Intron-based interspecific biocontainment

Abstract

The technology is directed to transgene constructs that use at least one intron to provide interspecies biocontainment. The transgene construct can comprise a polypeptide coding sequence interrupted by at least one heterologous intron sequence. Also described herein are nucleic acids, pharmaceutical compositions, and preparations comprising such transgene constructs. Method of uses of such transgene constructs and associated compositions are also disclosed herein.

Description

INTRON-BASED INTERSPECIFIC BIOCONTAINMENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 548,590 filed February 1, 2024, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on January 27, 2025, is named 002806-000128WOPT_SL.xml and is 21,293 bytes in size.TECHNICAL FIELD

[0003] The technology described herein relates to compositions and methods of interspecific biocontainment.BACKGROUND

[0004] Genetic engineering of organisms (e.g., bacteria, fungi) is a promising approach to endow cells with new functions, for example to create new live therapeutics. Many applications involve the potential release of these engineered organisms in a larger, natural population of micro-organisms (for example, in the soil or in the human gastrointestinal tract).

[0005] Horizontal gene transfer, a natural mechanism through which organisms acquire foreign genetic material, can potentially lead to transgenes being serendipitously transferred to unintended cells. This is cause for concern, as it can result in some of these genes being expressed in contexts where they can cause harm (for example, by favoring the survival of pathogens). There is therefore a need to create barriers to horizontal gene transfers between engineered and environmental organisms.

[0006] Yeasts are increasingly used as probiotics, and there is a need to prevent the expression of fungal transgenes in bacterial cells, which are known to take up and exchange foreign DNA at an appreciable rate.SUMMARY

[0007] The technology described herein is directed to transgene constructs that use at least one intron to provide interspecies biocontainment. The transgene construct can comprise a polypeptide coding sequence (e.g., a bacterial polypeptide coding sequence) interrupted by at least one heterologous intron sequence. For example, such a transgene construct can prevent or reduce horizontal transmission of the polypeptide from a fungal host cell to a non-host bystander cell, including but not limited to a bacterial cell. Also described herein are nucleic acids, pharmaceutical compositions, and preparations comprising such transgene constructs. Methods of use of such transgene constructs and associated compositions are also disclosed herein.

[0008] Accordingly, in one aspect described herein is a transgene construct comprising a bacterial polypeptide coding sequence, wherein the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence.

[0009] In some embodiments of any of the aspects, the at least one heterologous intron comprises: at least one in-frame stop codon; at least one stop codon in each of a plurality of reading frames; at least one stop codon in each reading frame; or at least two heterologous introns interrupting the bacterial polypeptide coding sequence.

[0010] In some embodiments of any of the aspects, the at least one heterologous intron is adapted for splicing out of the bacterial polypeptide coding sequence in a fungal host cell.

[0011] In some embodiments of any of the aspects, the bacterial polypeptide coding sequence is codon-optimized for expression in a fungal host cell.

[0012] In some embodiments of any of the aspects, the transgene construct comprises regulatory elements permitting expression of the bacterial polypeptide coding sequence in a fungal host cell; optionally wherein the regulatory elements comprise yeast-derived expression control elements operably linked to the bacterial polypeptide coding sequence; optionally wherein the expression control elements comprise a yeast-derived promoter and / or yeast-derived secretion signal; optionally wherein the yeast-derived secretion signal comprises or is derived from the secretion signal of S. cerevisiae mating factor alpha.

[0013] In some embodiments of any of the aspects, the fungal host cell is a hemiascomycetous yeast; or wherein the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia. Debaryomyces. Candida, Yarrowia, Kazachslania, I.achancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyerozyma, Hanseniaspora, Rhizophagus, andOgataea, or wherein the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boiilardii. Saccharomyces unisporiis. Saccharomyces servazzii, Saccharomyces kluyveri, Kluyveromyces marxianus. Pichia angusla. Debaryomyces hansenii. Candida tropicalis. Yarrowia lipolylica. Kluyveromyces laclis. Candida albicans, Candida glabrala, Candida krusei, Candida auris, Lachancea thermololerans, Malassezia furfur, Malassezia globosa, Malassezia pachydermatis, Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis, Schizosaccharomyces pombe, Komagataella pastoris, Pichia guilliermondii, Meyerozyma gruessi, Hanseniaspora osmophila, and Rhizophagus irregularis.

[0014] In some embodiments of any of the aspects, the at least one heterologous intron comprises a 5' splice site, a branch site, and a 3' splice site, optionally wherein the 5' splice site, the branch site, and the 3' splice site are each selected from Table 1.

[0015] In some embodiments of any of the aspects, the 5' splice site comprises GTATGT, the branch site comprises TACTAAC, and the 3' splice site comprises YAG.

[0016] In some embodiments of any of the aspects, the at least one heterologous intron is about 40 nucleotides to about 80 nucleotides long; about 90 nucleotides to about 300 nucleotides long; or about 40 nucleotides to about 1000 nucleotides long.

[0017] In some embodiments of any of the aspects, the distance between the 5' splice site and the branch site is between about 20 nt to about 950 nt; and / or wherein the distance between the branch site and the 3' splice site is between about 1 nt to about 50 nt.

[0018] In some embodiments of any of the aspects, the 5' splice site, the branch site, and the 3' splice site are derived from the same species or source; and / or wherein the at least one heterologous intron is derived from a natural fungal intron sequence, is synthetic, or is chimeric.

[0019] In some embodiments of any of the aspects, the at least one heterologous intron comprises an intron from the Saccharomyces cerevisiae TEF4 gene or from the Saccharomyces cerevisiae EFB 1 gene. In some embodiments of any of the aspects, the at least one heterologous intron comprises an intron from the Saccharomyces cerevisiae TEF4 gene, from the Saccharomyces cerevisiae EFB 1 gene, or from the Saccharomyces cerevisiae UBC4 gene.

[0020] In some embodiments of any of the aspects, the location of the at least one heterologous intron within the bacterial polypeptide coding sequence is: (a) in the center of the open reading frame (ORF); (b) in the central portion that is 45% to 55% of the waythrough the ORF; (c) in the central portion that is 40% to 60% of the way through the ORF; (d) in the central portion that is 30% to 70% of the way through the ORF; or (e) in the central portion that is 25% to 75% of the way through the ORF. In some embodiments of any of the aspects, the location of the at least one heterologous intron within the bacterial polypeptide coding sequence is: (a) in the center of the open reading frame (ORF); (b) in the central portion that is 45% to 55% of the way through the ORF; (c) in the central portion that is 40% to 60% of the way through the ORF; (d) in the central portion that is 30% to 70% of the way through the ORF; (e) in the central portion that is 25% to 75% of the way through the ORF; or (f) in an active site codon of the ORF.

[0021] In some embodiments of any of the aspects, the at least one heterologous intron comprises at least one functional RNA element, optionally a small nucleolar RNA (snoRNA).

[0022] In some embodiments of any of the aspects, the transgene construct is comprised by a plasmid or vector.

[0023] In some embodiments of any of the aspects, the bacterial polypeptide encoded by the bacterial polypeptide coding sequence comprises an antibiotic resistance enzyme or an antibiotic-degrading enzyme, optionally wherein the antibiotic-degrading enzyme comprises a beta-lactamase enzyme; optionally wherein the beta-lactamase enzyme is a Temoneira-type (TEM) beta-lactamase (e.g., TEM1) or a cefotaximase-Munich-type (CTX-M) beta-lactamase (e.g., CTX-M-15).

[0024] In some embodiments of any of the aspects, the bacterial polypeptide encoded by the bacterial polypeptide coding sequence can confer an advantage if horizontally transmitted from the fungal host cell to a non-host bystander cell and expressed in the non-host bystander cell; optionally wherein the advantage comprises increased virulence and / or increased fitness; optionally wherein the increased fitness comprises increased nutritional capacity or increased adhesion to another cell; optionally wherein the nutritional capacity comprises utilization of alternative carbon sources and / or nitrogen sources.

[0025] In some embodiments of any of the aspects, the non-host bystander cell is a bacterial cell.

[0026] In some embodiments of any of the aspects, the transgene construct produces a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a fungal host cell; and / or the transgene construct does not produce a spliced mRNA encoding the full- length bacterial polypeptide when introduced to a bacterial cell.

[0027] In one aspect described herein is a system comprising at least two transgene constructs, the system comprising: (a) a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and (b) a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence; wherein one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence.

[0028] In some embodiments of any of the aspects, the first portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence; the second portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence; or the first and second portions of the bacterial polypeptide coding sequence are each interrupted by at least one heterologous intron sequence.

[0029] In some embodiments of any of the aspects, the first and second portions of the bacterial polypeptide coding sequence are spliced and translated into functional first and second portions of the bacterial polypeptide, optionally wherein the bacterial polypeptide is an antibiotic-degrading enzyme, optionally wherein the antibiotic-degrading enzyme is a beta-lactamase enzyme; optionally wherein the beta-lactamase enzyme is a Temoneira-type (TEM) beta-lactamase (e.g., TEM1) or a cefotaximase-Munich-type (CTX-M) beta-lactamase (e.g., CTX-M-15).

[0030] In one aspect described herein is at least one fungal host cell comprising a transgene construct as described herein or a system as described herein.

[0031] In some embodiments of any of the aspects, the fungal host cell is a probiotic cell.

[0032] In some embodiments of any of the aspects, the fungal host cell is a hemiascomycetous yeast; or wherein the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Debaryomyces, Candida, Yarrowia, Kazachslania, I.achancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyerozyma, Hanseniaspora, Rhizophagus, and Ogataea, or wherein the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces unisporus, Saccharomyces servazzii, Saccharomyces kluyveri, Kluyveromyces marxianus, Pichia angusta, Debaryomyces hansenii, Candida tropicalis, Yarrowia lipolytica, Kluyveromyces lactis, Candida albicans, Candida glabrata, Candida krusei, Candida auris, Lachancea thermotolerans, Malassezia furfur, Malassezia globosa, Malassezia pachydermatis, Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis, Schizosaccharomycespombe, Komagataella pastoris, Pichia guilliermondii, Meyerozyma gruessi, Hanseniaspora os mophila, and Rhizophagus irregularis.

[0033] In some embodiments of any of the aspects, the at least one fungal host cell is dried and viable.

[0034] In some embodiments of any of the aspects, a first fungal host cell comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and wherein a second fungal host cell comprises a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence; wherein one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence.

[0035] In some embodiments of any of the aspects, the first and second portions of the bacterial polypeptide coding sequence are spliced and translated into functional first and second portions of the bacterial polypeptide, optionally wherein the bacterial polypeptide is an antibiotic-degrading enzyme, optionally wherein the antibiotic-degrading enzyme is a beta-lactamase enzyme; optionally wherein the beta-lactamase enzyme is a Temoneira-type (TEM) beta-lactamase (e.g., TEM1) or a cefotaximase-Munich-type (CTX-M) beta-lactamase (e.g., CTX-M-15).

[0036] In one aspect described herein is a composition comprising at least one fungal host cell as described herein, formulated for delivery to a subject.

[0037] In one aspect described herein is a food composition comprising at least one fungal host cell as described herein.

[0038] In one aspect described herein is a medical food comprising at least one fungal host cell as described herein.

[0039] In one aspect described herein is a supplement comprising at least one fungal host cell as described herein.

[0040] In one aspect described herein is a probiotic composition comprising at least one fungal host cell as described herein.

[0041] In one aspect described herein is a pharmaceutical composition comprising at least one fungal host cell as described herein and a pharmaceutically acceptable carrier.

[0042] In one aspect described herein is a preparation comprising at least one fungal host cell as described herein and a carrier permitting application to a plant or to soil.

[0043] In one aspect described herein is a method of limiting or eliminating potential for horizontal transmission of a bacterial transgene from a fungal host cell to a bacterial cell, themethod comprising introducing at least one heterologous intron into the bacterial polypeptide coding sequence of the bacterial transgene.

[0044] In some embodiments of any of the aspects, the method limits or eliminates horizontal transmission of the bacterial transgene nucleic acid and / or the bacterial polypeptide coding sequence to bacterial cells.

[0045] In one aspect described herein is a method of preventing or treating a disease or infection in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one fungal host cell as described herein, a composition as described herein, a food composition as described herein, a medical food as described herein, a supplement as described herein, a probiotic composition as described herein, or a pharmaceutical composition as described herein.

[0046] In one aspect described herein is a method of promoting heath in a subject, the method comprising administering to the subject an effective amount of at least one fungal host cell as described herein, a composition as described herein, a food composition as described herein, a medical food as described herein, a supplement as described herein, a probiotic composition as described herein, or a pharmaceutical composition as described herein.

[0047] In some embodiments of any of the aspects, the at least one fungal host cell is an agent for bio-control, bio-stimulation, and / or bio-nutrition in the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Fig. 1 shows a map of the pRGsb8 plasmid encoding a Saccharomyces -restricted expression cassette of the TEM1 P-lactamase.

[0049] Fig. 2 shows detection of P-lactamase activity in the engineered Saccharomyces cerevisiae iTEMl using a nitrocefin hydrolysis assay. Increased absorbance at 486 nm indicates activity of the enzyme in the culture supernatant. No P-lactamase activity is detected in the wild type strain of S. cerevisiae or in an E. coli strain that carries the TEM1 expression plasmid.

[0050] Fig. 3 shows detection of P-lactamase activity in the fecal pellets of mice dosed with S. boulardii iTEMl. S. boulardii iTEMl n=4 mice. S. boulardii control n=4 mice.

[0051] Fig. 4 shows the impact of yeast introns on the expression of functional payloads in yeast and bacteria. The transgene constructs were tested to determine if they were fully functional and fully inactive across species. The boxes represent expression levels forconstructs harboring one or two fungal introns (TEFlint ± EFBlint) in the Venus open reading frame, at positions specified in the key. The top-down order of the key corresponds to the left-right order of the bars in both tested host micro-organisms. Fluorescence was normalized to optical density, and the signals were linearly adjusted relative to the positive (wild-type, green) and negative (non fluorescent AY66 mutant, grey) controls. Box-and- whisker plot presents n=4 biological replicates (individual clones) for each condition. Results were analyzed with an ANOVA followed by post-hoc pairwise comparisons (Tukey); ns: non significant; ****: P-value < 0.001.DETAILED DESCRIPTION

[0052] The technology described herein is directed to transgene constructs that use at least one intron to provide interspecies biocontainment. The transgene construct can comprise a polypeptide coding sequence interrupted by at least one heterologous intron sequence. While transgenes comprising a bacterial polypeptide coding sequence are described herein, it is contemplated herein that a polypeptide coding sequence from archaea or eukarya (e.g., plant, fungal, animal) or a synthetic polypeptide coding sequence can also be used. As such, the term “bacterial polypeptide coding sequence” used throughout is merely exemplary and can be replaced with “archaea polypeptide coding sequence”, “eukaryotic polypeptide coding sequence”, “plant polypeptide coding sequence”, “fungal polypeptide coding sequence”, “animal polypeptide coding sequence”, and / or “synthetic polypeptide coding sequence”.

[0053] In exemplary embodiments, a transgene construct as described herein can prevent or reduce horizontal transmission of the bacterial polypeptide from a fungal host cell to a bacterial cell. Also described herein are nucleic acids, pharmaceutical compositions, and preparations comprising such transgene constructs. Method of uses of such transgene constructs and associated compositions are also disclosed herein. Non-limiting examples of such bacterial polypeptides, for which interspecies biocontainment is desirable, include any bacterial polypeptides that can confer an advantage to a non-host bystander (e.g., bacterial) cell if horizontally transmitted, such as increased virulence (e.g., virulence factors) and / or increased fitness (e.g., increased nutritional capacity, adhesion factors, antibiotic resistance enzymes, and / or antibiotic-degrading enzymes) in the bacterium.Transgene Construct

[0054] Described herein in multiple aspects are transgene constructs. As used herein, the term “transgene” refers to a gene which is artificially introduced into the genome of anotherorganism. In some embodiments, a bacterial gene is artificially introduced into the genome of a fungal host cell. In one aspect, described herein is a transgene construct comprising a bacterial polypeptide coding sequence, wherein the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence. In some aspects, the transgene construct is a fungal transgene construct. In one aspect, described herein is a transgene construct comprising a bacterial polypeptide coding sequence, wherein the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence, further comprising regulatory elements permitting expression of the bacterial polypeptide coding sequence in a fungal host cell.

