a fluorescent protein, including but not limited to a blue, cyan, green, red, or yellow fluorescent protein, a photoconvertible, photoswitchable, or optical highlighter fluorescent protein, or any of variant thereof, including, without limitation, codon-optimized, rapidly folding, monomeric, increased stability, and enhanced fluorescence variants.

[0045] Polypeptides of interest can be from any source or origin and can include a polypeptide found in prokaryotes, viruses, and eukaryotes, including fungi, plants, yeasts, insects, and animals, including mammals (e.g., humans). Polypeptides of interest also include synthetic polypeptides and de novo Al designed polypeptides. Polypeptides of interest include, but are not limited to any polypeptide sequences, known or hypothetical or unknown, which can be identified using common sequence repositories. Examples of such sequence repositories, include, but are not limited to GenBank EMBL, DDBJ and the NCBI. Other repositories can easily be identified by searching on the internet.

[0046] Exemplary polypeptides of interest include but are not limited to, cytokines, inflammatory molecules, growth factors, hormones, their receptors, receptor agonists/antagonists, and oncogene products or portions thereof. Examples of cytokines, inflammatory molecules, growth factors, their receptors, receptor agonists/antagonists, and oncogene products include, but are not limited to e.g., alpha- 1 antitrypsin, Angiostatin, Antihemolytic factor, amylases, antibodies (including an antibody or a functional fragment or derivative thereof selected from: Fab, Fab', F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody), angiogenic molecules, angiostatic molecules, Apolipopolypeptide, Apopolypeptide, Asparaginase, Adenosine deaminase, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, Angiotensin family members, Bone Morphogenic Polypeptide (BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP- 15, etc.); C — X — C chemokines (e.g., T39765, NAP -2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractant polypeptide- 1, Monocyte chemoattractant polypeptide-2, Monocyte chemoattractant polypeptide-3, Monocyte inflammatory polypeptide- 1 alpha, Monocyte inflammatory polypeptide-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262), CD40 ligand, C-kit Ligand, Ciliary Neurotrophic Factor, Collagen, Colony stimulating factor (CSF), Complement factor 5a, Complement inhibitor, Complement receptor 1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78, GRO alpha/MGSA, GRO beta, GRO gamma, MIP-1 alpha, MIP-1 delta, MCP-1), deoxyribonucleic acids, Epidermal Growth Factor (EGF), Erythropoietin (“EPO”, representing a preferred target for modification by the incorporation of one or more non-natural amino acid), Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF), Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factors, Hedgehog polypeptides (e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte Growth Factor (HGF), Hepatitis viruses, Hirudin, Human serum albumin, Hyalurin-CD44, Insulin, Insulin-like Growth Factor (IGF-I, IGF-II), interferons (e.g., interferon-alpha, interferon-beta, interferon-gamma, interferon-epsilon, interferon-zeta, interferon-eta, interferon-kappa, interferon-lambda, interferon-T, interferon-zeta, interferon- omega), glucagon-like peptide (GLP-1), GLP-2, GLP receptors, glucagon, other agonists of the GLP-1R, natriuretic peptides (ANP, BNP, and CNP), Fuzeon and other inhibitors of HIV fusion, Hurudin and related anticoagulant peptides, Prokineticins and related agonists including analogs of black mamba snake venom, TRAIL, RANK ligand and its antagonists, calcitonin, amylin and other glucoregulatory peptide hormones, and Fc fragments, exendins (including exendin-4), exendin receptors, interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, etc.), I-CAM-l/LFA-1, Keratinocyte Growth Factor (KGF), Lactoferrin, leukemia inhibitory factor, lipases, Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M, Osteogenic polypeptide, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones (e.g., Human Growth Hormone), Oncogene products (Mos, Rel, Ras, Raf, Met, etc.), Pleiotropin, Polypeptide A, Polypeptide G, proteases, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, ribonucleic acids, SCF/c-kit, Signal transcriptional activators and suppressors (p53, Tat, Fos, Myc, Jun, Myb, etc.), Soluble complement receptor 1, Soluble LCAM 1, Soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), soluble adhesion molecules, Soluble TNF receptor, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigens, i.e., Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), Steroid hormone receptors (such as those for estrogen, progesterone, testosterone, aldosterone, LDL receptor ligand and corticosterone), Superoxide dismutase (SOD), Toll-like receptors (such as Flagellin), Toxic shock syndrome toxin (TSST-1), Thymosin a 1, Tissue plasminogen activator, transforming growth factor (TGF-alpha, TGF-beta), Tumor necrosis factor beta (TNF beta), Tumor necrosis factor receptor (TNFR), Tumor necrosis factor-alpha (TNF alpha), transcriptional modulators (for example, genes and transcriptional modular polypeptides that regulate cell growth, differentiation and/or cell regulation), Vascular Endothelial Growth Factor (VEGF), virus-like particle, VLA-4/VCAM-1, Urokinase, signal transduction molecules, estrogen, progesterone, testosterone, aldosterone, LDL, corticosterone.

[0047] Additional polypeptides of interest include but are not limited to enzymes (e.g., industrial enzymes) or portions thereof. Examples of enzymes include, but are not limited to amidases, amylases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, haloperoxidases, monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile hydratases, nitrilases, proteases, phosphatases, subtilisins, transaminase, and nucleases.

[0048] Other polypeptides of interest include, but are not limited to, agriculturally related polypeptides such as insect resistance polypeptides (e.g., Cry polypeptides), starch and lipid production enzymes, plant and insect toxins, toxin-resistance polypeptides, Mycotoxin detoxification polypeptides, plant growth enzymes (e.g., Ribulose 1,5 -Bisphosphate Carboxylase/Oxygenase), lipoxygenase, and Phosphoenolpyruvate carboxylase.

[0049] Polypeptides of interest include, but are not limited to, antibodies, immunoglobulin domains of antibodies and their fragments. Examples of antibodies include, but are not limited to antibodies, antibody fragments, antibody derivatives, Fab fragments, Fab' fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-chain Fv fragments (scFv), diabodies, tribodies, tetrabodies, dimers, trimers, nanobodies, and minibodies.

[0050] Other polypeptides of interest include, but are not limited to, antibacterial or antifungal polypeptides such as low or high molecular weight bacteriocins, tailocins, ribosomal and non- ribosomal peptides, quorum sensing inhibitors, and lactonases.

[0051] In addition to polypeptides, the payload of interest can also include polypeptide complexes, non-proteinaceous molecules derived from polypeptide complexes, including but not limited to secondary metabolites produced from enzymatic pathways. Furthermore, the payload of interest can also include functional native or heterologous bacteriophage particles that are derived from the original prophage and are capable of infecting bacterial species/strains that are different from the original prophage host. For example, prophage residing in Bacillus can be modified to be released either as the native phage against related Bacillus strains or the payload can be a heterologous phage capable of targeting species of Staphylococcus or Streptococcus. [0052] In certain embodiments, the heterologous nucleic acid molecule encoding the payload is up to about 500 kilobases (kb) in length. In certain embodiments, the prophage of the disclosure comprises a payload nucleic acid molecule of up to about 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13kb, 14kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 25kb, 50 kb, 75 kb, lOOkb, 200 kb, 300 kb, 400 kb, or 500 kb in length. Payload nucleic acid molecules can range in length from any one of about 20 nucleotides (nt), 50 nt, 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, or 900 nt to any one of about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 25kb, 50 kb, 75 kb, lOOkb, 200 kb, 300 kb, 400 kb, or 500 kb in length in length.