[0055] In some embodiments, the transgene construct produces a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a fungal host cell. In some embodiments, the transgene construct does not produce a spliced mRNA encoding the full- length bacterial polypeptide when introduced to a bacterial cell. In some embodiments, the transgene construct produces a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a fungal host cell, and in addition the transgene construct does not produce a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a bacterial cell.Introns

[0056] Described herein are transgene constructs comprising at least one heterologous intron sequence. In some embodiments the intron is heterologous to the bacterial polypeptide coding sequence. As used herein, the term “heterologous” refers to that which is not endogenous to, or naturally occurring in, a cell, such as the bacterial cell. For example, a heterologous intron sequence of the present disclosure can be derived from a fungal species, such as a yeast species, non-limiting examples of which are provided herein. Where bacterial genes do not comprise introns, the term “heterologous intron” can be used interchangeably with “engineered intron” or “intron” or “fungal intron” or “yeast intron” and the like.

[0057] In some embodiments, the heterologous intron comprises at least one in-frame stop codon. In some embodiments, the heterologous intron comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more in-frame stop codons. A stop codon is a sequence of three nucleotides (a trinucleotide) in DNA or messenger RNA (mRNA) that signals a halt to protein synthesis in the cell. In some embodiments, the stop codon is TAA, TAG, or TGA. As used herein, the term “inframe” refers to the stop codon being in the same reading frame as the portion of the bacterial polypeptide coding sequence that is 5' from the stop codon. If the intron is retained in thetransgene construct, translation of the nucleic acid comprising the bacterial polypeptide coding sequence interrupted by at least one heterologous intron results in early termination of the bacterial polypeptide and a non-functional or reduced function bacterial polypeptide.

[0058] In some embodiments, the heterologous intron comprises at least one stop codon in each of a plurality of reading frames. In some embodiments, the heterologous intron comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more stop codons in each of a plurality of reading frames. In some embodiments, the heterologous intron comprises at least one stop codon in each reading frame. In some embodiments, the heterologous intron comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more stop codons in each reading frame. In some embodiments, the heterologous intron comprises at least one stop codon in the first forward reading frame, the second forward reading frame, and the third forward reading frame.

[0059] In some embodiments, the transgene construct comprises at least two heterologous introns interrupting the bacterial polypeptide coding sequence. In some embodiments, the transgene construct comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous introns interrupting the bacterial polypeptide coding sequence. In some embodiments, the transgene construct comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more heterologous introns interrupting the bacterial polypeptide coding sequence. In some embodiments, the transgene construct comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 heterologous introns interrupting the bacterial polypeptide coding sequence. In some embodiments, the at least two heterologous introns can each be the same heterologous intron. In some embodiments, at least one of the heterologous introns is different from another heterologous intron in the same gene or construct.

[0060] In some embodiments, the at least one heterologous intron is adapted for splicing out of the bacterial polypeptide coding sequence in a fungal host cell. In some embodiments, the at least one heterologous intron comprises (e.g., from 5' to 3') a 5' splice site, a branch site, and a 3' splice site. The 5' splice site can also be referred to as the 5' donor site (5'SS). The branch site (BS) can also be referred to as a branch potin (BP). The 3' splice site can also be referred to as the 3' acceptor site (3'SS).

[0061] Introns are removed by a spliceosome that catalyzes two stepwise transesterification reactions. The 5' splice-site (5' SS), 3' splice-site (3' SS), and branch point (BP) sequences directly base pair to partially complementary sequences within the spliceosome small nuclear RNAs (snRNAs). In the first transesterification reaction, the 5' splice site is cleaved and theintron 5' end is ligated to the branch site. In the second transesterification reaction, cleavage of the 3' splice site releases the intron as a lariat structure, and the 5' and 3' exons are ligated. For more details about yeast splicing, see e.g., Bon et al. “Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns,” Nucleic Acids Research, 2003, Vol. 31, No. 4 1121-1135; Schirman et al. “A broad analysis of splicing regulation in yeast using a large library of synthetic introns,” PLoS Genet 17(9): el009805 (2021); Gildea et al. “Transcript-specific determinants of pre-mRNA splicing revealed through in vivo kinetic analyses of the 1st and 2nd chemical steps,” Molecular Cell 82, 2967-2981, 2022; the contents of each of which is incorporated herein by reference in its entirety.

[0062] In some embodiments, the 5' splice site, the branch site, and the 3' splice site are each selected from Table 1. Any combination of 5' splice site, branch site, and 3' splice site are each selected from Table 1 for their ability to promote splicing in a fungal host cell. In some embodiments, 5' splice site, branch site, and 3' splice site are selected from the same strain, same species, or same genus of fungal host cell. In some embodiments, the 5' splice site comprises GTATGT. In some embodiments, the branch site comprises TACTAAC. In some embodiments, the 3' splice site comprises YAG. In some embodiments, the 5' splice site comprises GTATGT, the branch site comprises TACTAAC, and the 3' splice site comprises YAG. In some embodiments, the intron starts with GT and ends with AG. In some embodiments, the intron comprises a 5' splice-site (5'SS) starting with GT and a 3' splice-site (3' SS) ending with AG.

[0063] Table 1: Exemplary yeast introns; adapted from Bon et al. 2003, supra, Schirman et al. 2021, supra), the content of each of which is incorporated herein by reference in its entirety.

[0064] In some embodiments, the heterologous intron is about 40 nucleotides (nt) to about 300 nucleotides long. In some embodiments, the heterologous intron is about 40 nucleotides to about 80 nucleotides long. In some embodiments, the heterologous intron is about 90 nucleotides to about 300 nucleotides long. In some embodiments, the heterologous intron is about 40 nt, about 50 nt, about 60 nt, about 70 nt, about 80 nt, about 90 nt, about 100 nt, about 110 nt, about 120 nt, about 130 nt, about 140 nt, about 150 nt, about 160 nt, about 170 nt, about 180 nt, about 190 nt, about 200 nt, about 210 nt, about 220 nt, about 230 nt, about 240 nt, about 250 nt, about 260 nt, about 270 nt, about 280 nt, about 290 nt, about 300 nt long, about 350 nt, about 400 nt, about 450 nt, about 500 nt, about 550 nt, about 600 nt, about 650 nt, about 700 nt, about 750 nt, about 800 nt, about 850 nt, about 900 nt, about 950 nt, or about 1000 nt.

[0065] In some embodiments, the heterologous intron is at least 40 nt, at least 50 nt, at least 60 nt, at least 70 nt, at least 80 nt, at least 90 nt, at least 100 nt, at least 110 nt, at least 120 nt, at least 130 nt, at least 140 nt, at least 150 nt, at least 160 nt, at least 170 nt, at least 180 nt, at least 190 nt, at least 200 nt, at least 210 nt, at least 220 nt, at least 230 nt, at least 240 nt, at least 250 nt, at least 260 nt, at least 270 nt, at least 280 nt, at least 290 nt, at least 300 nt long, at least 350 nt, at least 400 nt, at least 450 nt, at least 500 nt, at least 550 nt, at least 600 nt, at least 650 nt, at least 700 nt, at least 750 nt, at least 800 nt, at least 850 nt, at least 900 nt, at least 950 nt, or at least 1000 nt.

[0066] In some embodiments, the heterologous intron is at most 40 nt, at most 50 nt, at most 60 nt, at most 70 nt, at most 80 nt, at most 90 nt, at most 100 nt, at most 110 nt, at most 120 nt, at most 130 nt, at most 140 nt, at most 150 nt, at most 160 nt, at most 170 nt, at most 180 nt, at most 190 nt, at most 200 nt, at most 210 nt, at most 220 nt, at most 230 nt, at most 240 nt, at most 250 nt, at most 260 nt, at most 270 nt, at most 280 nt, at most 290 nt, at most 300nt long, at most 350 nt, at most 400 nt, at most 450 nt, at most 500 nt, at most 550 nt, at most 600 nt, at most 650 nt, at most 700 nt, at most 750 nt, at most 800 nt, at most 850 nt, at most 900 nt, at most 950 nt, or at most 1000 nt.

[0067] In some embodiments, the distance between the 5' splice site and the branch site is between about 20 nt to about 950 nt. In some embodiments, the distance between the 5' splice site and the branch site is about 20 nt, about 30 nt, about 40 nt, about 50 nt, about 60 nt, about 70 nt, about 80 nt, about 90 nt, about 100 nt, about 110 nt, about 120 nt, about 130 nt, about 140 nt, about 150 nt, about 160 nt, about 170 nt, about 180 nt, about 190 nt, about 200 nt, about 210 nt, about 220 nt, about 230 nt, about 240 nt, about 250 nt, about 260 nt, about 270 nt, about 280 nt, about 290 nt, about 300 nt long, about 350 nt, about 400 nt, about 450 nt, about 500 nt, about 550 nt, about 600 nt, about 650 nt, about 700 nt, about 750 nt, about 800 nt, about 850 nt, about 900 nt, about 950 nt, or about 1000 nt.

[0068] In some embodiments, the distance between the branch site and the 3' splice site is between about 1 nt to about 50 nt. In some embodiments, the distance between the branch site and the 3' splice site is about 1 nt, about 2 nt, about 3 nt, about 4 nt, about 5 nt, about 6 nt, about 7 nt, about 8 nt, about 9 nt, about 10 nt, about 11 nt, about 12 nt, about 13 nt, about 14 nt, about 15 nt, about 16 nt, about 17 nt, about 18 nt, about 19 nt, about 20 nt, about 21 nt, about 22 nt, about 23 nt, about 24 nt, about 25 nt, about 26 nt, about 27 nt, about 28 nt, about 29 nt, about 30 nt, about 31 nt, about 32 nt, about 33 nt, about 34 nt, about 35 nt, about 36 nt, about 37 nt, about 38 nt, about 39 nt, about 40 nt, about 41 nt, about 42 nt, about 43 nt, about 44 nt, about 45 nt, about 46 nt, about 47 nt, about 48 nt, about 49 nt, or about 50 nt.

[0069] In some embodiments, the 5' splice site, the branch site, and the 3' splice site are derived from the same species (e.g., a fungal species, e.g., a yeast species) or source (e.g., synthetic). In some embodiments, at least one of the 5' splice site, the branch site, and the 3' splice site are derived from a different species or source than the other(s).

[0070] In some embodiments, the heterologous intron sequence is derived from a natural fungal intron sequence, for example a natural yeast intron sequence. In some embodiments, the heterologous intron sequence is derived from a natural yeast intron sequence selected from the group of species consisting of Saccharomyces cerevisiae. Saccharomyces boiilardii. Saccharomyces unisporiis. Saccharomyces servazzii (also referred to as Kazachstania servazzii), Saccharomyces kluyveri (also referred to as Lachancea khiyveri). Kluyveromyces marxianus. Pichia angusta (also referred to as Hansenula polymorpha or Ogataea polymorpha), Debaryomyces hansenii (also referred to as Candida famala). Candidatropicalis, and Yarrowia lipolytica. In some embodiments, the heterologous intron sequence is synthetic. In some embodiments, the heterologous intron sequence is chimeric, e.g., at least one portion is naturally derived and at least one portion is synthetic.

[0071] While any intron including sequences recognized by fungal splicing machinery and sufficient for splicing in a given fungal host species can be used, in some embodiments, the heterologous intron comprises an intron from the Saccharomyces cerevisiae TEF4 gene (any of the following names can be used interchangeably: formal name: TEF1; systematic name: YPR080W; common unofficial names: eEFla, EF-1 alpha).

[0072] In some embodiments, the intron from the Saccharomyces cerevisiae TEF4 gene comprises SEQ ID NO: 1. In some embodiments, the heterologous intron comprises SEQ ID NO: 1 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 1, that maintains its function (e.g., splicing in a fungal host cell).

[0073] SEQ ID NO: 1, intron from the Saccharomyces cerevisiae TEF4 gene (snoRNA SNR38 bolded, see e.g., SEQ ID NO: 3; the 5' splice site (GTATGT), the branch site (TACTAAC), and the 3' splice site (TAG) are each italicized) G 4ZG7TCCATATTTTATTTTAACACTTCCACATACATTGTTTTGCGCGTTTCCCGT TCGTTTATTTGGCACGTCATTTTTCTTCGAAACATAATGATGAAAAAAAATTTTA TCAAACAGTTATCCCTGTCTGAATGGGTAATAATAGGTAACCTCTCATATGT TGATATTTGTATTTCTGATATGTTTCTTAAAGAAAAATGAAAGTCAAAATAACA AAAAGGAAGTACAAAGCGTGTCCTAATCCAGGAAAAAATATAAAGGATTGTTT7 ztCZzL4CATTTCTTCAATTAATGGTTGTATAACCATCGAGATGATGTATATTZztG

[0074] In some embodiments, the heterologous intron comprises an intron from the Saccharomyces cerevisiae EFBI gene (any of the following names can be used interchangeably: formal name: EFBI; systematic name: YAL003W; common unofficial names: EFIB, eEFIBalpha, EF-1 beta, TEF5). In some embodiments, the intron from the Saccharomyces cerevisiae EFBI gene comprises SEQ ID NO: 2. In some embodiments, the heterologous intron comprises SEQ ID NO: 2 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 2, that maintains its function (e.g., splicing in a fungal host cell).

[0075] SEQ ID NO: 2, intron from the Saccharomyces cerevisiae EFB 1 gene (snoRNA SNR18 bolded see e.g., SEQ ID NO: 4; the 5' splice site (GTATGT), the branch site (TACTAAC), and the 3' splice site (TAG) are each italicized) G 47U7TCCGATTTAGTTTACTTTATAGATCGTTGTTTTTCTTTCTTTTTTTTTTTTC CTATGGTTACATGTAAAGGGAAGTTAACTAATAATGATTACTTTTTTTCGCTTATG TGAATGATGAATTTAATTCTTTGGTCCGTGTTTATGATGGGAAGTAAGACCC CCGATATGAGTGACAAAAGAGATGTGGTTGACTATCACAGTATCTGACGATA GCACAGAGCAGAGTATCATTATTAGTTATCTGTTATTTTTTTTTCCTTTTTTGTTCA AAAAAAGAAAGACAGAGTCTAAAGATTGCATTACAAGAAAAAAGTTCTCATZ4C 7A4CAAGCAAAATGTTTTGTTTCTCCTTTTAAAA 4G

[0076] In some embodiments, the heterologous intron comprises an intron from the Saccharomyces cerevisiae UBC4 gene (any of the following names can be used interchangeably: formal name: UBC4; systematic name: YBR082C). In some embodiments, the intron from the Saccharomyces cerevisiae UBC4 gene comprises SEQ ID NO: 6. In some embodiments, the heterologous intron comprises SEQ ID NO: 6 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 6, that maintains its function (e.g., splicing in a fungal host cell).

[0077] SEQ ID NO: 6, intron from the Saccharomyces cerevisiae UBC4 gene (the 5' splice site (GTATGT), the branch site (TACTAAC), and the 3' splice site (TAG) are each italicized) G 4ZGZCTAAAGTTATGGCCACGTTTCAAATGCGTGCTTTTTTTTTAAAACTTATG CTCTTATT 4C7A4CAAAATCAACATGCTATTGAAC 4G

[0078] In some embodiments, the position of the at least one heterologous intron in the polypeptide coding sequence is such that, in a bystander species that cannot splice out the at least one heterologous intron, translation of the coding sequence 5’ or 3’ of the at least one inserted, non-spliced heterologous intron does not produce a polypeptide with the biological function of the polypeptide produced in a species that can splice out the at least one heterologous intron (e.g., a fungal host species). Thus, any such partial translation product of the polypeptide coding sequence does not confer an advantage to the bystander species upon horizontal transmission.

[0079] In some embodiments, the at least one heterologous intron is located in the center of the open reading frame of the bacterial polypeptide coding sequence; in other words, the at least one heterologous intron is equidistant or approximately equidistant from the 5’ and 3’ ends of the ORF. In some embodiments, the at least one heterologous intron is located in the central portion that is 45% to 55% of the way through the ORF; for example, in an ORF that is 100 nucleotides long, the at least one heterologous intron is located between nucleotides 45 and 55 of the ORF. In some embodiments, the at least one heterologous intron is located in the central portion that is 40% to 60% of the way through the ORF. In some embodiments, the at least one heterologous intron is located in the central portion that is 30% to 70% of the way through the ORF. In some embodiments, the at least one heterologous intron is located in the central portion that is 25% to 75% of the way through the ORF.

[0080] In some embodiments, the at least one heterologous intron interrupts an active site codon of the bacterial polypeptide coding sequence; in other words, the at least one heterologous intron is positioned in the bacterial polypeptide coding sequence such that neither the 5’ and 3’ portions of the ORF contain an active site amino acid residue when translated. As used herein, the term “active site” or “catalytic site” are used interchangeably herein and refer to the at least one amino acid in an enzyme where a substrate of the enzyme binds and / or undergoes a chemical reaction; for example, an active site can include amino acid residues that stabilize substrate (e.g., by ionic bonds) without chemically reacting with them.

[0081] In some embodiments, at least one heterologous intron (e.g., Saccharomyces cerevisiae TEF4, EFBI, and / or UBC4 intron(s)) interrupts the sequence of TEM1 betalactamase (see e.g., SEQ ID NOs: 7-10). In some embodiments, at least one heterologous intron (e.g., Saccharomyces cerevisiae TEF4, EFBI, and / or UBC4 intron(s)) interrupts an active site codon of TEM1 beta-lactamase (see e.g., SEQ ID NOs: 7-10). In some embodiments, the nucleic acid sequence of a heterologous intron interrupting an active site residue in an enzyme comprises SEQ ID NO: 7 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 7, that maintains its function (e.g., splicing in a fungal host cell).

[0082] In some embodiments, the amino acid sequence of TEM1 beta-lactamase with a yeast signal peptide comprises SEQ ID NO: 8 or an amino acid sequence that is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 8, that maintains its function (e.g., beta-lactamase activity).

[0083] In some embodiments, the amino acid sequence of an inactive N-terminal portion of TEM1 beta-lactamase (e.g., when the heterologous intron in SEQ ID NO: 7 is not spliced out) with a yeast signal peptide comprises SEQ ID NO: 9 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 9, that maintains its lack function (e.g., no beta-lactamase activity).

[0084] In some embodiments, the amino acid sequence of an inactive C-terminal portion of TEM1 beta-lactamase (e.g., when the heterologous intron in SEQ ID NO: 7 is not spliced out) comprises SEQ ID NO: 10 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 10, that maintains its lack of function (e.g., no beta-lactamase activity).

[0085] SEQ ID NO: 7, DNA sequence of a heterologous intron (UBC4) interrupting an active site residue in an enzyme (TEM1 beta-lactamase); double-underlined: intron from S. cerevisiae UBC4 (systematic name YBR082C); uppercase: TEM1 beta-lactamase, soluble form + interrupting intron; bold: interrupted catalytic site Asnl70 (standard Ambler numbering) codon; lowercase: yeast signal peptide: atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggc acaaattcctgctgaagctgtcatcggttacttagatttagaaggagatttcgatgttgctgttttgccattttccaactctacaaataacggg ttattgtttataaatactactattgcctcaattgctgctaaagaagaaggtgtatctttggataaaagagaggctgaagctATGCATC CAGAAACCTTAGTCAAAGTCAAGGATGCAGAAGATCAATTAGGAGCAAGAGTCG GTTATATTGAGTTAGACTTGAATAGTGGTAAAATCTTAGAATCATTTAGACCAGA AGAGAGATTCCCCATGATGTCTACATTCAAAGTGTTATTGTGTGGTGCAGTGTTG TCAAGAGTTGACGCTGGCCAAGAACAATTAGGTAGGAGAATCCACTATTCTCAA AATGATTTAGTCGAATATTCACCAGTGACAGAAAAACATTTAACAGATGGAATG ACTGTTAGAGAATTGTGCTCCGCCGCTATCACTATGTCAGACAATACTGCAGCAA ACCTGCTTTTGACCACTATCGGAGGTCCCAAAGAATTAACCGCATTCTTGCACAA CATGGGTGACCACGTAACTAGATTAGACAGATGGGAACCAGAATTGAAGTATGT CTAAAGTTATGGCCACGTTTCAAATGCGTGCTTTTTTTTTAAAACTTATGCTCTTATTTACTAACAAAATCAACATGCTATTGAACTACTCCTACTCTCTATCCCTAACCTATCTAA AGGGACACCACCATGCCCGCTGCTATGGCAACTACCTTGAGAAAGTTGTTAACC GGAGAGTTATTAACATTAGCTTCAAGGCAGCAGTTAATAGATTGGATGGAAGCC GACAAAGTAGCTGGACCCTTATTGAGATCCGCTTTACCCGCTGGCTGGTTCATCG CCGACAAATCAGGCGCTGGCGAAAGAGGCTCAAGAGGAATCATTGCTGCTTTGG GTCCAGATGGTAAACCCAGTAGGATTGTCGTTATCTACACCACCGGCTCTCAAGC AACTATGGATGAAAGAAATAGACAGATCGCTGAAATAGGCGCCTCCTTAATTAA ACATTGGTAA

[0086] SEQ ID NO: 8, polypeptide sequence of a yeast-secreted Temoneira 1 (TEM1) betalactamase after correct splicing of a heterologous intron (e.g., UBC4), yielding a functional enzyme; see e.g., SEQ ID NO: 7; original yeast-secreted TEM1 sequence (as translated if the intron was spliced out, resulting in TEM1 beta-lactamase with an active catalytic site); double underlined: catalytic site Asnl70 (standard Ambler numbering) codon; lowercase: yeast signal peptide; uppercase: TEM1 beta-lactamase, soluble form: mrfpsiftavlfaassalaapvntttedetaqipaeavigyldlegdfdvavlpfsnstnngllfinttiasiaakeegvsldkreaeaM HPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLS RVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLL LTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLT GELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPD GKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW

[0087] SEQ ID NO: 9, N-terminal polypeptide half produced by the intron-interrupted sequence of SEQ ID NO: 7 (as translated if the intron was not spliced out, resulting in N- terminal and C-terminal portions of TEM1 beta-lactamase, both lacking an active catalytic site); double underlined: non-asparagine amino acid, the codon created by inserting the intron encodes a lysine where the asparagine (catalytic site Asnl70) should be; bold: extraneous amino acids introduced by the intron: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNST NNGLLFINTTIASIAAKEEGVSLDKREAEAMHPETLVKVKDAEDQLGARVGYIELDL NSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPV TEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR WEPELKYV

[0088] SEQ ID NO: 10, C-terminal polypeptide half produced by the intron-interrupted sequence of SEQ ID NO: 7 (assuming a productive translation initiation despite the absenceof methionine codon); double underlined: Non-asparagine amino acid, the codon created by inserting the intron encodes a serine where the asparagine (catalytic site Asnl70) should be; bold: extraneous amino acids from the intron:TSEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA SL1KHW

[0089] In some embodiments, at least one heterologous intron (e.g., Saccharomyces cerevisiae TEF4, EFBI, and / or UBC4 intron(s)) interrupts the sequence of an extended- spectrum beta-lactamase (ESBL; e.g., cefotaximase-Munich (CTX-M) beta-lactamases, e.g., CTX-M15) (see e.g., SEQ ID NOs: 11-12). In some embodiments, the nucleic acid sequence of a heterologous intron interrupting the ORF of an enzyme comprises SEQ ID NO: 11 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 11, that maintains its function (e.g., splicing in a fungal host cell).

[0090] In some embodiments, the amino acid sequence of CTX-M15 beta-lactamase with a yeast signal peptide comprises SEQ ID NO: 12 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 12, that maintains its function (e.g., betalactamase activity).

[0091] SEQ ID NO: 11, DNA sequence of a heterologous intron (UBC4) interrupting an enzyme (ESBL beta-lactamase); double-underlined: intron from S. cerevisiae UBC4 (systematic name YBR082C); uppercase: CTX-M15 beta-lactamase, soluble form; lowercase: yeast signal peptide: atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggc acaaattcctgctgaagctgtcatcggttacttagatttagaaggagatttcgatgttgctgttttgccattttccaactctacaaataacggg ttattgtttataaatactactattgcctcaattgctgctaaagaagaaggtgtatctttggataaaagagaggctgaagctATGCAAA CTGCTGATGTCCAACAGAAGCTGGCAGAACTAGAGCGTCAGAGTGGGGGGCGTT TAGGGGTCGCTTTAATCAACACGGCAGACAATTCCCAAATTCTGTATAGGGCGG ACGAGAGATTTGCTATGTGCAGGTATGTCTAAAGTTATGGCCACGTTTCAAATGC GTGCTTTTTTTTTAAAACTTATGCTCTTATTTACTAACAAAATCAACATGCTATTG AACTAGCACCAGCAAGGTTATGGCGGCGGCGGCCGTACTTAAAAAGTCTGAGTCCGAACCAAACCTGTTGAATCAGAGGGTCGAGATTAAAAAGTCTGACCTAGTCAA TTATAACCCAATAGCGGAAAAGCACGTAAATGGGACAATGTCATTGGCCGAGCT GTCTGCCGCCGCATTGCAATACAGCGATAACGTTGCAATGAATAAGTTAATCGCC CATGTGGGAGGACCTGCCAGTGTAACCGCTTTCGCGAGACAATTAGGGGATGAA ACCTTCCGTCTGGACCGTACGGAGCCTACTTTAAACACGGCGATACCTGGTGATC CTCGTGATACTACCTCCCCCAGGGCTATGGCACAGACTCTGCGTAACCTTACTCT AGGCAAAGCCTTAGGCGATTCCCAAAGAGCACAGCTAGTGACTTGGATGAAGGG CAACACAACAGGTGCAGCCTCCATCCAGGCTGGACTTCCGGCTTCCTGGGTAGTT GGGGATAAGACGGGTTCAGGCGGTTATGGTACAACGAATGATATAGCTGTGATC TGGCCTAAGGACCGTGCGCCCCTAATCTTAGTCACGTATTTCACACAACCCCAAC CGAAGGCTGAATCCAGAAGGGATGTTCTTGCTAGCGCAGCGAAAATAGTGACAG ATGGTCTATAA

[0092] SEQ ID NO: 12, polypeptide sequence of a yeast-secreted ESBL Beta-lactamase after correct splicing of a heterologous intron (e.g., UBC4), yielding a functional enzyme; see e.g., SEQ ID NO: 11; original yeast-secreted CTX-M15 sequence (as translated if the intron was spliced out, resulting in active ESBL Beta-lactamase); lowercase: yeast signal peptide; uppercase: CTX-M15 beta-lactamase, soluble form: mrfpsiftavlfaassalaapvntttedetaqipaeavigyldlegdfdvavlpfsnstnngllfinttiasiaakeegvsldkreaeaM QTADVQQKLAELERQSGGRLGVALINTADNSQILYRADERFAMCSTSKVMAAAAVL KKSESEPNLLNQRVEIKKSDLVNYNPIAEKHVNGTMSLAELSAAALQYSDNVAMNK LIAHVGGPASVTAFARQLGDETFRLDRTEPTLNTAIPGDPRDTTSPRAMAQTLRNLTL GKALGDSQRAQLVTWMKGNTTGAASIQAGLPASWVVGDKTGSGGYGTTNDIAVIW PKDRAPLILVTYFTQPQPKAESRRDVLASAAKIVTDG

[0093] In some embodiments, the at least one heterologous intron is within a 5’ region of the bacterial polypeptide coding sequence. In some embodiments, the at least one heterologous intron is within a 3’ region of the bacterial polypeptide coding sequence. In some embodiments, the 5’ end of the at least one heterologous intron is within 350 nucleotides (nt) of the 5’ start of the bacterial polypeptide coding sequence. In some embodiments, the 5’ end of the at least one heterologous intron is within 350 nucleotides (nt) of the 3’ end of the bacterial polypeptide coding sequence. In some embodiments, the 5’ end of the at least one heterologous intron is about 50 nt, about 100 nt, about 150 nt, about 200 nt, about 250 nt, about 300 nt, about 350 nt from the 5’ start and / or 3’ end of the bacterial polypeptide coding sequence. In some embodiments, the 5’ end of the at least one heterologous intron is at least50 nt, at least 100 nt, at least 150 nt, at least 200 nt, at least 250 nt, at least 300 nt, at least 350 nt from the 5’ start and / or 3’ end of the bacterial polypeptide coding sequence. In some embodiments, the 5’ end of the at most one heterologous intron is at most 50 nt, at most 100 nt, at most 150 nt, at most 200 nt, at most 250 nt, at most 300 nt, at most 350 nt from the 5’ start and / or 3’ end of the bacterial polypeptide coding sequence. In some embodiments, the 5’ end of the at least one heterologous intron is at most 50 nt, at most 100 nt, at most 150 nt, at most 200 nt, at most 250 nt, at most 300 nt, at most 350 nt from the 5’ start and / or 3’ end of the bacterial polypeptide coding sequence.

[0094] In some embodiments, the at least one heterologous intron comprises at least one functional RNA element. In some embodiments, the at least one functional RNA element comprises a small nucleolar RNA (snoRNA). In some embodiments, the at least one heterologous intron comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more snoRNAs. In some embodiments, the snoRNA is a U24 or U18 snoRNA, for example from Saccharomyces cerevisiae. In some embodiments, snoRNAs assemble into small ribonucleoprotein particles (snoRNPs), which are active in rRNA processing and modification and in ribosome assembly. For more details about snoRNAs comprised by yeast introns, see e.g., Vincenti et al. “The position of yeast snoRNA-coding regions within host introns is essential for their biosynthesis and for efficient splicing of the host pre-mRNA,” RNA (2007), 13: 138-150; Ooi et al. “Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariatdebranching enzyme: Intron length effects and activity of a precursor snoRNA,” RNA (1998), 4: 1096-1110; the contents of each of which is incorporated herein by reference in its entirety.

[0095] In some embodiments, the snoRNA comprises SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 3 or SEQ ID NO: 4, that maintains its function (e.g., snoRNP, rRNA processing and modification, ribosome assembly).

[0096] SEQ ID NO: 3, snoRNA SNR38 from the intron in the Saccharomyces cerevisiae TEF4 gene (see e g., SEQ ID NO: 1) CATAATGATGAAAAAAAATTTTATCAAACAGTTATCCCTGTCTGAATGGGTAATA ATAGGTAACCTCTCATATGTTGATATTTGTATTTCTGATA

[0097] SEQ ID NO: 4, snoRNA SNR 18 from the intron in the Saccharomyces cerevisiae EFBI gene (see e.g., SEQ ID NO: 2) GTGAATGATGAATTTAATTCTTTGGTCCGTGTTTATGATGGGAAGTAAGACCCCC GATATGAGTGACAAAAGAGATGTGGTTGACTATCACAGTATCTGACG

[0098] In some embodiments, the at least one heterologous intron provides at least one benefit to the fungal host cell, including but not limited to increased expression of the bacterial polypeptide coding sequence. In some embodiments, the at least one heterologous intron comprises at least one sequence coding for a functional polypeptide. As a non-limiting example, fungal introns can comprise at least one sequence coding for a functional polypeptide, such as the S. cerevisiae homing endonuclease Seel. In some embodiments, the at least one heterologous intron comprises at least one synthetic RNA structure, including but not limited to an RNA switch or a riboswitch. In some embodiments, the RNA switch can turn on or off expression (e.g., transcription, translation) of the RNA comprising it, depending on the presence or absence of at least one environmental cue or compound; see e.g., US Patent 10494646, the contents of which are incorporated herein by reference in their entirety.Polypeptides

[0099] As described herein, the transgene construct comprises a polypeptide coding sequence interrupted by at least one heterologous intron sequence. In some embodiments, the polypeptide coding sequence is derived from prokarya, archaea, or eukarya. In some embodiments, the polypeptide coding sequence is bacterial, plant, fungal, animal, and / or synthetic in origin.