[0053] A prophage of the present disclosure can further include one or more additional modifications to maximize and/or fine-tune the payload expression and release. Modifications can occur in any useful portion of the prophage, including modifications to an integrase gene, a repressor gene, an anti-repressor gene, an operator, a repressor-binding site, a virulence gene, a resistance gene, a toxin gene, a lysis gene, a host receptor recognition gene, a structural phage protein gene, a DNA replication gene, a nuclease gene, a nucleotide synthesis gene, a tRNA gene, a chaperone gene, or a DNA packaging gene. Such modifications can include deletions (e.g., deletion of a gene or a portion thereof), mutations (e.g., one or more mutations that provide changes in expression of the gene, that provides structural mutants having changed activity, or that provide changes in binding, transcription, or translation).

[0054] Exemplary integrase genes to be modified can include genes encoding for, e.g., a tyrosine integrase (e.g., X integrase, HP1 integrase, or Cre phage recombinase), a tyrosine recombinase (e.g., FLP yeast invertase or XerC bacterial recombinase), a serine integrase (e.g., R4 phage integrase, a TP901 integrase, or cpC31 phage integrase), a serine recombinase (including integrases and transposases, e.g., Gin invertase or y5 resolvase), an XerD recombinase, or a transposase.

[0055] Exemplary repressor genes to be modified can include genes encoding for a CI protein, a Cl-like protein, a MarR-like protein, or a Cro protein. Exemplary operators to be modified including a site on a nucleic acid configured to bind a repressor protein (e.g., any repressor protein described herein). In certain embodiments, the modified repressor gene comprises a helix-tum-helix domain, wherein the helix-turn helix domain comprises a mutation of at least one acidic residue. Acidic residues of the repressor which interact with host transcription machinery to dictate lysogen stability are conserved in certain phage repressor homologs. In certain embodiments, the modified repressor gene has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148. In certain embodiments, the modified repressor gene encodes a modified repressor protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149. The modified repressor protein set forth in SEQ ID NO: 149 comprises an aspartic acid to asparagine substitution at position 39 (D39N) relative to the wild-type repressor protein set forth in SEQ ID NO: 124. In certain embodiments, the prophage comprises a mutation in the operator region that controls the expression of the repressor. In certain embodiments, the modified operator region comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 150.

[0056] Exemplary structural phage protein genes include genes encoding for a portal protein, a scaffold protein, a tail protein (e.g., a tail fiber protein, a major tail protein, a minor tail protein, a tail tape measure protein, a phage endopeptidase, a lipase, or a hydrolase, including amidase protein K), a capsid protein, a head protein (e.g., a portal protein, a minor head protein, a major head protein, a scaffold protein, a connector protein), a head morphogenesis protein, a baseplate protein, a coat protein, or a virion structural protein.

[0057] Exemplary lysis genes include genes encoding for a holin, an antiholin, an endolysin, an inner-membrane spanin, an outer-membrane spanin, a unimolecular spanin, a mycolic acid esterase, or any gene which could affect lysis timing and mechanisms. In certain embodiments, the prophage of the disclosure comprises a modified holin gene. In certain embodiments, the modified holin gene comprises at least one amino acid substitution set forth in Table 2.

[0058] TABLE 2 shows lysis times and dominant or recessive phenotypes of Lambda holin (5) mutants with substitutions at various amino acid positions.

[0059] Exemplary virulence genes include genes encoding for the LuxR family transcription factor, lectin (e.g., galactophilic lectin), elastase (e.g., elastase precursor protein), leucocidin, exfoliative toxin (e.g., exfoliative toxin A), staphylokinase, chemotaxis inhibitory protein, staphylococcal complement inhibitor, or enterotoxin (e.g., enterotoxin S).

[0060] Other exemplary genes to be modified can include packaging genes (e.g., gene(s) encoding for a terminase), transcriptional regulator genes (e.g., gene(s) encoding for the XRE family transcriptional regulator), transcription factor genes (e.g., gene(s) encoding for the LuxR family transcription factor), a nuclease gene (e.g., gene(s) encoding for an endonuclease), a helicase gene (e.g., gene(s) encoding for a DNA helicase), an excisionase gene, or a transferase gene (e.g., gene(s) encoding for an acetyl transferase).

[0061] In certain embodiments, the heterologous nucleic acid is inserted within or adjacent to an integrase gene, a repressor gene, an anti-repressor gene, a virulence gene, a resistance gene, a toxin gene, a lysis gene, a phage receptor recognition gene, a structural phage protein gene, a DNA replication gene, a nuclease gene, a nucleotide synthesis gene, a tRNA gene, a chaperone gene, or a DNA packaging gene of the prophage. In certain embodiments, the heterologous nucleic acid is randomly inserted in the prophage.

[0062] In certain embodiments, the prophage comprises a deletion of one or more genes. The prophage may comprise a reduction of genes to the minimal components required for maximal protein expression and release of the payload. In certain embodiments, the prophage comprises a deletion of an integrase gene, a repressor gene, an anti-repressor gene, a virulence gene, a resistance gene, a toxin gene, a lysis gene, a phage receptor recognition gene, a structural phage protein gene, a DNA replication gene, a nuclease gene, a nucleotide synthesis gene, a tRNA gene, a chaperone gene, or a DNA packaging gene of the prophage. In certain embodiments, the prophage comprises a deletion of one or more genes set forth in Table 1. In certain embodiments, the deletion of one or more genes reduces the size of the genome of the prophage by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to the size of the genome of the corresponding wild-type prophage. In certain embodiments, the size of the genome of the prophage comprising the deletion of one or more genes is from about 1 kb to about 200 kb, 5 kb to about 100 kb, or about 10 kb to about 50 kb. In certain embodiments, the size of the genome of the prophage comprising the deletion of one or more genes is from about 1 kb to about 40 kb, 5 kb to about 30 kb, or about 10 kb to about 20 kb. In certain embodiments, the size of the genome of the prophage comprising the deletion of one or more genes is less than 200 kb, less than 100 kb, less than 75 kb, less than 50 kb, less than 25 kb, less than 20kb, less than 15 kb, less than 14 kb, less than 13 kb, less than 12 kb, less than 11 kb, less than 10 kb, less than 9 kb, less than 8 kb, less than 7 kb, less than 6 kb, or less than 5 kb.

[0063] In certain embodiments, the prophage, or host bacterial cell, is modified to render the bacterial cell responsive to a signal when expression of the payload is no longer needed. Such a signal may act as a response regulator for the expression of a gene that is toxic or repressive to the bacterial cell, or cause the expression of a gene essential for cell survival to cease, resulting in the elimination or substantial reduction of the cell population. Modifications of the bacterial cell include increased cell sensitivity to antibiotics (e.g., overexpression of antibiotic import porins) or removal of key metabolic genes in the bacterial chromosome.