[0100] In some embodiments, the polypeptide coding sequence is non-bacterial or non- prokaryotic in origin. A transgene of interest that comes from the two non-bacterial domains of life (e.g., archaea or eukarya) or that is synthetic can have adverse effects if transferred to an unintended bystander cell (e.g., a bacterium). For example, sweet protein genes are derived from plants, GFP is derived from jellyfish, and the Venus protein is synthetic. It is contemplated herein that such gene products can give an unwanted advantage if transferred to an unintended bystander cell (e.g., a bacterium). For example, a transgene related to the metabolism of a given substrate can change cellular physiology if transferred to a bystander cell. Therefore, the transgenes described herein prevent a transgene (of any origin) from being transferred to other organisms in any domain of life that cannot splice the at least one heterologous (e.g., yeast) intron. In some embodiments of any of the aspects, the bystandercell which cannot splice the heterologous intron out of the polypeptide sequence include all bacteria, all archaea, and any species that are phylogenetically distant from the species from which at least one of the heterologous introns (or intron motifs, e.g., 5' splice site, branch site, and / or 3' splice site) was derived.

[0101] As such, the term “bacterial polypeptide coding sequence” used throughout is merely exemplary and can be replaced with “archaea polypeptide coding sequence”, “eukaryotic polypeptide coding sequence”, “plant polypeptide coding sequence”, “fungal polypeptide coding sequence”, “animal polypeptide coding sequence”, and / or “synthetic polypeptide coding sequence”.

[0102] In non-limiting embodiments, the transgene construct comprises a bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence. In some embodiments, the bacterial polypeptide coding sequence is from or derived from any bacterial species. In some embodiments, the bacterial polypeptide coding sequence is from or derived from a probiotic bacterial species. In some embodiments, the bacterial polypeptide coding sequence is from or derived from Lactococcus lactis. In some embodiments, the bacterial polypeptide coding sequence is from or derived from E. coli.

[0103] In some embodiments, the bacterial polypeptide coding sequence is codon-optimized for expression in a fungal host cell. In some embodiments of any of the aspects, the bacterial polypeptide coding sequence is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system (e.g., a fungal host cell). In some embodiments of any of the aspects, the expression system is an organism (e.g., a fungal host cell) other than the source of the native / wild-type sequence (e.g., a bacterial polypeptide coding sequence).

[0104] In some embodiments, the bacterial polypeptide encoded by the bacterial polypeptide coding sequence comprises an antibiotic resistance enzyme. In some embodiments, the bacterial polypeptide encoded by the bacterial polypeptide coding sequence comprises an antibiotic-degrading enzyme. In some embodiments, the antibiotic-degrading enzyme comprises a beta-lactamase enzyme. In some embodiments, the beta-lactamase enzyme comprises TEM1 beta-lactamase (see e.g., SEQ ID NOs: 7-10). In some embodiments, the beta-lactamase enzyme comprises an extended-spectrum beta-lactamase (ESBL). In some embodiments, the extended-spectrum beta-lactamase comprises a CTX-M beta-lactamase. Insome embodiments, the CTX-M beta-lactamase comprises CTX-M15 (see e.g., SEQ ID NOs: 11-12). In some embodiments, the beta-lactamase enzyme comprises TEM1 betalactamase (see e.g., SEQ ID NOs: 7-10) or CTX-M15 beta-lactamase (see e.g., SEQ ID NOs: 11-12). In some embodiments, the antibiotic-degrading enzyme comprises a macrolide esterase (e.g., EreA, EreC). In some embodiments, the antibiotic is selected from the group consisting of aminoglycosides, ansamycins, beta-lactams, bis-biguanides, carbacephems, carbapenems, cationic polypeptides, cephalosporins, fluoroquinolones, glycopeptides, iron- sequestering glycoproteins, linosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quaternary ammonium compounds, quinolones, silver compounds, sulfonamides, tetracyclines, and any combinations thereof.

[0105] Some exemplary specific antibiotics include broad penicillins, amoxicillin (e.g., Ampicillin, Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, Ticarcillin), Penicillins and Beta Lactamase Inhibitors (e.g., Amoxicillin-Clavulanic Acid, Ampicillin- Sulbactam, Benzylpenicillin, Cioxacillin, Dicloxacillin, Methicillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin Tazobactam, Ticarcillin Clavulanic Acid, Nafcillin), Cephalosporins (e.g., Cephalosporin I Generation, Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine), Cephalosporin II Generation (e.g., Cefaclor, Cefamandole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Cefmetazole, Cefuroxime, Loracarbef), Cephalosporin III Generation (e.g., Cefdinir, Ceftibuten, Cef operazone, Cefixime, Cefotaxime, Cefpodoxime proxetil, Ceftazidime, Ceftizoxime, Ceftriaxone), Cephalosporin IV Generation (e.g., Cefepime), Macrolides and Lincosamides (e.g., Azithromycin, Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin, Troleandomycin), Quinolones and Fluoroquinolones (e.g., Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid, Gemifloxacin, Perfloxacin), Carbapenems (e.g., Imipenem-Cilastatin, Meropenem), Monobactams (e.g., Aztreonam), Aminoglycosides (e.g., Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin), Glycopeptides (e.g., Teicoplanin, Vancomycin), Tetracyclines (e.g., Demeclocycline, Doxycycline, Methacycline, Minocycline, Oxytetracycline, Tetracycline, Chlortetracycline), Sulfonamides (e.g., Mafenide, Silver Sulfadiazine, Sulfacetamide, Sulfadiazine, Sulfamethoxazole, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Sulfamethizole), Rifampin (e.g., Rifabutin, Rifampin, Rifapentine), Oxazolidinones (e.g., Linezolid, Streptogramins, Quinupristin Dalfopristin),Bacitracin, Chloramphenicol, Fosfomycin, Isoniazid, Methenamine, Metronidazole, Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin, Spectinomycin, Trimethoprim, Colistin, Cycloserine, Capreomycin, Ethionamide, Pyrazinamide, Paraaminosalicylic acid, Erythromycin ethyl succinate, and the like.

[0106] In some embodiments, the bacterial polypeptide encoded by the bacterial polypeptide coding sequence can confer an advantage to a non-host bystander cell if horizontally transmitted from the fungal host cell to the non-host bystander cell and expressed as a polypeptide in the non-host bystander cell. In some embodiments of any of the aspects, the non-host bystander cell is any cell that cannot splice the at least one heterologous intron out of the polypeptide sequence. In some embodiments, the non-host bystander cell is a bacterial cell. In some embodiments of any of the aspects, the non-host bystander cell is an archaea cell. In some embodiments of any of the aspects, the non-host bystander cell is phylogenetically distant from the species from which at least one of the heterologous introns (or intron motifs, e.g., 5' splice site, branch site, and / or 3' splice site) was derived, such that it cannot splice the at least one heterologous intron. In some embodiments of any of the aspects, the non-host bystander cell is a fungus that is phylogenetically distant from the fungal species from which at least one of the heterologous fungal introns (or intron motifs, e.g., 5' splice site, branch site, and / or 3' splice site) was derived, such that it cannot splice the at least one heterologous fungal intron.

[0107] In some embodiments, the advantage comprises increased virulence of the non-host bystander cell. In some embodiments, the advantage comprises increased fitness of the non- host bystander cell. In some embodiments, the increased fitness comprises increased nutritional capacity. In some embodiments, the nutritional capacity comprises utilization of alternative carbon sources and / or nitrogen sources. In some embodiments, the polypeptide conferring a nutritional capacity advantage. Non-limiting examples of bacterial polypeptides conferring a nutritional capacity advantage include acetamidase (AmdS; e.g., AmdS from Aspergillus species); carbohydrate metabolism proteins (e.g., beta-glucosidases, cellobiohydrolases, endoglucanases, beta-fructosidase, sulfatases, beta-galactosidases, and the like); enzymes for the metabolism of compounds such as inulin, porphyran, cellulose; opine utilization proteins (e.g., arginases, opine dehydrogenases); membrane transporters (e.g., amino acid transporters, nucleotide transporters, permeases, metallophore transporters); proteases (e.g., keratin hydrolases); disulfide reductases; lignin modifying enzymes (e.g., peroxidases); or detoxifying enzymes (e.g., laccase). In some embodiments, the increasedfitness comprises increased adhesion to another cell. In some embodiments, the bacterial polypeptide conferring increased adhesion comprises an adhesion factor. In some embodiments, the adhesion factor comprises an adhesin.

[0108] In some embodiments, the bacterial polypeptide coding sequence encodes at least one portion of a bacterial polypeptide. Accordingly, in one aspect, described herein is a system comprising at least two transgene constructs. In some embodiments, the system comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more transgene constructs.

[0109] In one aspect, described herein is a system comprising: (a) a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and (b) a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence. In some embodiments, one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence. In some embodiments, the first portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence. In some embodiments, the second portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence. In some embodiments, the first and second portions of the bacterial polypeptide coding sequence are each interrupted by at least one heterologous intron sequence.

[0110] In some embodiments, the system comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence and (b) a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence. In some embodiments, the system comprises (a) a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence and (b) a second transgene construct comprising a second portion of said bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence. In some embodiments, the system comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence and (b) a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence.

[0111] The at least one heterologous intron sequence in the first portion of the bacterial polypeptide coding sequence can be the same as the at least one heterologous intron sequence in the second portion of the bacterial polypeptide coding sequence. In some embodiments, atleast one heterologous intron sequence in the first portion of the bacterial polypeptide coding sequence is different from at least one heterologous intron sequence in the second portion of the bacterial polypeptide coding sequence.

[0112] In some embodiments, the first and second portions of the bacterial polypeptide coding sequence are spliced in at least one fungal host cell to remove the heterologous intron sequences from the first and second portions of the bacterial polypeptide coding sequence. In some embodiments, the first and second portions of the bacterial polypeptide coding sequence are translated into functional first and second portions of the bacterial polypeptide that associate to provide the same function as the full-length bacterial polypeptide. In other words, the interaction of first and second portions of the bacterial polypeptide results in the same function as the wild-type full-length bacterial polypeptide.

[0113] In some embodiments, the functional first and second portions of the bacterial polypeptide comprise functional first and second portions of an antibiotic-degrading enzyme. In some embodiments, the functional first and second portions of the bacterial polypeptide comprise functional first and second portions of a beta-lactamase enzyme. See e.g., international patent publication WO 2022119926 Al, the contents of which are incorporated herein by reference in their entirety.Regulatory Elements

[0114] In some embodiments, the transgene construct comprises at least one regulatory element permitting expression of the bacterial polypeptide coding sequence in a fungal host cell. In some embodiments, the at least one regulatory element permits activity of the bacterial polypeptide coding sequence and encoded bacterial polypeptide in the fungal host cell. In some embodiments, expression includes, but is not limited to, transcription, splicing, translation, post-translational processing, and secretion of the bacterial polypeptide coding sequence or encoded bacterial polypeptide in the fungal host cell.

[0115] In some embodiments, the regulatory element comprises at least one yeast-derived expression control element, which directs or permits expression of the transgene in a yeast host cell. In some embodiments, the at least one yeast-derived expression control element is operably linked to the bacterial polypeptide coding sequence. In some embodiments, the at least one yeast-derived expression control element comprises a yeast-derived promoter. In some embodiments, the at least one yeast-derived expression control element comprises a yeast-derived secretion signal. In some embodiments, the yeast-derived secretion signal comprises the secretion signal of S. cerevisiae mating factor alpha. In some embodiments, theyeast-derived secretion signal is derived from the secretion signal of S. cerevisiae mating factor alpha. In some embodiments, a bacterial secretion signal in the bacterial polypeptide coding sequence is replaced with a yeast-derived secretion signal, to allow for secretion of the bacterial polypeptide in fungal host cells. In some embodiments, the yeast-derived secretion signal comprises SEQ ID NO: 13 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 13, that maintains its function (e.g., protein secretion in yeast cells).

[0116] SEQ ID NO: 13, secretion signal of Saccharomyces cerevisiae mating factor alpha; see e.g., amino acids 1-89 of SEQ ID NOs: 8, 9, or 12 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNST NNGLLFINTTIASIAAKEEGVSLDKREAEA

[0117] In some embodiments of any of the aspects, a DNA molecule comprising a transgene construct as described herein comprises at least one fungal-derived regulatory sequence upstream of the encoded bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence. In some embodiments of any of the aspects, a DNA molecule encoding a transgene construct as described herein comprises a fungal-derived promoter for transcription of the bacterial polypeptide coding sequence using an RNA polymerase in the fungal host cell.

[0118] When the nucleic acid molecule that comprises a transgene construct described herein is expressed in a fungal host cell, a variety of transcription control sequences (e.g., promoter / enhancer sequences) can be used to direct its expression. The promoter can be a promoter native to the fungal host cell. The promoter can be a promoter derived from another fungal species that is functional in the fungal host cell. In some embodiments the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of the transgene construct in the fungal host cell. A variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule or condition.

[0119] The precise nature of the regulatory sequences needed for expression can vary between species or cell types, but in general can include, as necessary, 5' non-transcrib ed and 5' non-translated sequences involved with the initiation of transcription and translation respectively. In particular, such 5' non-transcribed regulatory sequences can include a promoter region which includes a promoter sequence for transcriptional control of thetransgene construct. Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired.

[0120] As used herein, transgene construct and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the bacterial polypeptide coding sequence under the influence or control of the regulatory sequences. If it is desired that at least one bacterial polypeptide coding sequence encoded in the transgene construct be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the bacterial polypeptide coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the bacterial polypeptide coding sequence, or (3) interfere with the ability of the bacterial polypeptide coding sequence in the transgene construct to be translated into a protein.Nucleic Acids and Vectors

[0121] The transgene construct described herein can be comprised by nucleic acids and / or vectors. Accordingly, in one aspect described herein is a nucleic acid encoding or comprising a transgene construct described herein. In another aspect described herein is a vector encoding or comprising a transgene construct as described herein.

[0122] In some embodiments of any of the aspects, the nucleic acid encoding or comprising a transgene construct as described herein comprises DNA. In some embodiments of any of the aspects, the nucleic acid encoding or comprising a transgene construct as described herein consists essentially of DNA. In some embodiments of any of the aspects, the nucleic acid encoding or comprising a transgene construct as described herein consists of DNA.

[0123] In some embodiments of any of the aspects, the nucleic acid encoding or comprising a transgene construct as described herein comprises RNA. In some embodiments of any of the aspects, the nucleic acid encoding or comprising a transgene construct as described herein consists essentially of RNA. In some embodiments of any of the aspects, the nucleic acid encoding or comprising a transgene construct as described herein consists of RNA.

[0124] A nucleic acid molecule that encodes a transgene construct as described herein can be introduced into a cell or cells using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particlebombardment, etc. Expressing the nucleic acid molecule encoding a transgene construct as described herein can also be accomplished by integrating the nucleic acid molecule into the genome.

[0125] In some embodiments, one or more of the transgene constructs described herein is expressed in a recombinant expression vector or plasmid. Fig. 1 contains a schematic of an exemplary vector comprising a transgene construct as described herein. In some embodiments, one or more of the transgene constructs described herein is expressed in a shuttle plasmid that can express the at least one transgene in multiple species, such as yeast and bacteria. In some embodiments, the shuttle plasmid comprises SEQ ID NO: 5 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or more to SEQ ID NO: 5, that maintains its function (e.g., shuttle plasmid, expression plasmid).