[0064] In certain embodiments, the bacterial cell may have an existing prophage that is engineered to deliver the payload of interest. In certain embodiments, the cell may be free of prophage prior to receiving an engineered prophage ex vivo. In certain embodiments, the cell may have one or more native prophage in addition to the engineered prophage. In certain embodiments, the cell may have the engineered prophage and be otherwise free of prophage.

Bacterial Cells

[0065] The bacterial cell can be generally any bacterial cell including Gram-positive and Gramnegative bacterial cells. In certain embodiments, the bacterial cell is a Gram-positive bacterial cell. In certain embodiments, the bacterial cell is a Gram-negative bacterial cell.

[0066] In certain embodiments, the bacterial cell is a species suitable for use as a probiotic. “Probiotic” is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. Some species, strains, and/or subtypes of non-pathogenic microorganisms are currently recognized as probiotic. Examples of probiotics include, but are not limited to, Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, and Lactobacillus plantarum. [0067] In certain embodiments, Lactobacillus sp. may include, without limitation, a Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei (such as Lactobacillus casei Shirota), Lactobacillus salivarius, Lactobacillus paracasei, Lactobacillus lactis, Lactobacillus acidophilus, Lactobacillus sakei, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus garvieae, Lactobacillus acetotolerans, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus aviarus. Lactobacillus bifermenlans. Lactobacillus bulgaricus, Lactobacillus carnis. Lactobacillus calernaformis. Lactobacillus cellobiosis, Lactobacillus collinoides, Lactobacillus confuses, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus divergens, Lactobacillus farciminis, Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus haiotoierans, Lactobacillus hamster, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus iners, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kandleri, Lactobacillus kefiri, Lactobacillus kefuranofaciens, Lactobacillus kefirgranum, Lactobacillus kunkeei, Lactobacillus leichmannii, Lactobacillus llndnerl, Lactobacillus malefermentans, Lactobacillus mall, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus minor, Lactobacillus minutus, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus oris, Lactobacillus panis, Lactobacillus parabuchneri, Lactobacillus paracasei, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus piscicola, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus rhamnosus, Lactobacillus rhamnosus GG, Lactobacillus rimae, Lactobacillus rogosae, Lactobacillus ruminis, Lactobacillus sanfranciscensis, Lactobacillus sharpeae, Lactobacillus suebicus, Lactobacillus trichodes, Lactobacillus uli, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus xylosus, Lactobacillus yamanashiensis, or Lactobacillus zeae.

[0068] In certain embodiments, ^Bifidobacterium sp. may be Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium animalis subsp animalis, Bifidobacterium longum, Bifidobacterium fidobacterium breve, Bifidobacterium bifidum, Bifidobacterium animalis subsp. lactis o Bifidobacterium lactis, such as Bifidobacterium lactis DN-173 010. [0069] In certain embodiments, a Bacillus sp. may be Bacillus coagulans. In certain embodiments, a Lactococcus may be Lactococcus lactis subsp. lactis such as Lactococcus lactis subsp. lactis CV56. In some embodiments, an Enterococcus may be Enterococcus durans. In certain embodiments, a Streptococcus may be Streptococcus thermophilus.

[0070] In certain embodiments, the Escherichia coli may be E. coli Nissle 1917 or “EcN”. Escherichia is a genus of Gram-negative, non-spore forming, facultatively anaerobic, rodshaped bacteria from the family Enterob acteriaceae. The genus Escherichia includes various species, such as Escherichia coli. E. coli Nissle 1917 has evolved into one of the best characterized probiotics. The strain is characterized by its complete harmlessness and has GRAS (generally recognized as safe) status. E. coli strain Nissle 1917 lacks defined virulence factors such as alpha-hemolysin, other toxins, and mannose-resistant hemagglutinating adhesins, P- fimbrial adhesins, and the semirough lipopolysaccharide phenotype and expresses fitness factors such as microcins, ferritins, six different iron uptake systems, adhesins, and proteases, which support its survival and successful colonization of the human gut. As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called MUTAFLOR®, for therapeutic use. [0071] The probiotic may be a variant or a mutant strain of bacterium. Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Bacterial cells may be genetically engineered to enhance or improve probiotic properties, e.g., enhance gut colonization.

[0072] In certain embodiments, the bacterial cell is capable of colonizing the gastrointestinal tract, skin, nasal cavity, or other site of a human or animal.

[0073] In certain embodiments, the bacterial cell is a species suitable for use in industrial fermentation. In certain embodiments, the bacterial cell is selected from Escherichia spp., Bacillus spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp., and Pseudomonas spp. In certain embodiments, the bacterial cell is a species selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulates, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens, and Pseudomonas putida. [0074] In certain embodiments, the bacterial cell is a species suitable for attaching to and/or invading eukaryotic cells including cancer cells.

Methods for Producing Payload

[0075] In certain embodiments, the present disclosure provides methods for producing one or more payloads of interest. The payloads of interest according to the present disclosure can be generally any polypeptide, complexes of polypeptides, and bacteriophage particles, and can be, for example recombinant proteins and peptides suitable for pharmaceutical, nutraceutical, or industrial compositions. Non-limiting examples of suitable recombinant polypeptides include therapeutic polypeptides, prophylactic polypeptides, diagnostic polypeptides, nutraceutical polypeptides, industrial enzymes, and reporter polypeptides. The expressed polypeptides may be composed of a single polypeptide chain, multiple polypeptide chains, or a complex of multiple polypeptide chains (e.g., a homomultimeric or heteromultimeric complex). In certain embodiments, polypeptides of interest made in accordance with the present disclosure have a variety of uses including, but not limited to, use as vaccines, antimicrobials and other therapeutic compounds, use as diagnostic agents and use as antigens in the production of polyclonal or monoclonal antibodies.

[0076] In certain embodiments, the bacterial cells can be in culture (e.g., ex vivo). In certain embodiments, the bacterial cells can be in a living subject (e.g., in vivo). Accordingly, in certain embodiments, the method for producing a polypeptide of interest can include the cultivation of a bacterial cell including a prophage comprising a heterologous nucleic acid encoding a payload of interest. To produce one or more payloads of interest according to the present disclosure, a bacterial cell is cultured in an effective medium, using any one of cell culturing techniques known in the art. As used herein, an effective medium refers to any medium in which the bacterial cells can produce one or more polypeptides of interest. An effective medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins, growth factors and other hormones. The medium may comprise complex nutrients or may be a defined medium. Bacterial cells of the present disclosure can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, cell recycle and continuous fermenters. Culturing can also be conducted in shake flasks, test tubes, microtiter dishes and petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the bacterial cell. Such culturing conditions are well within the expertise of one of ordinary skill in the art.

[0077] In certain embodiments, the bacterial cell produces from about 5,000 to about 500,000 copies of the payload per induced cell. In certain embodiments, the bacterial cells produce about 10⁹ to about 10¹¹ copies of the payload per ml of culture per bacterial generation, based on a culture density of about 10⁷ CFU/ml.

[0078] In certain embodiments, the bacterial cell can be modified to respond to a signal that acts as a response regulator for the expression of a gene that is toxic to the bacterial cell, or causes expression of a gene essential for cell survival to cease, causing elimination or substantial reduction of the cell population. This is applicable both in batch cultures ex vivo and continuous production in vivo.

[0079] As used herein, the phrase “recovering the payload” refers simply to collecting the whole fermentation medium containing the payload and can, but need not, entail additional steps of separation or purification. Payloads of interest of the present disclosure can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing, and differential solubilization.

[0080] Isolated payloads of interest of the present disclosure are preferably retrieved in “substantially pure” form. As used herein, “substantially pure” refers to a purity that allows for the effective use of the compound as a therapeutic composition or diagnostic.

Pharmaceutical Compositions and Formulations

[0081] Pharmaceutical compositions comprising the bacterial cells of the disclosure may be used to treat, manage, ameliorate, and/or prevent a disease or disorder. Pharmaceutical compositions comprising one or more bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.

[0082] The pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA). In certain embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.

[0083] The bacteria may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, immediate-release, pulsatile- release, delayed-release, or sustained release). Suitable dosage amounts for the bacteria may range from about 10⁵ to 10¹² bacteria, e.g., approximately 10⁵ bacteria, approximately 10⁶ bacteria, approximately 10⁷ bacteria, approximately 10⁸ bacteria, approximately 10⁹ bacteria, approximately 10¹⁰ bacteria, approximately 10¹¹ bacteria, or approximately 10¹² bacteria. The composition may be administered once or more daily, weekly, or monthly. The bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents.

[0084] The bacteria may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

[0085] The bacteria may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

[0086] Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate-polylysine-alginate (APA), alginate- polymethylene-co-guanidine-alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallyl sulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co- glycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, polyamino acids, and enteric coating polymers.

[0087] In certain embodiments, the bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the small or large intestines. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In certain embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In certain embodiments, at least two coatings are used. In certain embodiments, the outside coating and the inside coating are degraded at different pH levels.

[0088] Materials used for enteric coatings include Cellulose acetate phthalate (CAP), Poly(methacrylic acid-co-methyl methacrylate), Cellulose acetate trimellitate (CAT), Poly(vinyl acetate phthalate) (PVAP) and Hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, Shellac (esters of aleurtic acid), plastics and plant fibers. Additionally, Zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch and starch derivatives, and dextrins (e.g., maltodextrin) are also used. Other known enteric coatings include ethylcellulose, methylcellulose, hydroxypropyl methylcellulose, amylose acetate phthalate, cellulose acetate phthalate, hydroxyl propyl methyl cellulose phthalate, an ethyl acrylate, and a methylmethacrylate.

[0089] Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the bacteria.

[0090] In certain embodiments, the bacteria may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. In certain embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

[0091] In certain embodiments, the pharmaceutical composition comprising the bacteria of the disclosure may be a comestible product, for example, a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In another embodiment, the bacteria of disclosure are combined in a preparation containing other live bacterial cells intended to serve as probiotics. In another embodiment, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is a jelly or a pudding. Other food products suitable for administration of the bacteria of the disclosure are well known in the art. See, e.g., US 2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical composition of the disclosure is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.

[0092] The bacteria may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0093] The bacteria may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0094] In certain embodiments, the disclosure provides pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion.

[0095] Single dosage forms of the pharmaceutical composition may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.

[0096] Dosage regimens may be adjusted to provide a therapeutic response. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician.

[0097] The bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0098] The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0099] The pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2 °C and 8 °C and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.

[0100] Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LDso, EDso, ECso, and ICso may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

Embodiments

[0101] The following numbered embodiments also form part of the present disclosure: [0102] 1. A bacterial cell comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a payload of interest. [0103] 2. The bacterial cell of embodiment 1, wherein the payload of interest comprises a polypeptide, a complex of polypeptides, and/or a bacteriophage particle.

[0104] 3. The bacterial cell of embodiment 1 or embodiment 2, wherein the bacterial cell or prophage comprises one or more modifications that increase spontaneous prophage induction. [0105] 4. The bacterial cell of any one of embodiments 1-3, wherein the prophage comprises a modification of a repressor coding region, repressor promoter, or repressor operator binding site, or wherein the bacterial cell comprises a hyperactive recA allele, optionally wherein the prophage comprises a modification of a repressor ligand binding domain, a repressor dimerization domain, a DNA binding domain, an auto-cleavage domain, or a RecA interacting domain.

[0106] 5. The bacterial cell of any one of embodiments 1-4, wherein the prophage comprises one or more modifications that delay lysis or increase payload accumulation; or wherein the prophage comprises a deletion of one or more genes, optionally wherein the prophage comprises a deletion of an integrase gene, a repressor gene, an anti-repressor gene, a virulence gene, a resistance gene, a toxin gene, a lysis gene, a phage receptor recognition gene, a structural phage protein gene, a DNA replication gene, a nuclease gene, a nucleotide synthesis gene, a tRNA gene, a chaperone gene, or a DNA packaging gene.

[0107] 6. The bacterial cell of any one of embodiments 1-5, wherein the bacterial cell is a Bacillus sp., Bacteroides sp., Bifidobacterium sp., Corynebacterium sp., Escherichia sp., Lactobacillus sp., Lactococcus sp., Pseudomonas sp., Rhodobacter sp., Vibrio sp., Zymomonas sp., Burkholderia sp., Stenotrophomonas sp., Brucella sp., Salmonella sp., Prevotella sp., Streptococcus sp., Staphylococcus sp., Desulfovibrio sp., Cellulosimicrobium sp., Microbacterium sp., Acinetobacter sp., Akkermansia sp., Gluconacetobacter sp., Acetobacter sp., or Fusobacterium sp.

[0108] 7. The bacterial cell of any one of embodiments 1-6, wherein the prophage is a Lambda, Lambda-like, P2, 186, P22, Pasto, ProddE, P335, phiETA, 77, 80, TP901-1, PBSX, or SP-beta like prophage.

[0109] 8. The bacterial cell of any one of embodiments 1-7, wherein the payload of interest comprises an immunomodulator, a cytotoxin, an antimicrobial, an enzyme, an antigen, an antibody, a nanobody, a single-chain variable fragment (scFv), a peptide hormone, a high molecular weight bacteriocin, or a heterologous lytic phage.

[0110] 9. The bacterial cell of any one of embodiments 1-8, wherein the heterologous nucleic acid is inserted within or adjacent to an integrase gene, a repressor gene, an anti-repressor gene, a virulence gene, a resistance gene, a toxin gene, a lysis gene, a phage receptor recognition gene, a structural phage protein gene, a DNA replication gene, a nuclease gene, a nucleotide synthesis gene, a tRNA gene, a chaperone gene, or a DNA packaging gene of the prophage; or wherein the heterologous nucleic acid is randomly inserted in the prophage.

[OHl] 10. The bacterial cell of embodiment 1-9, wherein the bacterial cell or prophage is modified to render the bacterial cell responsive to a signal when expression of the payload is no longer needed.

[0112] 11. A cell culture comprising one or more bacterial cells of any one of embodiments 1- 10.

[0113] 12. The cell culture of embodiment 11, wherein from about 0.5% to about 40% of bacterial cells undergo spontaneous prophage induction per generation.