[0126] SEQ ID NO: 5, shuttle plasmid expressing the Venus protein in both S. boulardii and E. coli. The Venus open reading frame is in bold. The mixed fungal -bacterial promoter, based on the Saccharomyces cerevisiae. THD3 promoter and a synthetic bacterial promoter (BBa_J23102) followed by a strong bacterial ribosome binding site, is double-underlined. Asterisks (*) mark the location of tested insertion points for the TEF4 and EFBI introns. Residues TAT that were removed to create the non -fluorescent AY66 mutant are italicized.GCTGGAAATCTGCTCGTCAGTGGTGCTCACACTGACGAATCATGTACAGATCATA CCGATGACTGCCTGGCGACTCACAACTAAGCAAGACAGCCGGAACCAGCGCCGG CGAACACCACTGCATATATGGCATATCACAACAGTCCACGTCTCAAGCAGTTACA GAGATGTTACGAACCACTAGTGCACTGCAGTACAGTTTAGCTTGCCTCGTCCCCG CCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTT GACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATAC ATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAG CAAAAATTACGGCTCCTCGCTCCAGACCTGCGAGCAGGGAAACGCTCCCCTCAC AGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTA GGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGAT ACAGTTCTCACATCACATCCGAACATAAACAAAAATGGGTAAGGAAAAGACTCA CGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAA TGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGG AAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTGGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAAGTAACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGGAGTATCACGTGCTATAAAAATAATTATAATTTAAATTTTTTAATATAAATATATAAATTAAAAATAGAAAGTAAAAAAAGAAATTAAAGAAAAAATAGTTTTTGTTTTCCGAAGATGTAAAAGACTCTAGGGGGATCGCCAACAAATACTACCTTTTATCTTGCTCTTCCTGCTCTCAGGTATTAATGCCGAATTGTTTCATCTTGTCTGTGTAGAAAACCACACACGAAAATCCTGTGATTTTACATTTTACTTATCGTTAATCGAATGTATATCTATTTAATCTGCTTTTCTTGTCTAATAAATATATATGTAAAGTACGCTTTTTGTTGAAATTTTTTAAACCTTTGTTTATTTTTTTTTCTTCATTCCGTAACTCTTCTACCTTCTTTATTTACTTTCTAAAATCCAAATACAAAACATAAAAATAAATAAACACAGAGTAAATTCCCAAATTATTCCATCATTAAAAGATACGAGGCGCGTGTAAGTTACAGGCAAGCGATCCGTCGCTGGTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGG GTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTT TATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT CCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTC TGTGGATAACCGTGCGGCCGCCCCTGAATTCGCATCTAGACTGATGAGACGTGGT AGAGCCACAAACAGCCGGTACAAGCAACGATCTCCAGGACCATCTGAATCATGC GCGGATGACACGAACTCACGACGGCGATCACAGACATTAACCCACAGTACAGAC ACTGCGAC A ACGTGGC A ATTCGTCGC A AT AC A ACGCAGTTCGAGTTTATC ATT AT CAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAAC TTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAA ATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGT TTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAG AAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCA TAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACA AGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTC TGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGA AATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAA TTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTT TTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAAAAA AGATTGACAGCTAGCTCAGTCCTAGGTACTGTGCTAGCCCAGGTACCAAAGAGG AGAAATAAATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAA GGTGAAGGTGATGCTACTTACGGTAAATTGACCTTAAAATTGATT*TGTACT ACTGGTAAATTGCCAGTTCCATGGCCAACCTTAGTCACTACTTTAGGTZ47G GTTTGCAATGTTT*TGCTAGATACCCAGATCATATGAAACAACATGACTTTTT CAAGTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAACTATTTTTTTCAAA GATGACGGTAACTACAAGACCAGAGCTGAAGTCAAGTT*TGAAGGTGATACC TTAGTTAATAGAATCGAATTAAAAGGTATTGATTTTAAAGAAGGTGGTAACA TTTTAGGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTACATCACT GCTGACAAACAAAAGAATGG*TATCAAAGCTAACTTCAAAATTAGACACAAC ATTGAAGATGGTGGTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATTGGTGATGGTCCAGTCTTGTTACCAGACAACCATTACTTATCCTATCAATC TGCCTTATCCAAAGATCCAAACGAAAAGAGAGATCACATGGTCTTGTTAGAA TTTGTTACTGCTGCTGGTATTACCCATGGTATGGATGAATTGTACAAAGGAT CCTAACTCGAGAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTC ATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTA AAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCAT AATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTT GCAGTTTGAAAAAT

[0127] As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring nucleic acids (e.g., a transgene construct as described herein) into a host cell, such as a fungal host cell. The vector can encompass any genetic element that is capable of replication when associated with the proper control elements and that can transfer nucleic acid sequences to cells. The term “vector” includes a plasmid, a cloning vector, an expression vector, naked DNA, a mini-chromosome, a chromosome, a transposon, a cosmid, a virus, virion, phage, and the like. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, wherein additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the technology is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0128] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequencesoriginating from at least two different genes, e.g., it comprises a transgene construct operably linked to at least one genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).

[0129] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for reduced expression in a bacterial cell.

[0130] A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector can be cut in a determinable fashion and into which a desired DNA sequence (e.g., a transgene construct as described herein) can be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.

[0131] An expression vector is one into which a desired DNA sequence (e.g., a transgene construct as described herein) can be inserted by restriction and ligation such that it is operably joined to regulatory sequences and can be expressed as an RNA transcript. Vectors can further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds (e.g., ampicillin resistance), genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., P-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., greenfluorescent protein). In certain embodiments, the vectors used herein are capable of autonomous replication and expression of the transgene construct present in the DNA segments to which they are operably joined. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in fungal cells for expression and in a prokaryotic host for cloning and amplification.

[0132] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (or RNA). That heterologous DNA (or RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.

[0133] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the transgene construct in place of non-essential viral genes. The vector and / or particle can be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Non-limiting examples of a viral vector of this invention include an AAV vector, an adenovirus vector, a lenti virus vector, a retrovirus vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector, a baculovirus vector, and a chimeric virus vector.

[0134] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleic acid of interest (e.g., a transgene construct as described herein) in the fungal host cell in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.Fungal Host Cells

[0135] Described herein in multiple aspects are fungal host cells comprising a transgene construct as described herein. In some embodiments, the fungal host cell comprises a nucleic acid comprising a transgene construct as described herein. In some embodiments, the fungal host cell comprises a vector comprising a transgene construct as described herein. In someembodiments, the fungal host cell comprises a transgene construct system as described herein.

[0136] In some embodiments, the fungal host cell is capable of expressing the bacterial polypeptide coding sequence. In some embodiments, the transgene construct comprises regulatory elements permitting expression of the bacterial polypeptide coding sequence in the fungal host cell.

[0137] In some embodiments, the fungal host cell is capable of transcribing, splicing, and translating the bacterial polypeptide coding sequence. In some embodiments, the fungal host cell is capable of splicing the at least one heterologous intron out of the bacterial polypeptide coding sequence in the transgene construct. In some embodiments, the fungal host cell is capable of post-translational processing of the encoded bacterial polypeptide. In some embodiments, the fungal host cell is capable of secreting the encoded bacterial polypeptide.

[0138] In some embodiments, the fungal host cell is a probiotic cell. In some embodiments, the fungal host cell is a budding yeast. In some embodiments, the fungal host cell is a hemiascomycetous yeast.

[0139] In some embodiments, the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Debaryomyces, Candida, andYarrowia. In some embodiments, the fungal host cell belongs to the Saccharomyces genus. In some embodiments, the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Debaryomyces, Candida, Yarrowia, Kazachstania, Lachancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyerozyma, Hanseniaspora, Rhizophagus, and Ogataea.

[0140] In some embodiments, the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces unisporus, Saccharomyces servazzii (also referred to as Kazachstania servazzii), Saccharomyces kluyveri (also referred to as Lachancea kluyveri), Kluyveromyces marxianus, Pichia angusta (also referred to as Hansenula polymorpha or Ogataea polymorpha), Debaryomyces hansenii (also referred to as Candida famata), Candida tropicalis, Yarrowia lipolytica, Kluyveromyces lactis, Candida albicans, Candida glabrata, Candida krusei, Candida auris, Lachancea thermotolerans, Malassezia furfur, Malassezia globosa, Malassezia pachydermatis, Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis, Schizosaccharomyces pombe, Komagataella pastoris, Pichia guilliermondii, Meyerozyma gruessi, Hanseniasporaosmophila. and Rhizophagus irregularis. In some embodiments, the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae. Saccharomyces boulardii. Saccharomyces unisporiis. Saccharomyces servazzii, and Saccharomyces kluyveri. In some embodiments, the fungal host cell is Saccharomyces cerevisiae. In some embodiments, the fungal host cell is Saccharomyces boulardii.

[0141] In some embodiments, the at least one fungal host cell is dried and viable. In some embodiments, the at least one fungal host cell is freeze-dried. In some embodiments, the at least one fungal host cell is spray-dried. In some embodiments, the viable, dried fungal host cell is minimally metabolically active when in the dried state. In some embodiments, the viable, dried fungal host cell becomes metabolically active once reconstituted in an aqueous liquid.

[0142] In some embodiments, the at least one fungal host cell is capable of sporulation. Accordingly, in some embodiments the at least one fungal host cell is in a spore form.

[0143] In some aspects, described herein are systems comprising at least two fungal host cells. Each fungal host cell can comprise at least one transgene construct or system as described herein. In one aspect, a first fungal host cell comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and a second fungal host cell comprises a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence. In some embodiments, one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence.

[0144] In one aspect, (a) a first fungal host cell comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence, and (b) a second fungal host cell comprises a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence. In another aspect, (a) a first fungal host cell comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence, and (b) a second fungal host cell comprises a second transgene construct comprising a second portion of said bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence. In another aspect, (a) a first fungal host cell comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence, and (b) a second fungal host cell comprises a secondtransgene construct comprising a second portion of said bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence.

[0145] The at least one heterologous intron sequence in the first portion of the bacterial polypeptide coding sequence can be the same as the at least one heterologous intron sequence in the second portion of the bacterial polypeptide coding sequence. In some embodiments, at least one heterologous intron sequence in the first portion of the bacterial polypeptide coding sequence is different from at least one heterologous intron sequence in the second portion of the bacterial polypeptide coding sequence.

[0146] In some embodiments, the first fungal host cell is capable of splicing the first portion of the bacterial polypeptide coding sequence and translating the spliced nucleic acid into a functional first portion of the bacterial polypeptide. In some embodiments, the second fungal host cell is capable of splicing the second portion of the bacterial polypeptide coding sequence and translating the spliced nucleic acid into a functional second portion of the bacterial polypeptide. In other words, the interaction of first and second portions of the bacterial polypeptide results in the same function, such as catalyzing the same enzymatic reaction, with the same level of activity or at least 50% activity as compared to the wild-type full-length bacterial polypeptide.

[0147] In some embodiments, the first fungal host cell is capable of post-transcriptional processing and secretion of the functional first portion of the bacterial polypeptide. In some embodiments, the second fungal host cell is capable of post-transcriptional processing and secretion of the functional second portion of the bacterial polypeptide.

[0148] In some embodiments, the functional first and second portions of the bacterial polypeptide comprise functional first and second portions of an antibiotic-degrading enzyme. In some embodiments, the functional first and second portions of the bacterial polypeptide comprise functional first and second portions of a beta-lactamase enzyme.Formulations and Preparations

[0149] In one aspect, described herein is a composition comprising a fungal host cell as described herein, formulated for delivery to a subject. The formulation can, for example, deliver the fungal host cell as a probiotic, and can comprise, for example, one or more carriers. In one aspect, described herein is a pharmaceutical composition comprising the at least one fungal host cell as described herein and a pharmaceutically acceptable carrier. In one aspect, described herein is a formulation comprising the at least one fungal host cell as described herein and a pharmaceutically acceptable carrier. In one aspect, described herein isa preparation comprising at least one fungal host cell as described herein and a carrier permitting application to a plant or to soil.

[0150] In some embodiments of any of the aspects, the at least one fungal host cell is formulated in a food composition. In some embodiments of any of the aspects, the food composition comprises a yogurt or a yogurt beverage. In some embodiments of any of the aspects, the at least one fungal host cell is formulated in a medical food. As used herein, "medical food" is understood to mean a food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation. In some embodiments of any of the aspects, the at least one fungal host cell is formulated in a supplement.

[0151] In one aspect, described herein is a food composition comprising at least one fungal host cell as described herein. In one aspect, described herein is a medical food comprising at least one fungal host cell as described herein. In one aspect, described herein is a supplement comprising at least one fungal host cell as described herein.Formulations

[0152] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising at least one fungal host cell as described herein, and optionally a pharmaceutically acceptable carrier.

[0153] In some embodiments, the active ingredients of the pharmaceutical composition comprise the transgene construct as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of the transgene construct as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of the transgene construct as described herein.

[0154] In some embodiments, the active ingredients of the pharmaceutical composition comprise the transgene product (e.g., expressed bacterial polypeptide) as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of the transgene product as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of the transgene product as described herein.

[0155] In some embodiments, the active ingredients of the pharmaceutical composition comprise the at least one fungal host cell as described herein. In some embodiments, theactive ingredients of the pharmaceutical composition consist essentially of the at least one fungal host cell as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of the at least one fungal host cell as described herein.

[0156] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum component, such as serum albumin, HDL and LDL; (24) C2-C12 alcohols; and (25) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g., the transgene construct, the transgene product, or fungal host cell as described herein.

[0157] Pharmaceutical compositions comprising at least one fungal host cell can be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the at least one fungal host cell or thepharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).Dosing

[0158] In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) to a subject in order to prevent or alleviate a symptom of a disease or disorder. As used herein, "alleviating a symptom of a disease or disorder " is ameliorating any condition or symptom associated with the disease or disorder. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art.

[0159] Subjects can be administered an effective (e.g., prophylactic and / or therapeutic) amount of a composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) comprising at least one fungal host cell, such as, about 101colony forming units (CFUs), about 102CFUs, about 103CFUs, about 104CFUs, about 105CFUs, about 106CFUs, about 107CFUs, about 108CFUs, about 109CFUs, about 1010CFUs, about 1011CFUs, about 1012CFUs, about 1013CFUs, or more of the at least one fungal host cell as described herein. In one embodiment, the subject is administered about 108CFUs of the at least one fungal host cell as described herein.

[0160] In embodiments related to compositions such as food compositions, medical foods, supplements, and / or probiotics, the subject can be administered a predetermined serving size. As non-limiting examples, the serving size can be about 1 mL, about 5 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, about 100 mL, or more, about 1 g, about 5 g, about 10 g, about 20 g, about 30 g, about 40 g, about 50 g, about 60 g, about 70 g, about 80 g, about 90 g, about 100 g, or more, or 1 pill, 2 pills, 3 pills, 4 pills, 5 pills, or more.

[0161] The term “effective amount" as used herein refers to the amount of the at least one fungal host cell as described herein needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, apharmaceutical composition) to provide the desired effect. As used herein, the phrase “effective amount” means an amount sufficient to achieve a meaningful benefit (including, but not limited to, e.g., reducing antibiotic concentration in the gut; reduced risk and / or incidence of C. difficile infection; maintenance of commensal bacteria in the gastrointestinal tract).

[0162] In embodiments in which the bacterial transgene encodes an antibiotic-degrading enzyme, the term “effective amount" as used herein refers to the amount of a population of engineered cells (e.g., fungal host cells) needed to locally degrade at least 50% of an administered antibiotic in the gastrointestinal tract (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100% (below detectable levels) of the antibiotic).

[0163] Alternatively, the “effective amount” can refer to the amount of a population of engineered cells (e.g., fungal host cells) needed to maintain commensal bacteria in the gut (e.g., to preserve diversity or abundance of the commensal bacteria). An effective amount as used herein can also include an amount sufficient to prevent or delay the development of a symptom of a disease (e.g., C. difficile infection), alter the course of a symptom of the disease (for example, reducing diarrhea), or reverse a symptom of the disease. It is understood that for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using routine experimentation. Given the intricacies of the body and the nature of cell establishment, the “effective amount” of cells can vary among different patients; however, one can easily determine in hindsight if the amount of cells administered was indeed an “effective amount.” Thus, further treatments can be modified accordingly. Note that long-term colonization or establishment, while often desirable, is not necessary for effective treatment as regular administration can achieve effective treatment as well.

[0164] Where a loss of species diversity is generally the trigger for opportunistic infection, e.g., productive or symptomatic infection with C. difficile, one measure of efficacy of treatment with an engineered microbe as described herein is maintenance of gut microbial diversity in a subject receiving antibiotic treatment to an extent greater than normally occurs upon treatment with that antibiotic in the absence of an engineered microbe as described herein. There are a number of different measures of microbial diversity, but one commonly looks at two parameters for a community: the number of different microbes, e.g., gut bacteria, in a sample, and the relative abundance of the different microbes. A significant change in either of these parameters can change the ability of the microbiota community to suppress orresist overgrowth or over-activity of a given species, such as C. difficile. While other indices can be used, the Shannon diversity index is commonly used to express gut microbiota diversity. In one embodiment, then, an effective treatment is one that maintains the microbial diversity of the gut of a given subject, as measured by the Shannon diversity index, upon antibiotic treatment, or throughout a course of antibiotic treatment for an infection.

[0165] One of skill in the art will appreciate that there is a great degree of variance of diversity status among individuals. Thus, in highly diverse individuals, a reduction of less than 50% of the starting diversity can still permit colonization resistance to pathogenic bacteria. In subjects initially having lower diversity, a smaller decrease in diversity can have greater physiological effects.

[0166] Alternatively, one of skill in the art can use the efficacy measurement, e.g., as described in international patent publication WO 2022119926 Al, the contents of which are incorporated herein by reference in their entirety. Assuming that the initial status of the microbiota is 100% effective at excluding C. difficile, sufficient efficacy can be assessed as a combination of (i) smaller reduction in the microbial diversity, (ii) conservation of the initial composition of the microbiota, and (iii) preservation of bacterial families that are known to have effects on colonization resistance, in particular Lachnospiraceae and Ruminococcaceae. In one embodiment, efficacious treatment is determined by scoring an individual’s microbial diversity as “no change in score relative to initiation of treatment” or “minimal change in score relative to initiation of treatment.” By “minimal” in this context is meant, for example, less than 5% change, e.g., less than 4% change, less than 3% change, less than 2% change, or less than 1% change.

[0167] In one embodiment, effective treatment is determined by a reduction in one or more symptoms associated with an opportunistic bacterial infection in the gastrointestinal tract.

[0168] The term "therapeutically effective amount" therefore refers to an amount of the at least one fungal host cell as described herein that is sufficient to provide a particular therapeutic effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0169] Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0170] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. A dose can be formulated in animal models. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the administration.

[0171] The dosage ranges for the administration of the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition), according to the methods described herein depend upon, for example, the form of the composition, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced. The dosage should not be so large as to cause adverse side effects, such as yeast infection. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0172] The efficacy of the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) in, e.g., the prevention and / or treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, an administration is considered “effective," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following administration according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the diseaseis halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein.

[0173] Prevention keeps a subject from developing a disease or disorder or condition, such as dysbiosis, and includes prophylactic administration. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms; or (2) relieving the severity of the disease, e.g., causing regression of symptoms. Prevention or treatment can also include delaying the onset of a disease or condition in the subject.

[0174] An effective amount for the prevention or treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective prevention or treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein. When using an experimental animal model, efficacy of prevention or treatment is evidenced when a statistically significant change in a marker is observed. In vitro and animal model assays allow the assessment of a given dose of the composition.

[0175] With respect to duration and frequency of administration, it is typical for skilled clinicians to monitor subjects in order to determine when the administration is providing a benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue administration, resume administration, or make other alterations to the administration regimen.

[0176] In certain embodiments, an effective dose of a composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) can be administered to a subject once. In certain embodiments, an effective dose of a composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) can be administered to a subject repeatedly.

[0177] The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the fungal host cell. The desired dose or amount can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments, administration can be one or more doses and / or administrations daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

[0178] In some embodiments, after an initial regimen, the doses and / or treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer.Administration

[0179] A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral or intrarectal.

[0180] In some embodiments of any of the aspects, the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) as described herein is administered as a monotherapy, e.g., another treatment for the disease or disorder is not administered to the subject.

[0181] The methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g., as part of a combinatorial therapy.

[0182] By way of non-limiting example, if a microbial infection is to be prevented or treated in a subject according to the methods described herein, the subject can also be administered at least one antimicrobial agent effective against the specific microbe of the microbial infection, in addition to the composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition).

[0183] As used herein, the term “antimicrobial agent” (also referred to herein as an antimicrobial, antimicrobial therapeutic, antibiotic and the like) refers to a molecule or composition which destroys microbes (i.e., bacteria, fungi, viruses, parasites, and / or microbial spores) or prevents or inhibits their development, proliferation and / or pathogenic action. The term “antimicrobial” thus comprises antibacterials, antifungals, and antivirals. Exemplary antimicrobial agents include, but are not limited to, small organic or inorganic molecules; peptides; proteins; peptide analogs and derivatives; peptidomimetics; antibodies(polyclonal or monoclonal) that target a microbe; antigen binding fragments of such antibodies; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof.

[0184] In some embodiments of any of the aspects, the antimicrobial agent can be selected from aminoglycosides, ansamycins, beta-lactams, bis-biguanides, carbacephems, carbapenems, cationic polypeptides, cephalosporins, fluoroquinolones, glycopeptides, iron- sequestering glycoproteins, linosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quaternary ammonium compounds, quinolones, silver compounds, sulfonamides, tetracyclines, and any combinations thereof. In some embodiments of any of the aspects, the antimicrobial agent can comprise an antibiotic.

[0185] Some exemplary specific antimicrobial agents include broad penicillins, amoxicillin (e.g., Ampicillin, Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, Ticarcillin), Penicillins and Beta Lactamase Inhibitors (e.g., Amoxicillin-Clavulanic Acid, Ampicillin- Sulbactam, Benzylpenicillin, Cioxacillin, Dicloxacillin, Methicillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin Tazobactam, Ticarcillin Clavulanic Acid, Nafcillin), Cephalosporins (e.g., Cephalosporin I Generation, Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine), Cephalosporin II Generation (e.g., Cefaclor, Cefamandole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Cefmetazole, Cefuroxime, Loracarbef), Cephalosporin III Generation (e.g., Cefdinir, Ceftibuten, Cef operazone, Cefixime, Cefotaxime, Cefpodoxime proxetil, Ceftazidime, Ceftizoxime, Ceftriaxone), Cephalosporin IV Generation (e.g., Cefepime), Macrolides and Lincosamides (e.g., Azithromycin, Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin, Troleandomycin), Quinolones and Fluoroquinolones (e.g., Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid, Gemifloxacin, Perfloxacin), Carbapenems (e.g., Imipenem-Cilastatin, Meropenem), Monobactams (e.g., Aztreonam), Aminoglycosides (e.g., Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin), Glycopeptides (e.g., Teicoplanin, Vancomycin), Tetracyclines (e.g., Demeclocycline, Doxycycline, Methacycline, Minocycline, Oxytetracycline, Tetracycline, Chlortetracycline), Sulfonamides (e.g., Mafenide, Silver Sulfadiazine, Sulfacetamide, Sulfadiazine, Sulfamethoxazole, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Sulfamethizole), Rifampin (e.g., Rifabutin, Rifampin,Rifapentine), Oxazolidinones (e.g., Linezolid, Streptogramins, Quinupristin Dalfopristin), Bacitracin, Chloramphenicol, Fosfomycin, Isoniazid, Methenamine, Metronidazole, Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin, Spectinomycin, Trimethoprim, Colistin, Cycloserine, Capreomycin, Ethionamide, Pyrazinamide, Paraaminosalicylic acid, Erythromycin ethyl succinate, and the like.

[0186] In some embodiments of any of the aspects, the antifungal is selected from the group consisting of: polyene antifungals, Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, imidazole antifungals, triazole antifungals, thiazole antifungals, Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Triazolesfedit], Albaconazole, Efinaconazole, Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Propi conazole, Ravuconazole, Terconazole, Voriconazole, Abafungin, Allylamines, amorolfm, butenafine, naftifine, terbinafine, Echinocandins, Anidulafungin, Caspofungin, Micafungin, Aurones, Benzoic acid, Ciclopirox, Flucytosine, 5-fluorocytosin, Griseofulvin, Haloprogin, Tolnaftate, Undecylenic acid, Triacetin, Crystal violet, Castellani's paint, Orotomide, Miltefosine, Potassium iodide, Coal tar, Copper(II) sulfate, Selenium disulfide, Sodium thiosulfate, Piroctone olamine, lodoquinol, clioquinol, Acrisorcin, Zinc pyrithione, and Sulfur.Additional antifungals known in the art can also be used.

[0187] In some embodiments of any of the aspects, the antiviral is selected from the group consisting of: Abacavir, Acyclovir, Adefovir, Amantadine, Ampligen, Amprenavir, antiretroviral, Arbidol, Atazanavir, Atripla, Cidofovir, Combivir, Darunavir, Delavirdine, Didanosine, Docosanol, Dolutegravir, Ecoliever, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Famciclovir, Fomivirsen, Fosamprenavir, Foscamet, Fosfonet, Fusion inhibitor, Ibacitabine, Idoxuridine, Imiquimod, Imunovir, Indinavir, Inosine, Integrase inhibitor, Interferon, Interferon type I, Interferon type II, Interferon type III, Lamivudine, Lopinavir, Loviride, Maraviroc, Methisazone, Moroxydine, Nelfinavir, Nevirapine, Nexavir, Nitazoxanide, Norvir, Nucleoside analogues, Oseltamivir (Tamiflu), Peginterferon alfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor, Pyramidine, Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine, Ritonavir, Saquinavir, Sofosbuvir, Stavudine, Synergistic enhancer (antiretroviral), Telaprevir, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex),Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir (Relenza), Zidovudine. Additional antivirals known in the art can also be used.

[0188] By way of another non-limiting example, if pain or inflammation is to be prevented or treated in a subject according to the methods described herein, the subject can also be administered a second agent and / or treatment known to be beneficial for subjects suffering from pain or inflammation. Examples of such agents and / or treatments include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g., cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclometasone); methotrexate; sulfasalazine; leflunomide; anti-TNF medications; cyclophosphamide; proresolving drugs; my cophenolate; or opiates (e.g., endorphins, enkephalins, and dynorphin), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.

[0189] In some embodiments of any of the aspects, the subject is co-administered at least one prebiotic in addition to the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition). Nonlimiting examples of such prebiotics include amino acid (e.g., arginine, glutarate, and ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose-enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectin (e.g., xylogal actouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, com bran, and oat fiber), xylooligosaccharide, and polyamine (e.g., spermidine, putrescine).

[0190] In some embodiments of any of the aspects, the subject is co-administered at least one probiotic in addition to the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition). In some embodiments of any of the aspects, the subject is co-administered with the composition at least one fungal probiotic. In some embodiments of any of the aspects, the subject is coadministered with the composition at least one bacterial probiotic.Methods of Use

[0191] In one aspect, described herein is a method of limiting or eliminating potential for horizontal transmission of a bacterial transgene from a fungal host cell to a bacterial cell. Insome embodiments, the method comprises introducing at least one heterologous intron into the bacterial polypeptide coding sequence of the bacterial transgene.

[0192] In some embodiments, the method limits or eliminates horizontal transmission of the bacterial transgene nucleic acid to bacterial cells. In some embodiments, the method limits or eliminates horizontal transmission of the bacterial polypeptide coding sequence to bacterial cells. While a nucleic acid or a vector comprising the transgene construct might be transmitted to a bacterial cell, a bacterial polypeptide coding sequence interrupted by at least one heterologous intron sequence will not be expressed in the bacterial cell, nor will the bacterial polypeptide coding sequence confer a growth or selective advantage to the bacterial cell.

[0193] In one aspect, described herein is a method of preventing or treating a disease or infection in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of at least one fungal host cell as described herein or a composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition).

[0194] In one aspect, described herein is a method of promoting heath in a subject. In some embodiments, the method comprises administering to the subject an effective amount of at least one fungal host cell as described herein or a composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition).

[0195] In some embodiments, the at least one fungal host cell is an agent for bio-control, biostimulation, and / or bio-nutrition in the subject. In some embodiments, the at least one fungal host cell is a probiotic.

[0196] In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the non-human mammal is a livestock animal. In some embodiments, the livestock animal is selected from the group consisting of cow, chicken, pig, horse, and goat.

[0197] In one aspect, described herein is a method of preventing or treating a disease or infection in a plant. In some embodiments, the method comprises contacting the plant with an effective amount of at least one fungal host cell as described herein or a preparation as described herein (e.g., comprising a carrier permitting application to a plant or to soil). As a non-limiting example, a fungal host cell, such as a yeast, can be used for agricultural remediation by secreting into the soil and / or plant at least one antibiotic-degrading enzyme.As another non-limiting example, a fungal host cell, such as an engineered soil fungus, can express a protein effector (e.g., SP7 or RiNLEl, e.g., from Rhizophagus irregularis) to modulate the plant immune system and / or promote mycorrhizal symbioses to promote plant growth. As another non-limiting example, a fungal host cell can be engineered to secrete at least one enzyme that breaks down soil to increase nutrient bioavailability, for example lignin-modifying enzymes (e.g., peroxidases, laccases).

[0198] In one aspect, described herein is a method of promoting health in a plant. In some embodiments, the method comprises contacting the plant with an effective amount of at least one fungal host cell as described herein or a preparation as described herein (e.g., comprising a carrier permitting application to a plant or to soil). In some embodiments, the at least one fungal host cell is an agent for bio-remediation in the plant.

[0199] In one aspect described herein is a method of removing or reducing at least one toxin from soil. In some embodiments, the method comprises contacting the soil with an effective amount of at least one fungal host cell as described herein or a preparation as described herein (e.g., comprising a carrier permitting application to a plant or to soil).

[0200] The compositions described herein can be administered to a subject in need thereof, for instance for the prevention and / or treatment of dysbiosis. In some embodiments, the method of prevention or treatment can comprise first determining and / or diagnosing a subject or patient who can benefit from treatment by a composition described herein. In some embodiments, such determination and / or diagnosis comprises detecting or measuring a low level of at least one probiotic species in a sample from the subject or patient, or detecting or measuring a high level of at least one pathogenic microbial species in a sample from the subject or patient, each of which are examples of an abnormal level of each analyte. In some embodiments, the method further comprises administering to the patient a composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition).

[0201] In some embodiments, the subject has previously been determined to have an abnormal level of an analyte described herein relative to a reference. In some embodiments, the reference level can be the level in a sample of similar cell type, sample type, sample processing, and / or obtained from a subject of similar age, sex and other demographic parameters as the sample / subject. In some embodiments, the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g., the same number and type of cells.

[0202] The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood or plasma sample from a subject. In some embodiments of any of the aspects, the technology described herein encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, or bodily fluid. In some embodiments of any of the aspects, the biological sample comprises feces. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; semen; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and / or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject.

[0203] In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise performing or having performed an assay on a sample obtained from the subject to determine / measure the level of analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise ordering or requesting an assay on a sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise receiving the results of an assay on a sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise receiving a report, results, or other means of identifying the subject as a subject with a decreased level of the analyte.

[0204] In one aspect of any of the embodiments, described herein is a method of preventing or treating a disease or disorder (e.g., dysbiosis) in a subject in need thereof, the method comprising: a) determining if the subject has an abnormal level of an analyte described herein; and b) instructing or directing that the subject be administered a compositioncomprising at least one fungal host cell as described herein if the level of the analyte is abnormal relative to a reference. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and / or treatment recommendations in view of the assay results.Definitions

[0205] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0206] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal, e.g., for an individual without a given disorder.

[0207] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0208] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0209] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease or disorder. A subject can be male or female.

[0210] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for a disease or disorder or the one or more complications related to the disease or disorder. Alternatively, a subject can also be one who has not been previously diagnosed as having the disease or disorder or one or more complications related to the disease or disorder. For example, a subject can be one who exhibits one or more risk factors for the disease or disorder or one or more complications related to the disease or disorder or a subject who does not exhibit risk factors.

[0211] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0212] As used herein, the terms “protein" and “polypeptide" are used interchangeably to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and"polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0213] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0214] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested to confirm that a desired activity, e.g., activity and specificity of a native or reference polypeptide is retained.