[0114] 13. A method for producing a payload of interest, the method comprising: culturing one or more bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a payload of interest. [0115] 14. The method of embodiment 13, further comprising recovering the payload of interest. [0116] 15. The method of embodiment 13 or embodiment 14, wherein the bacterial cell or prophage comprises one or more modifications that increase spontaneous prophage induction. [0117] 16. The method of any one of embodiments 13-15, wherein the prophage comprises a modification of a repressor coding region, repressor promoter, or repressor operator binding site, or wherein the bacterial cell comprises a hyperactive recA allele, optionally wherein the prophage comprises a modification of a repressor ligand binding domain, a repressor dimerization domain, a DNA binding domain, an auto-cleavage domain, or a RecA interacting domain.

[0118] 17. The method of any one of embodiments 13-16, wherein from about 0.5% to about 40% of bacterial cells undergo spontaneous prophage induction per generation.

[0119] 18. The method of any one of embodiments 13-17, wherein the prophage comprises one or more modifications that delay lysis or increase payload accumulation; or wherein the prophage comprises a deletion of one or more genes.

[0120] 19. The method of any one of embodiments 13-18, wherein the bacterial cell is ^Bacillus sp., Bacteroides sp., Bifidobacterium sp., Corynebacterium sp., Escherichia sp., Lactobacillus sp., Lactococcus sp., Pseudomonas sp., Rhodobacter sp., Vibrio sp., Zymomonas sp., Burkholderia sp., Stenotrophomonas sp., Brucella sp., Salmonella sp., Prevotella sp., Streptococcus sp., Staphylococcus sp., Desulfovibrio sp., Cellulosimicrobium sp., Microbacterium sp., Acinetobacter sp., Akkermansia sp., Gluconacetobacter sp., Acetobacter sp., or Fusobacterium sp.

[0121] 20. The method of any one of embodiments 13-19, wherein the prophage is a Lambda, Lambda-like, P2, 186, P22, Pasto, ProddE, P335, phiETA, 77, 80, TP901-1, PBSX, or SP-beta like prophage.

[0122] 21. The method of any one of embodiments 13-20, wherein the payload of interest comprises an immunomodulator, a cytotoxin, an antimicrobial, an enzyme, an antigen, an antibody, a nanobody, a ScFV, a peptide hormone, a high molecular weight bacteriocin, or a heterologous lytic phage.

[0123] 22. The method of any of embodiment 13-21, wherein the bacterial cell or prophage is modified to render the bacterial cell responsive to a signal when expression of the payload is no longer needed.

[0124] 23. A method for producing a payload of interest in a subject, the method comprising administering to the subject one or more bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a payload of interest.

[0125] 24. The method of embodiment 23, wherein the bacterial cell or prophage comprises one or more modifications that increase spontaneous prophage induction.

[0126] 25. The method of embodiment 23 or embodiment 24, wherein the prophage comprises a modification of a repressor coding region, repressor promoter, or repressor operator binding site, or wherein the bacterial cell comprises a hyperactive recA allele, optionally wherein the prophage comprises a modification of a repressor ligand binding domain, a repressor dimerization domain, a DNA binding domain, an auto-cleavage domain, or a RecA interacting domain.

[0127] 26. The method of any one of embodiments 23-25, wherein from about 0.5% to about 40% of bacterial cells undergo spontaneous prophage induction per generation.

[0128] 27. The method of any one of embodiments 23-26, wherein the prophage comprises one or more modifications that delay lysis or increase payload accumulation; or wherein the prophage comprises a deletion of one or more genes.

[0129] 28. The method of any one of embodiments 23-27, wherein the bacterial cell is a Bacillus sp., Bacteroides sp., Bifidobacterium sp., Corynebacterium sp., Escherichia sp., Lactobacillus sp., Lactococcus sp., Pseudomonas sp., Rhodobacter sp., Vibrio sp., Zymomonas sp., Burkholderia sp., Stenotrophomonas sp., Brucella sp., Salmonella sp., Prevotella sp., Streptococcus sp., Staphylococcus sp., Desulfovibrio sp., Cellulosimicrobium sp., Microbacterium sp., Acinetobacter sp., Akkermansia sp., Gluconacetobacter sp., Acetobacter sp., or Fusobacterium sp.

[0130] 29. The method of any one of embodiments 23-28, wherein the prophage is a Lambda, Lambda-like, P2, 186, P22, Pasto, ProddE, P335, phiETA, 77, 80, TP901-1, PBSX, or SP-beta like prophage.

[0131] 30. The method of any one of embodiments 23-29, wherein the payload of interest comprises an immunomodulator, a cytotoxin, an antimicrobial, an enzyme, an antigen, an antibody, a nanobody, a ScFV, a peptide hormone, a high molecular weight bacteriocin, or a heterologous lytic phage.

[0132] 31. The method of any of embodiments 23-30, wherein the bacterial cell or prophage is modified to render the bacterial cell responsive to a signal when expression of the payload is no longer needed. [0133] 32. The method of any one of embodiments 23-31, wherein the composition is administered orally, rectally, intravaginally, intranasally, inhalationally, intravenously, or topically.

[0134] 33. A pharmaceutical composition comprising one or more bacterial cells of any one of embodiments 1-10; and a pharmaceutically acceptable carrier.

[0135] 34. A method of treating or preventing a disorder in a subject in need thereof, the method comprising: administering to the subject a pharmaceutical composition comprising one or more bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a therapeutic polypeptide.

[0136] 35. The method of embodiment 34, wherein the bacterial cell or prophage comprises one or more modifications that increase spontaneous prophage induction.

[0137] 36. The method of embodiment 34 or embodiment 35, wherein the prophage comprises a modification of a repressor coding region, repressor promoter, or repressor operator binding site, or wherein the bacterial cell comprises a hyperactive recA allele, optionally wherein the prophage comprises a modification of a repressor ligand binding domain, a repressor dimerization domain, a DNA binding domain, an auto-cleavage domain, or a RecA interacting domain.

[0138] 37. The method of any one of embodiments 34-36, wherein from about 0.5% to about 40% of bacterial cells undergo spontaneous prophage induction per generation.

[0139] 38. The method of any one of embodiments 34-37, wherein the prophage comprises one or more modifications that delay lysis or increase payload accumulation; or wherein the prophage comprises a deletion of one or more genes.

[0140] 39. The method of any one of embodiments 34-38, wherein the bacterial cell is a Bacillus sp., Bacteroides sp., Bifidobacterium sp., Corynebacterium sp., Escherichia sp., Lactobacillus sp., Lactococcus sp., Pseudomonas sp., Rhodobacter sp., Vibrio sp., Zymomonas sp., Burkholderia sp., Stenotrophomonas sp., Brucella sp., Salmonella sp., Prevotella sp., Streptococcus sp., Staphylococcus sp., Desulfovibrio sp., Cellulosimicrobium sp., Microbacterium sp., Acinetobacter sp., Akkermansia sp., Gluconacetobacter sp., Acetobacter sp., or Fusobacterium sp.

[0141] 40. The method of any one of embodiments 34-39, wherein the prophage is a Lambda, Lambda-like, P2, 186, P22, Pasto, ProddE, P335, phiETA, 77, 80, TP901-1, PBSX, or SP-beta like prophage. [0142] 41. The method of any one of embodiments 34-40, wherein the therapeutic polypeptide comprises an immunomodulator, a cytotoxin, an antimicrobial, an enzyme, an antigen, an antibody, a nanobody, a ScFV, a peptide hormone, a high molecular weight bacteriocin, or a heterologous lytic phage.