[0215] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively,naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into lie or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into lie; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0216] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a polypeptide which retains at least 50% of the wild-type reference polypeptide’s activity. A functional fragment can comprise conservative substitutions relative to a wild-type reference polypeptide’s activity.

[0217] In some embodiments, the polypeptide described herein can be a variant of a subject bacterial polypeptide sequence. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a protein or fragment thereof that retains activity of the native or reference polypeptide. A wide variety of, for example, PCR-based, site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan to generate and test artificial variants.

[0218] A variant amino acid or DNA sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can bedetermined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTp or BLASTn with default settings).

[0219] A variant amino acid sequence can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to a native or reference sequence. As used herein, “similarity” refers to an identical amino acid or a conservatively substituted amino acid, as described herein. Accordingly, the percentage of “sequence similarity” is the percentage of amino acids which is either identical or conservatively changed; e.g., “sequence similarity” = (% sequence identity)+(% conservative changes). It should be understood that a sequence that has a specified percent similarity to a reference sequence necessarily encompasses a sequence with the same specified percent identity to that reference sequence. The skilled person will be aware of various computer programs, using different mathematical algorithms, that are available to determine the identity or similarity between two sequences. For instance, use can be made of a computer program employing the Needleman and Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego U.S.A.); the algorithm of E. Meyers and W. Miller (Meyers et al. (1989)) which has been incorporated into the ALIGN program (version 2.0); or more preferably the BLAST (Basic Local Alignment Tool using default parameters); see e.g., US Patent 10,023,890, the content of which is incorporated by reference herein in its entirety.

[0220] As used herein, the phrase “maintains the same function”, when used in reference to an enzyme, catalyzes the same reaction as a reference enzyme.

[0221] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (e.g., mRNA) or antisense RNA derived from a nucleic acid fragment or fragments and / or to the translation of mRNA into a polypeptide.

[0222] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regionspreceding and following a coding region, e.g., 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0223] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0224] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g., dysbiosis. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0225] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments ofany of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in or within nature.

[0226] As used herein, the term "administering," refers to the placement of a composition or compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the composition, compound, or metabolite thereof at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0227] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, transfection, transduction, perfusion, injection, or other delivery method known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0228] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0229] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0230] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0231] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0232] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0233] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0234] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0235] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in cell biology, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton &Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0236] Other terms are defined herein within the description of the various aspects of the invention.

[0237] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0238] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein canbe applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0239] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0240] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:1. A transgene construct comprising a bacterial polypeptide coding sequence, wherein the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence.2. The transgene construct of paragraph 1, wherein the at least one heterologous intron comprises: at least one in-frame stop codon; at least one stop codon in each of a plurality of reading frames; at least one stop codon in each reading frame; or at least two heterologous introns interrupting the bacterial polypeptide coding sequence.3. The transgene construct of any one of paragraphs 1-2, wherein the at least one heterologous intron is adapted for splicing out of the bacterial polypeptide coding sequence in a fungal host cell.4. The transgene construct of any one of paragraphs 1-3, wherein the bacterial polypeptide coding sequence is codon-optimized for expression in a fungal host cell.5. The transgene construct of any one of paragraphs 1-4, wherein the transgene construct comprises regulatory elements permitting expression of the bacterial polypeptide coding sequence in a fungal host cell; optionally wherein the regulatory elements comprise yeast-derived expression control elements operably linked to the bacterial polypeptide coding sequence; optionally wherein the expression control elementscomprise a yeast-derived promoter and / or yeast-derived secretion signal; optionally wherein the yeast-derived secretion signal comprises or is derived from the secretion signal of S. cerevisiae mating factor alpha. The transgene construct of any one of paragraphs 3-5, wherein the fungal host cell is a hemiascomycetous yeast; or wherein the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Debaryomyces, Candida, Yarrowia, Kazachslania, I.achancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyer ozyma, Hanseniaspora, Rhizophagus, and Ogataea, or wherein the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces unisporus, Saccharomyces servazzii, Saccharomyces kluyveri, Kluyveromyces marxianus, Pichia angusta, Debaryomyces hansenii, Candida tropicalis, Yarrowia lipolytica, Kluyveromyces lactis, Candida albicans, Candida glabrata, Candida krusei, Candida auris, Lachancea thermotolerans, Malassezia furfur, Malassezia globosa, Malassezia pachydermatis, Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis, Schizosaccharomyces pombe, Komagataella pastoris, Pichia guilliermondii, Meyer ozyma gruessi, Hanseniaspora osmophila, and Rhizophagus irregularis. The transgene construct of any one of paragraphs 1-6, wherein the at least one heterologous intron comprises a 5' splice site, a branch site, and a 3' splice site, optionally wherein the 5' splice site, the branch site, and the 3' splice site are each selected from Table 1. The transgene construct of paragraph 7, wherein the 5' splice site comprises GTATGT, the branch site comprises TACTAAC, and the 3' splice site comprises YAG. The transgene construct of any one of paragraphs 1-8, wherein the at least one heterologous intron is about 40 nucleotides to about 80 nucleotides long; about 90 nucleotides to about 300 nucleotides long; or about 40 nucleotides to about 1000 nucleotides long. The transgene construct of any one of paragraphs 1-9, wherein the distance between the 5' splice site and the branch site is between about 20 nt to about 950 nt; and / orwherein the distance between the branch site and the 3' splice site is between about 1 nt to about 50 nt. The transgene construct of any one of paragraphs 1-10, wherein the 5' splice site, the branch site, and the 3' splice site are derived from the same species or source; and / or wherein the at least one heterologous intron is derived from a natural fungal intron sequence, is synthetic, or is chimeric. The transgene construct of any one of paragraphs 1-11, wherein the at least one heterologous intron comprises an intron from the Saccharomyces cerevisiae TEF4 gene, from the Saccharomyces cerevisiae EFB 1 gene, or from the Saccharomyces cerevisiae UBC4 gene. The transgene construct of any one of paragraphs 1-12, wherein the location of the at least one heterologous intron within the bacterial polypeptide coding sequence is:(a) in the center of the open reading frame (ORF);(b) in the central portion that is 45% to 55% of the way through the ORF;(c) in the central portion that is 40% to 60% of the way through the ORF;(d) in the central portion that is 30% to 70% of the way through the ORF;(e) in the central portion that is 25% to 75% of the way through the ORF or(f) in an active site codon of the ORF. The transgene construct of any one of paragraphs 1-13, wherein the at least one heterologous intron comprises at least one functional RNA element, optionally a small nucleolar RNA (snoRNA). The transgene construct of any one of paragraphs 1-14, which is comprised by a plasmid or vector. The transgene construct of any one of paragraphs 1-15, wherein the bacterial polypeptide encoded by the bacterial polypeptide coding sequence comprises an antibiotic resistance enzyme or an antibiotic-degrading enzyme, optionally wherein the antibiotic-degrading enzyme comprises a beta-lactamase enzyme; optionally wherein the beta-lactamase enzyme is a Temoneira-type (TEM) beta-lactamase (e.g., TEM1) or a cefotaximase-Munich-type (CTX-M) beta-lactamase (e.g., CTX-M-15). The transgene construct of any one of paragraphs 1-16, wherein the bacterial polypeptide encoded by the bacterial polypeptide coding sequence can confer an advantage if horizontally transmitted from the fungal host cell to a non-host bystander cell and expressed in the non-host bystander cell; optionally wherein the advantagecomprises increased virulence and / or increased fitness; optionally wherein the increased fitness comprises increased nutritional capacity or increased adhesion to another cell; optionally wherein the nutritional capacity comprises utilization of alternative carbon sources and / or nitrogen sources. The transgene construct of paragraph 17, wherein the non-host bystander cell is a bacterial cell. The transgene construct of any one of paragraphs 1-18, which produces a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a fungal host cell; and / or which does not produce a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a bacterial cell. A system comprising at least two transgene constructs, the system comprising:(a) a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and(b) a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence; wherein one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence. The system of paragraph 20, wherein:(a) the first portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence;(b) the second portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence; or(c) the first and second portions of the bacterial polypeptide coding sequence are each interrupted by at least one heterologous intron sequence. The system of paragraph 20 or 21, wherein the first and second portions of the bacterial polypeptide coding sequence are spliced and translated into functional first and second portions of the bacterial polypeptide, optionally wherein the bacterial polypeptide is an antibiotic-degrading enzyme, optionally wherein the antibioticdegrading enzyme is a beta-lactamase enzyme; optionally wherein the beta-lactamase enzyme is a Temoneira-type (TEM) beta-lactamase (e.g., TEM1) or a cefotaximase- Muni ch-type (CTX-M) beta-lactamase (e.g., CTX-M-15).At least one fungal host cell comprising the transgene construct of any one of paragraphs 1-19 or the system of any one of paragraphs 20-22. The at least one fungal host cell of paragraph 23, wherein the fungal host cell is a probiotic cell. The at least one fungal host cell of paragraph 23 or 24, wherein the fungal host cell is a hemiascomycetous yeast; or wherein the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Debaryomyces, Candida, Yarrowia, Kazachslania, I.achancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyer ozyma, Hanseniaspora, Rhizophagus, and Ogataea, or wherein the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces unisporus, Saccharomyces servazzii, Saccharomyces kluyveri, Kluyveromyces marxianus, Pichia angusta, Debaryomyces hansenii, Candida tropicalis, Yarrowia lipolytica, Kluyveromyces lactis, Candida albicans, Candida glabrata, Candida krusei, Candida auris, Lachancea thermotolerans, Malassezia furfur, Malassezia globosa, Malassezia pachydermatis, Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis, Schizosaccharomyces pombe, Komagataella pastoris, Pichia guilliermondii, Meyer ozyma gruessi, Hanseniaspora osmophila, and Rhizophagus irregularis. The at least one fungal host cell of any one of paragraphs 23-25, wherein the at least one fungal host cell is dried and viable. The at least one fungal host cell of any one of paragraphs 23-26, wherein a first fungal host cell comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and wherein a second fungal host cell comprises a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence; wherein one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence. The at least one fungal host cell of paragraph 27, wherein the first and second portions of the bacterial polypeptide coding sequence are spliced and translated into functional first and second portions of the bacterial polypeptide, optionally wherein the bacterial polypeptide is an antibiotic-degrading enzyme, optionally wherein the antibiotic-degrading enzyme is a beta-lactamase enzyme; optionally wherein the beta-lactamase enzyme is a Temoneira-type (TEM) beta-lactamase (e.g., TEM1) or a cefotaximase- Muni ch-type (CTX-M) beta-lactamase (e.g., CTX-M-15). A composition comprising the at least one fungal host cell of any one of paragraphs 23-28, formulated for delivery to a subject. A food composition comprising the at least one fungal host cell of any one of paragraphs 23-28. A medical food comprising the at least one fungal host cell of any one of paragraphs 23-28. A supplement comprising the at least one fungal host cell of any one of paragraphs 23-28. A probiotic composition comprising the at least one fungal host cell of any one of paragraphs 23-28. A pharmaceutical composition comprising the at least one fungal host cell of any one of paragraphs 23-28 and a pharmaceutically acceptable carrier. A preparation comprising the at least one fungal host cell of any one of paragraphs 23-28 and a carrier permitting application to a plant or to soil. A method of limiting or eliminating potential for horizontal transmission of a bacterial transgene from a fungal host cell to a bacterial cell, the method comprising introducing at least one heterologous intron into the bacterial polypeptide coding sequence of the bacterial transgene. The method of paragraph 36, wherein the method limits or eliminates horizontal transmission of the bacterial transgene nucleic acid and / or the bacterial polypeptide coding sequence to bacterial cells. A method of preventing or treating a disease or infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the at least one fungal host cell of any one of paragraphs 23-28, the composition of paragraph 29, the food composition of paragraph 30, the medical food of paragraph 31, the supplement of paragraph 32, the probiotic composition of paragraph 33, or the pharmaceutical composition of paragraph 34. A method of promoting heath in a subject, the method comprising administering to the subject an effective amount of the at least one fungal host cell of any one of paragraphs 23-28, the composition of paragraph 29, the food composition ofparagraph 30, the medical food of paragraph 31, the supplement of paragraph 32, the probiotic composition of paragraph 33, or the pharmaceutical composition of paragraph 34.40. The method of paragraphs 38 or 39, wherein the at least one fungal host cell is an agent for bio-control, bio-stimulation, and / or bio-nutrition in the subject.