[0143] 42. The method of any one of embodiments 34-41, wherein the bacterial cell or prophage is modified to render the bacterial cell responsive to a signal when expression of the payload is no longer needed.

[0144] 43. The method of any one of embodiments 34-42, wherein the composition is administered orally, rectally, intravaginally, intranasally, inhalationally, intravenously, or topically.

[0145] 44. The method of any one of embodiments 34-43, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0146] 45. A bacteriophage particle produced by the bacterial cell of any one of embodiments 1- 10.

[0147] 46. The bacteriophage particle of embodiment 45, wherein the particle is purified.

[0148] 47. The bacteriophage particle of embodiment 45 or embodiment 46, wherein the particle is infectious.

[0149] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0150] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

[0151] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1: Selection of protein delivery hosts for prophage-based protein delivery

[0152] Protein overproduction and release by prophage induction systems will be developed in bacterial strains suitable for biotechnological applications. This includes but is not limited to human colonizing strains for in vivo drug delivery, strains for enzyme production, and industrial fermentations. The prototype system is based on the Lambda prophage (the paradigm prophage system) residing in an E. coli host.

[0153] As proof of concept, Lambda prophage was engineered to harbor a lacZ gene encoding a large enzyme P-Galactosidase, a glycoside hydrolase enzyme also known as lactase or P-gal. After the prophage in E. coli was thermally induced (by shifting the incubating temperature from 30 °C to 37 °C), the P-Galactosidase enzyme was released from the bacterial host cells, degrading the substrate X-Gal (5-bromo-4-chloro-3-indolyl-P-d-galactoside) embedded in the agar plates and resulting in visible blue color surrounding the producing cells (FIG. 3).

Example 2: Modification of the phage lysogeny control system to achieve high levels of spontaneous phage induction

[0154] Prophage induction rates in the host strains will be monitored by inserting a fluorescent reporter (such as GFP or mCherry) for protein expression. In addition to fluorescent reporter, induction rates can be measured by release of phage particles and enumeration of plaque forming units. By thorough characterization of several prophage systems and understanding of the precision control of their induction mechanisms, derivatives will be developed to achieve spontaneous prophage induction at high frequencies (from 0.5% to 40% of cells per generation) in bulk culture. Specifically, changes will be made to the bacterial host cell and the lysogeny control region to improve spontaneous induction rates. Modifications include expression of a hyperactive bacterial recA allele, alterations to the promoter that controls repressor expression, alterations to repressor operator binding sites, and alterations to the repressor protein itself. This approach can be expanded to all Lambda-like lysogeny control systems.

[0155] As a proof of concept, a mutation was introduced into the lambda repressor protein (part of the lysogeny control system) and the rates of prophage induction were compared when E. coli cells harboring prophage were incubated at 30°C. The lambda prophage carrying the mutation was spontaneously induced at a significantly higher rate than the lambda prophage without the mutation (FIG. 4). The increased spontaneous induction rate results in an increased release of the P-galactosidase enzyme carried on the prophage from the E. coli cells, as evidenced by the development of a deeper blue color due to the degradation of the substrate X-Gal (5-bromo-4- chloro-3-indolyl-P-d-galactoside). Example 3: Determine optimal location for transgene insertion in the prophage

[0156] Transcriptomics/ribosome profiling can be utilized to help determine the most desirable locations for the payload. Alternatively, a reporter gene such as GFP will be randomly inserted at various locations in prophage. The modified prophages will be induced, and the reporter signal will be measured to identify sites that confer maximal protein expression. Obtained optimal location information will be expanded to other similar prophage types.

Example 4: High throughput screening of large numbers of prophage mutant clones for the best prophage design

[0157] After random mutations are introduced to the prophage, either to modify the lysogeny control system or to probe the optimal location for protein expression, high throughput systems capable of detecting reporter signals (such as fluorescence, luminescence, or colorimetric signals) will be utilized to assess prophage mutant clones for their expression of reporter constructs to identify mutants with the desired characteristics. This is a generalized method that will be used in diverse hosts and prophages.

Example 5: Minimizing the prophage element for portability and safety

[0158] The engineered prophage elements will have genes not used for protein expression and release deleted to increase portability of the element between strains and prevent the release of functional phages into the medium during protein production. These deletions will generate a miniPIPER system. This improved prophage element will be transferred into new hosts using methods similar to those used for transferring DNA mobile elements. This will allow the desired protein to be expressed in a diverse range of bacterial strains tailored to specific applications. An illustration of this concept can be seen in FIG. 6, where in from intact PIPER systems, infectious phage particles can be used for dissemination into new hosts, while the miniPIPER can be transferred following traditional DNA mobile element transfer, including but not limited to, plasmid or transposon-based methods.

Example 6: Implement payload overproduction and release system in bacterial hosts via a desired triggering mechanism

[0159] The prophage control system will be modified to be inducible by an exogenous signal such as temperature or addition of a chemical compound to the culture. This inducing signal would effectively override the spontaneous induction signal and cause all cells in the system to undergo the program of prophage induction, protein expression and release.

Example 7: “Kill Switch” of the bacterial cell to turn off the payload production [0160] The bacterial cell harboring the prophage is modified to render the bacterial cell responsive to a signal when expression of the payload is not needed. Such a signal acts as a response regulator for the expression of a gene that is toxic or repressive to the bacterial cell, or causes the expression of a gene essential for cell survival to cease, resulting in the elimination or substantial reduction of the cell population. Modifications of the bacterial cell include increased cell sensitivity to antibiotics (e.g., overexpression of antibiotic import porins) or removal of key metabolic genes in the bacterial chromosome. This is applicable both in batch cultures ex vivo and continuous production in vivo.

Example 8: Spontaneous induction and effect of growth

[0161] As a further example of the ability to increase the rate of spontaneous induction for high levels of cargo release, the actual induction events over time were calculated via monitoring of released PFU from the PIPER prototype and parental prophage (Lambda), rather than the monitoring of ZacZ cargo as in FIG. 5. By mutation of the Ci-repressor protein, the PIPER prophage was able to achieve a ~200x increase in lysis events per CFU per 30 min compared to the parental prophage underlying an increase in the spontaneous induction rate.

[0162] TABLE 3 To calculate the alteration of induction events of time PIPER prophage and the parental prophage were cultured at low density to avoid secondary absorption of released phage and monitored over time under non-inducing conditions. At 30-minute time intervals, free phage particles quantified (PFU/ml) and host level of bacterial host quantified (CFU/ml). These values were used to determine the number of lysis events per CFU per 30 min intervals. These values demonstrate increase in lysis events via mutation to CI mutation. [0163] In support of the ability of PIPER platform for sustained delivery and release from long term cultures in an in vitro setting (e.g., a chemostat or turbidostat for industrial protein production use) or from an in vivo setting (e.g., therapeutic cargo delivery from colonizing PIPER probiotics), the impact of PIPER prophages on growth of the host bacterium was also monitored. To this end, growth of the bacterial host containing either PIPER prophage or parental prophage was tracked over a 3.5 hour period via direct plating and quantification of CFU (FIG. 7). The doubling time of the parental prophage and the PIPER prophage host was approximately 20 min and 30 min, respectively. These results indicate that despite an increased number of induction events per generation, growth of the host, while reduced slightly compared to WT, retains a positive trajectory indicating a stable population with an altered spontaneous induction rate is possible.

Example 9: Generation of miniPIPER prototype achievable without majorly disrupting lysis timing

[0164] This example demonstrates that the PIPER prophage can be substantially reduced in size without major disruption to induction and parental prophage lysis and lysis timing. While Example 5 above describes the reduction in size of a generalized hypothetical prophage as this process is applicable to a range of prophages, FIG. 8A shows this process applied to the parental prophage lambda, highlighting both the unnecessary genes and the beneficial and/or essential genes for the PIPER system. The intact lambda genome is approximate 48 kb in length, and through the removal of non-essential gene content, the miniPIPER derived from the parental phage lamba can be reduced to approximately 10 kb in length (FIG. 8B). This increases portability, eliminates viable phage particles, minimizes safety concerns, and increases the already high capacity for large gene cargo.

[0165] As a proof-of-concept that large deletions can be introduced to eliminate non-essential genes to the PIPER system without disrupting phage-mediated lysis and lysis timing. A recombineering approach was used to introduce a series of successive deletions eliminating the majority of structural genes reducing the parental prophage genome size by -40% (FIG. 9A). To confirm that these deletions did not interrupt phage lysis, these deletions were performed in the thermally inducible parental Lambda prophage (cI857) to form miniPIPER prototype (miniPIPER W-stf) (FIG. 9A). The parental prophage and miniPIPER prototype were then thermally induced and growth monitored via OD for lysis activity (FIG. 9B). Parental prophage lysis begins approximately 30min post induction. While in the miniPIPER prototype, the initiation of lysis occurs at approximately 45min post-induction. This indicates that while lysis timing has slightly been affected occurring about 15 minutes later, lysis of the miniPIPER prototype follows a similar rapid and synchronized drop in OD compared to induction of the parental prophage. While this prototype of miniPIPER retains a number of non-essential regions, it demonstrates that such major deletions are achievable to eliminate phage particles, increase portability and increase safety.

Example 10: Comparable genetic switches exist in distant bacteria and can be converted to the PIPER system through similar mutations

[0166] The PIPER system of repressor modification, cargo insertion, and prophage reduction can be broadly applied to distantly related bacteria and their prophage elements, not just Lambda. A temperate phage was bioinformatically identified that infects the distantly related genus of bacteria, Lactobacillus, that is highly compatible for conversion to the PIPER system. This characterized temperate Lactobacillus phage A2 contains a similar master lysis/lysogeny to that of Lambda characterized by divergent promoters driving transcription of homologous repressor/ antirepressor proteins (FIG. 10A). This conserved lysis/lysogeny switch arrangement indicates it can be similarly adapted to the PIPER system through a similar mutational approach to increase its rate of spontaneous induction. Further evidence that phage A2 could be easily converted into the PIPER system is indicated by the conservation of amino acid residues in the phage A2 CI repressor compared to the Lambda CI repressor (FIG. 10B). Critical acidic residues of the Ci-repressor which interact with host transcription machinery to dictate lysogen stability are conserved in the phage A2 Ci-repressor homolog. This indicates similar mutations, including but not limited to those, used in the conversion of phage Lambda to the PIPER system could be used to convert more distantly relate temperate phages to the PIPER system.

Example 11: Holin mutants can be utilized for delayed or early lysis timing as an additional layer of cargo dosage control

[0167] The amount of cargo accumulated per induction event (i.e., dosage in a therapeutic application) can be further modulated by delaying the lysis timing of the host cell. This example describes systematic mutations of the lambda holin and their effect on the timing of the lysis event (FIG. 11A-D). Amino acid substitutions to key amino acids in the 2^nd transmembrane domain of the lambda holin (S protein). The effect of these substitutions on lambda lysis timing was observed after thermal induction of phage Lambda. As can be seen, mutations at residues T49, M50, C51, or A52 have drastic effects on lysis timing, in some cases, shortening or lengthening the duration to phage mediated lysis. Delays in lysis allow the continuous accumulation of phage particles (or cargo) before lysing the host cells. More rapid lysis results in a reduction cargo accumulated before lysing the host cell. As a demonstration of lysis timings correlation with cargo accumulation, FIG. 12 shows the accumulation of phage particles over time in a bacterial lysogen host, E. coli MC4100 (CI857 Sam7 which does not undergo lysis due to a mutation in the holin gene. In the case of wild-type lambda after approximately 45min post induction, -100 new virions are released; however, as can be seen in FIG. 12, without holin activity, phage particles continue to accumulate over time reaching >600 phage particles per cell. These modifications to lysis timing allow a large dynamic range of cargo production depending on the dosage required by a specific therapeutic delivery application or industrial use case.

Claims

What is claimed is:

1. A bacterial cell comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a payload of interest.

2. The bacterial cell of claim 1, wherein payload of interest comprises a polypeptide, a complex of polypeptides, and/or a bacteriophage particle.

3. The bacterial cell of claim 1, wherein the bacterial cell or prophage comprises one or more modifications that increase spontaneous prophage induction.

4. The bacterial cell of claim 3, wherein the prophage comprises a modification of a repressor coding region, repressor promoter, or repressor operator binding site, or wherein the bacterial cell comprises a hyperactive recA allele.

5. The bacterial cell of claim 1, wherein the prophage comprises one or more modifications that delay lysis or increase payload accumulation; or wherein the prophage comprises a deletion of one or more genes.

6. The bacterial cell of claim 1, wherein the bacterial cell is a Bacillus sp., Bacteroides sp., Bifidobacterium sp., Corynebacterium sp., Escherichia sp., Lactobacillus sp., Lactococcus sp., Pseudomonas sp., Rhodobacter sp., Vibrio sp., Zymomonas sp., Burkholderia sp., Stenotrophomonas sp., Brucella sp., Salmonella sp., Prevotella sp., Streptococcus sp., Staphylococcus sp., De sulfovibrio sp., Cellulosimicrobium sp ., Microbacterium sp., Acinetobacter sp., Akkermansia sp., Gluconacetobacter sp., Acetobacter sp., or Fusobacterium sp.

7. The bacterial cell of claim 1, wherein the prophage is a Lambda, Lambda-like, P2, 186, P22, Pasto, ProddE, P335, phiETA, 77, 80, TP901-1, PBSX, or SP-beta like prophage.

8. The bacterial cell of claim 1, wherein the payload of interest comprises an immunomodulator, a cytotoxin, an antimicrobial, an enzyme, an antigen, an antibody, a nanobody, a single-chain variable fragment (scFv), a peptide hormone, a high molecular weight bacteriocin, or a heterologous lytic phage.

9. The bacterial cell of claim 1, wherein the heterologous nucleic acid is inserted within or adjacent to an integrase gene, a repressor gene, an anti-repressor gene, a virulence gene, a resistance gene, a toxin gene, a lysis gene, a phage receptor recognition gene, a structural phage protein gene, a DNA replication gene, a nuclease gene, a nucleotide synthesis gene, a tRNA gene, a chaperone gene, or a DNA packaging gene of the prophage; or wherein the heterologous nucleic acid is randomly inserted in the prophage.

10. The bacterial cell of claim 1, wherein the bacterial cell or prophage is modified to render the bacterial cell responsive to a signal when expression of the payload is no longer needed.

11. A cell culture comprising one or more bacterial cells of any one of claims 1-10.

12. The cell culture of claim 11, wherein from about 0.5% to about 40% of bacterial cells undergo spontaneous prophage induction per generation.

13. A method for producing a payload of interest, the method comprising: culturing one or more bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a payload of interest.

14. The method of claim 13, further comprising recovering the payload of interest.

15. The method of claim 13, wherein the bacterial cell or prophage comprises one or more modifications that increase spontaneous prophage induction.

16. The method of claim 15, wherein the prophage comprises a modification of a repressor coding region, repressor promoter, or repressor operator binding site, or wherein the bacterial cell comprises a hyperactive recA allele.

17. The method of claim 15, wherein from about 0.5% to about 40% of bacterial cells undergo spontaneous prophage induction per generation.

18. The method of claim 13, wherein the prophage comprises one or more modifications that delay lysis or increase payload accumulation; or wherein the prophage comprises a deletion of one or more genes.

19. The method of claim 13, wherein the bacterial cell is Bacillus sp., Bacteroides sp., Bifidobacterium sp., Corynebacterium sp., Escherichia sp., Lactobacillus sp., Lactococcus sp., Pseudomonas sp., Rhodobacter sp., Vibrio sp., Zymomonas sp., Burkholderia sp., Stenotrophomonas sp., Brucella sp., Salmonella sp., Prevotella sp., Streptococcus sp., Staphylococcus sp., De sulfovibrio sp., Cellulosimicrobium sp ., Microbacterium sp., Acinetobacter sp., Akkermansia sp., Gluconacetobacter sp., Acetobacter sp., or Fusobacterium sp.

20. The method of claim 13, wherein the prophage is a Lambda, Lambda-like, P2, 186, P22, Pasto, ProddE, P335, phiETA, 77, 80, TP901-1, PBSX, or SP-beta like prophage.

21. The method of claim 13, wherein the payload of interest comprises an immunomodulator, a cytotoxin, an antimicrobial, an enzyme, an antigen, an antibody, a nanobody, a ScFV, a peptide hormone, a high molecular weight bacteriocin, or a heterologous lytic phage.

22. The method of claim 13, wherein the bacterial cell or prophage is modified to render the bacterial cell responsive to a signal when expression of the payload is no longer needed.

23. A method for producing a payload of interest in a subject, the method comprising administering to the subject one or more bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a payload of interest.

24. The method of claim 23, wherein the bacterial cell or prophage comprises one or more modifications that increase spontaneous prophage induction.

25. The method of claim 24, wherein the prophage comprises a modification of a repressor coding region, repressor promoter, or repressor operator binding site, or wherein the bacterial cell comprises a hyperactive recA allele.

26. The method of claim 24, wherein from about 0.5% to about 40% of bacterial cells undergo spontaneous prophage induction per generation.

27. The method of claim 23, wherein the prophage comprises one or more modifications that delay lysis or increase payload accumulation; or wherein the prophage comprises a deletion of one or more genes.

28. The method of claim 23, wherein the bacterial cell is a Bacillus sp., Bacteroides sp., Bifidobacterium sp., Corynebacterium sp., Escherichia sp., Lactobacillus sp., Lactococcus sp., Pseudomonas sp., Rhodobacter sp., Vibrio sp., Zymomonas sp., Burkholderia sp., Stenotrophomonas sp., Brucella sp., Salmonella sp., Prevotella sp., Streptococcus sp., Staphylococcus sp., De sulfovibrio sp., Cellulosimicrobium sp ., Microbacterium sp., Acinetobacter sp., Akkermansia sp., Gluconacetobacter sp., Acetobacter sp., or Fusobacterium sp.

29. The method of claim 23, wherein the prophage is a Lambda, Lambda-like, P2, 186, P22, Pasto, ProddE, P335, phiETA, 77, 80, TP901-1, PBSX, or SP-beta like prophage.

30. The method of claim 23, wherein the payload of interest comprises an immunomodulator, a cytotoxin, an antimicrobial, an enzyme, an antigen, an antibody, a nanobody, a ScFV, a peptide hormone, a high molecular weight bacteriocin, or a heterologous lytic phage.

31. The method of claim 23, wherein the bacterial cell or prophage is modified to render the bacterial cell responsive to a signal when expression of the payload is no longer needed.

32. The method of claim 23, wherein the composition is administered orally, rectally, intravaginally, intranasally, inhalationally, intravenously, or topically.

33. A pharmaceutical composition comprising one or more bacterial cells of any one of claims 1-10; and a pharmaceutically acceptable carrier.

34. A method of treating or preventing a disorder in a subject in need thereof, the method comprising: administering to the subject a pharmaceutical composition comprising one or more bacterial cells comprising a prophage integrated into the bacterial chromosome, wherein the prophage comprises a heterologous nucleic acid encoding a therapeutic polypeptide.

35. The method of claim 34, wherein the bacterial cell or prophage comprises one or more modifications that increase spontaneous prophage induction.

36. The method of claim 35, wherein the prophage comprises a modification of a repressor coding region, repressor promoter, or repressor operator binding site, or wherein the bacterial cell comprises a hyperactive recA allele.

37. The method of claim 35, wherein from about 0.5% to about 40% of bacterial cells undergo spontaneous prophage induction per generation.

38. The method of claim 34, wherein the prophage comprises one or more modifications that delay lysis or increase payload accumulation; or wherein the prophage comprises a deletion of one or more genes.

39. The method of claim 34, wherein the bacterial cell is a Bacillus sp., Bacteroides sp., Bifidobacterium sp., Corynebacterium sp., Escherichia sp., Lactobacillus sp., Lactococcus sp., Pseudomonas sp., Rhodobacter sp., Vibrio sp., Zymomonas sp., Burkholderia sp., Stenotrophomonas sp., Brucella sp., Salmonella sp., Prevotella sp., Streptococcus sp., Staphylococcus sp., De sulfovibrio sp., Cellulosimicrobium sp ., Microbacterium sp., Acinetobacter sp., Akkermansia sp., Gluconacetobacter sp., Acetobacter sp., or Fusobacterium sp.

40. The method of claim 34, wherein the prophage is a Lambda, Lambda-like, P2, 186, P22, Pasto, ProddE, P335, phiETA, 77, 80, TP901-1, PBSX, or SP-beta like prophage.

41. The method of claim 34, wherein the therapeutic polypeptide comprises an immunomodulator, a cytotoxin, an antimicrobial, an enzyme, an antigen, an antibody, a nanobody, a ScFV, a peptide hormone, a high molecular weight bacteriocin, or a heterologous lytic phage.

42. The method of claim 34, wherein the bacterial cell or prophage is modified to render the bacterial cell responsive to a signal when expression of the payload is no longer needed.

43. The method of claim 34, wherein the composition is administered orally, rectally, intravaginally, intranasally, inhalationally, intravenously, or topically.

44. The method of claim 34, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

45. A bacteriophage particle produced by the bacterial cell of claim 1.

46. The bacteriophage particle of claim 45, wherein the particle is purified.

47. The bacteriophage particle of claim 45, wherein the particle is infectious.