[0241] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:1. A transgene construct comprising a bacterial polypeptide coding sequence, wherein the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence.2. The transgene construct of paragraph 1, wherein the at least one heterologous intron comprises: at least one in-frame stop codon; at least one stop codon in each of a plurality of reading frames; at least one stop codon in each reading frame; or at least two heterologous introns interrupting the bacterial polypeptide coding sequence.3. The transgene construct of any one of paragraphs 1-2, wherein the at least one heterologous intron is adapted for splicing out of the bacterial polypeptide coding sequence in a fungal host cell.4. The transgene construct of any one of paragraphs 1-3, wherein the bacterial polypeptide coding sequence is codon-optimized for expression in a fungal host cell.5. The transgene construct of any one of paragraphs 1-4, wherein the transgene construct comprises regulatory elements permitting expression of the bacterial polypeptide coding sequence in a fungal host cell; optionally wherein the regulatory elements comprise yeast-derived expression control elements operably linked to the bacterial polypeptide coding sequence; optionally wherein the expression control elements comprise a yeast-derived promoter and / or secretion signal.6. The transgene construct of any one of paragraphs 3-5, wherein the fungal host cell is a hemiascomycetous yeast; or wherein the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia. Debaryomyces. Candida, Yarrowia, Kazachslania, I.achancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyer ozyma, Hanseniaspora, Rhizophagus, and Ogataea, orwherein the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boiilardii. Saccharomyces unisporiis.Saccharomyces servazzii, Saccharomyces kluyveri, Kluyveromyces marxianus. Pichia angusta, Debaryomyces hansenii. Candida tropicalis. Yarrowia lipolylica.Kluyveromyces laclis. Candida albicans, Candida glabrala, Candida krusei, Candida auris, Lachancea thermololerans, Malassezia furfur, Malassezia globosa, Malassezia pachydermatis, Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis, Schizosaccharomyces pombe, Komagataella pastoris, Pichia guilliermondii, Meyer ozyma gruessi, Hanseniaspora osmophila, and Rhizophagus irregularis. The transgene construct of any one of paragraphs 1-6, wherein the at least one heterologous intron comprises a 5' slice site, a branch site, and a 3' slice site, optionally wherein the 5' slice site, the branch site, and the 3' slice site are each selected from Table 1. The transgene construct of paragraph 7, wherein the 5' slice site comprises GTATGT, the branch site comprises TACTAAC, and the 3' slice site comprises YAG. The transgene construct of any one of paragraphs 1-8, wherein the at least one heterologous intron is about 40 nucleotides to about 80 nucleotides long; about 90 nucleotides to about 300 nucleotides long; or about 40 nucleotides to about 1000 nucleotides long. The transgene construct of any one of paragraphs 1-9, wherein the distance between the 5' slice site and the branch site is between about 20 nt to about 950 nt; and / or wherein the distance between the branch site and the 3' slice site is between about 1 nt to about 50 nt. The transgene construct of any one of paragraphs 1-10, wherein the 5' slice site, the branch site, and the 3' slice site are derived from the same species or source; and / or wherein the at least one heterologous intron is derived from a natural fungal intron sequence, is synthetic, or is chimeric. The transgene construct of any one of paragraphs 1-11, wherein the at least one heterologous intron comprises an intron from the Saccharomyces cerevisiae TEF4 gene or from the Saccharomyces cerevisiae EFB 1 gene. The transgene construct of any one of paragraphs 1-12, wherein the location of the at least one heterologous intron within the bacterial polypeptide coding sequence is:(a) in the center of the open reading frame (ORF);(b) in the central portion that is 45% to 55% of the way through the ORF;(c) in the central portion that is 40% to 60% of the way through the ORF;(d) in the central portion that is 30% to 70% of the way through the ORF; or(e) in the central portion that is 25% to 75% of the way through the ORF. The transgene construct of any one of paragraphs 1-13, wherein the at least one heterologous intron comprises at least one functional RNA element, optionally a small nucleolar RNA (snoRNA). The transgene construct of any one of paragraphs 1-14, which is comprised by a plasmid or vector. The transgene construct of any one of paragraphs 1-15, wherein the bacterial polypeptide encoded by the bacterial polypeptide coding sequence comprises an antibiotic resistance enzyme or an antibiotic-degrading enzyme, optionally wherein the antibiotic-degrading enzyme comprises a beta-lactamase enzyme. The transgene construct of any one of paragraphs 1-16, wherein the bacterial polypeptide encoded by the bacterial polypeptide coding sequence can confer an advantage if horizontally transmitted from the fungal host cell to a non-host bystander cell and expressed in the non-host bystander cell; optionally wherein the advantage comprises increased virulence and / or increased fitness; optionally wherein the increased fitness comprises increased nutritional capacity or increased adhesion to another cell; optionally wherein the nutritional capacity comprises utilization of alternative carbon sources and / or nitrogen sources. The transgene construct of paragraph 17, wherein the non-host bystander cell is a bacterial cell. The transgene construct of any one of paragraphs 1-18, which produces a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a fungal host cell; and / or which does not produce a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a bacterial cell. A system comprising at least two transgene constructs, the system comprising:(a) a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and(b) a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence;wherein one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence. The system of paragraph 20, wherein:(a) the first portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence;(b) the second portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence; or(c) the first and second portions of the bacterial polypeptide coding sequence are each interrupted by at least one heterologous intron sequence. The system of paragraph 20 or 21, wherein the first and second portions of the bacterial polypeptide coding sequence are spliced and translated into functional first and second portions of the bacterial polypeptide, optionally wherein the bacterial polypeptide is an antibiotic-degrading enzyme, optionally wherein the antibioticdegrading enzyme is a beta-lactamase enzyme. At least one fungal host cell comprising the transgene construct of any one of paragraphs 1-19 or the system of any one of paragraphs 20-22. The at least one fungal host cell of paragraph 23, wherein the fungal host cell is a probiotic cell. The at least one fungal host cell of paragraph 23 or 24, wherein the fungal host cell is a hemiascomycetous yeast; or wherein the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Debaryomyces, Candida, Yarrowia, Kazachslania, I.achancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyer ozyma, Hanseniaspora, Rhizophagus, and Ogataea, or wherein the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces unisporus, Saccharomyces servazzii, Saccharomyces kluyveri, Kluyveromyces marxianus, Pichia angusta, Debaryomyces hansenii, Candida tropicalis, Yarrowia lipolytica, Kluyveromyces lactis, Candida albicans, Candida glabrata, Candida krusei, Candida auris, Lachancea thermotolerans, Malassezia furfur, Malassezia globosa, Malassezia pachydermatis, Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis,Schizosaccharomyces pombe, Komagataella pastoris, Pichia guilliermondii, Meyer ozyma gruessi. Hanseniaspora os mophila, and Rhizophagus irregularis. The at least one fungal host cell of any one of paragraphs 23-25, wherein the at least one fungal host cell is dried and viable. The at least one fungal host cell of any one of paragraphs 23-26, wherein a first fungal host cell comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and wherein a second fungal host cell comprises a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence; wherein one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence. The at least one fungal host cell of paragraph 27, wherein the first and second portions of the bacterial polypeptide coding sequence are spliced and translated into functional first and second portions of the bacterial polypeptide, optionally wherein the bacterial polypeptide is an antibiotic-degrading enzyme, optionally wherein the antibioticdegrading enzyme is a beta-lactamase enzyme. A composition comprising the at least one fungal host cell of any one of paragraphs 23-28, formulated for delivery to a subject. A food composition comprising the at least one fungal host cell of any one of paragraphs 23-28. A medical food comprising the at least one fungal host cell of any one of paragraphs 23-28. A supplement comprising the at least one fungal host cell of any one of paragraphs 23-28. A probiotic composition comprising the at least one fungal host cell of any one of paragraphs 23-28. A pharmaceutical composition comprising the at least one fungal host cell of any one of paragraphs 23-28 and a pharmaceutically acceptable carrier. A preparation comprising the at least one fungal host cell of any one of paragraphs 23-28 and a carrier permitting application to a plant or to soil. A method of limiting or eliminating potential for horizontal transmission of a bacterial transgene from a fungal host cell to a bacterial cell, the method comprisingintroducing at least one heterologous intron into the bacterial polypeptide coding sequence of the bacterial transgene.37. The method of paragraph 36, wherein the method limits or eliminates horizontal transmission of the bacterial transgene nucleic acid and / or the bacterial polypeptide coding sequence to bacterial cells.38. A method of preventing or treating a disease or infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the at least one fungal host cell of any one of paragraphs 23-28, the composition of paragraph 29, the food composition of paragraph 30, the medical food of paragraph 31, the supplement of paragraph 32, the probiotic composition of paragraph 33, or the pharmaceutical composition of paragraph 34.39. A method of promoting heath in a subject, the method comprising administering to the subject an effective amount of the at least one fungal host cell of any one of paragraphs 23-28, the composition of paragraph 29, the food composition of paragraph 30, the medical food of paragraph 31, the supplement of paragraph 32, the probiotic composition of paragraph 33, or the pharmaceutical composition of paragraph 34.40. The method of paragraphs 38 or 39, wherein the at least one fungal host cell is an agent for bio-control, bio-stimulation, and / or bio-nutrition in the subject.

[0242] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1

[0243] Described herein is a fungal expression cassette that is not functional in prokaryotic species due to the presence of an intron sequence that disrupts the coding sequence of the encoded protein.

[0244] This strategy can be implemented, for example, to prevent the dissemination of antibiotic resistance genes from an antibiotic-degrading strain of yeast to bacteria that can get a selective advantage from acquiring an antibiotic resistance gene. The yeast codon- optimized TEM1 P-lactamase was expressed in Saccharomyces cerevisiae (strain S288c) using an episomal plasmid (pRGsb8, see e.g., Fig. 1). The TEMl-coding sequence harbors the full-length signal pro-peptides and pre-peptides from yeast mating factor a (MAT a)upstream of TEM1 to allow the extracellular secretion of the enzyme from yeast. The TEM1 coding sequence is interrupted by an intron, taken from the TEF4 gene (5. cerevisiae reference strain S288c); this gene was termed iTEMl. Several elements prevent the expression of the iTEMl antibiotic resistance gene if it were to be transferred to a bacterial species: the promoter and the secretion signal are not of bacterial origin; none of them are expected to work in bacteria. In addition, bacteria do not possess the splicing machinery required to excise introns, since pre-mRNA splicing requires multiple accessory proteins and small RNAs not found in bacteria. In this example, in a bacterium the TEM1 -coding gene would be interrupted by multiple in-frame stop codons preventing the expression of the C- terminal half of the enzyme. Expression and secretion of a bioactive P-lactamase enzyme in yeast but not in bacteria is demonstrated by the detection of the hydrolysis of the chromogenic P-lactam substrate nitrocefin (see e.g., Fig. 2). Since yeast species are naturally resistant to most bacterial antibiotics (including P-lactams), the expression of these enzymes is not expected to create an added fitness advantage to the engineered yeast strain. This is one relevant example of kingdom restriction using the TEM1 beta-lactamase enzyme due to its easy readout. The intron-biocontainment strategy can also be used to prevent undesired transmission of any genetically engineered trait into prokaryotic species and distantly related eukaryotic species.

[0245] The functionality of the intron-modified transgene was also demonstrated in an in vivo setting. For this, the pRGsb8 vector was transferred to Saccharomyces boulardii. a closely related yeast to S. cerevisiae that has been used as a probiotic in humans. 108CFUs of the S. boulardii iTEMl or a control S. boulardii strain was orally dosed to 8-week-old C57BL / 6J mice, and fecal samples were collected 5 hours after dosing. To demonstrate the production of the iTEM enzyme after the transit through the mouse GI tract, the fecal pellets were incubated with the P-lactamase colorimetric substrate nitrocefin for 30 mins and the liquid fraction was recovered for absorbance measurement (see e.g., Fig. 3). In contrast to the control treatment, the fecal pellets of the mice that were dosed with S. boulardii iTEMl were able to hydrolyze the nitrocefin substrate demonstrating active production of the iTEM P- lactamase and functionality of the engineered intron decoding in an in vivo setting.

[0246] Restricting the expression of genes to their intended host is a safeguard against the dissemination of transgenes in natural microbial populations. This built-in safety feature allows for safer microbe-based technologies, such as probiotics, soil microbes, etc.Example 2

[0247] As discussed in Example 1, the intron found in S. cerevisiae TEF1 can be inserted in the TEM1 beta-lactamase gene without compromising expression in yeast cells. At least one heterologous intron, such as the intron found in S. cerevisiae UBC4, can be inserted in the TEM1 beta-lactamase gene to interrupt its active site codon (see e.g., SEQ ID NOs: 7-10); without wishing to be bound by theory, it is expected that such insertion does not compromise expression of the TEM1 beta-lactamase in fungal cells (when the at least heterologous intron is spliced out), but it is expected to comprise expression of the TEM1 beta-lactamase in non-host bystander cells that cannot splice out the heterologous intron. As another example, at least one heterologous intron, such as the intron found in S. cerevisiae UBC4, can be inserted into other beta-lactamase genes, such as an extended-spectrum betalactamase (ESBL; e.g., CTX-M beta-lactamases, e.g., CTX-M15) (see e.g., SEQ ID NOs: 11-12); without wishing to be bound by theory, it is expected that such insertion does not compromise expression of the beta-lactamase (e.g., ESBL; e.g., CTX-M beta-lactamases, e.g., CTX-M15) in fungal cells (when the at least heterologous intron is spliced out), but it is expected to comprise expression of the beta-lactamase (e.g., ESBL; e.g., CTX-M betalactamases, e.g., CTX-M15) in non-host bystander cells that cannot splice out the heterologous intron.

[0248] Introns are removed from pre-mRNAs during RNA maturation in eukaryotes, using a complex and specialized splicing mechanism involving many eukaryote-specific genes. The splicing machinery is absent in bacteria, therefore the presence of an intron in the TEM1 transgene restricts its expression to the intended fungal expression host and prevents the expression of a functional protein in bacteria. This prevents the undesired expression of the genetic payload in case of unintended transfer of this potential bacterial virulence factor.

[0249] These observations from Example 1 above were generalized to other transgenes, hosts and introns. A shuttle plasmid (yeast-bacteria; see e.g., SEQ ID NO: 5) was created carrying the gene for the fluorescent protein Venus, under the control of a strong hybrid promoter; this construct allows the expression of cytosolic Venus in both S. boulardii (probiotic yeast) and E. coli (human gut bacterium). The Venus transgene is a synthetic gene derived from a eukaryotic sequence. Variants of the plasmid were created in which the intron of the S. cerevisiae TEF1 gene was inserted at three different locations (after nucleotides 141, 212, 341 of the Venus open reading frame). Excluding stop codons, Venus protein open reading frame (ORF) is typically about 714 base pairs (bp) to 720bp long. The intron insertions were chosen in the middle of the ORF, avoiding positions that were too close to the 5’ or 3’ end ofthe ORF. The rationale is that if either the upstream or downstream exons gets translated, each exon would produce a half-protein that is not functional. If one were to put the intron in the ORF, for example too close to the 3’ end of the ORF (e.g., 12bp away from the stop codon), it is contemplated herein that the upstream uninterrupted sequence would be long enough to produce at least partially functional protein fragment. Thus, the intron or introns should be located such that any partial translation product produced if the sequence is horizontally transmitted to a species that cannot splice the intron is not functional.

[0250] Using one of the inserted introns described above as a starting point (n.341 insertion), a second intron (from S. cerevisiae EFBI) was added in the open reading frame to test whether other introns were also functional and whether a double layer of containment would compromise expression in the original host. In a last construct, a variant of the wild-type Venus harbored a deletion of the Tyr66 residue necessary for fluorescence to define the baseline signal for autofluorescence.

[0251] The six plasmids were inserted in both S. boulardii and E. coh. and the cells was grown in their preferred conditions (YPD medium with G418 at 30°C or LB medium with kanamycin at 37°C, respectively). After 24h of growth, the expression of Venus in the saturated cultures was measured by fluorescence spectroscopy (BMG CLARIOSTAR instrument with GFP settings). The results shown in Fig. 4 show that (i.) the presence of one or multiple different introns in the Venus transgene does not prevent expression in the probiotic yeast, (ii.) at all tested locations, introns are sufficient to completely abolish protein expression in bacteria.

[0252] This experiment shows at least the following: (1) the intron-based biocontainment approach can be generalized to transgenes other than TEM1 beta-lactamase; (2) the approach works in probiotic fungal strains (here, S. boulardiiy, (3) multiple locations along the transgene sequence are suitable for insertion of a biocontainment intron; (4) other introns can be used for the purpose of biocontainment; (5) the technique is not restricted to the intron found in S. cerevisiae TEF1; (6) multiple introns can be inserted in the same transgene to reinforce the containment even further; and (7) the intron is sufficient to completely abolish transgene expression in bacteria, even in the absence of other genetic barriers (e.g., speciesspecific gene promoter, secretion signals etc.).

Claims

CLAIMSWhat is claimed herein is:

1. A transgene construct comprising a bacterial polypeptide coding sequence, wherein the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence.

2. The transgene construct of claim 1, wherein the at least one heterologous intron comprises: at least one in-frame stop codon; at least one stop codon in each of a plurality of reading frames; at least one stop codon in each reading frame; or at least two heterologous introns interrupting the bacterial polypeptide coding sequence.

3. The transgene construct of any one of claims 1-2, wherein the at least one heterologous intron is adapted for splicing out of the bacterial polypeptide coding sequence in a fungal host cell.

4. The transgene construct of any one of claims 1-3, wherein the bacterial polypeptide coding sequence is codon-optimized for expression in a fungal host cell.

5. The transgene construct of any one of claims 1-4, wherein the transgene construct comprises regulatory elements permitting expression of the bacterial polypeptide coding sequence in a fungal host cell; optionally wherein the regulatory elements comprise yeast-derived expression control elements operably linked to the bacterial polypeptide coding sequence; optionally wherein the expression control elements comprise a yeast-derived promoter and / or yeast-derived secretion signal; optionally wherein the yeast-derived secretion signal comprises or is derived from the secretion signal of S. cerevisiae mating factor alpha.

6. The transgene construct of any one of claims 3-5, wherein the fungal host cell is a hemiascomycetous yeast; or wherein the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kliiyveromyces. Pichia, Debaryomyces. Candida, Yarrowia, Kazachslania, I.achancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyer ozyma, Hanseniaspora, Rhizophagus, and Ogataea, or wherein the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces unisporus, Saccharomyces servazzii, Saccharomyces kluyveri, Kluyveromyces marxianus, Pichiaangusta, Debaryomyces hansenii, Candida tropicalis, Yarrowia lipolytica, Kluyveromyces laclis. Candida albicans, Candida glabrala, Candida krusei, Candida auris, Lachancea thermotolerans, Malassezia furfur, Malassezia globosa, Malassezia pachydermatis, Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis, Schizosaccharomyces pombe, Komagataella pastoris, Pichia guilliermondii, Meyer ozyma gruessi, Hanseniaspora osmophila, and Rhizophagus irregularis.

7. The transgene construct of any one of claims 1-6, wherein the at least one heterologous intron comprises a 5' splice site, a branch site, and a 3' splice site, optionally wherein the 5' splice site, the branch site, and the 3' splice site are each selected from Table 1.

8. The transgene construct of claim 7, wherein the 5' splice site comprises GTATGT, the branch site comprises TACTAAC, and the 3' splice site comprises YAG.

9. The transgene construct of any one of claims 1-8, wherein the at least one heterologous intron is about 40 nucleotides to about 80 nucleotides long; about 90 nucleotides to about 300 nucleotides long; or about 40 nucleotides to about 1000 nucleotides long.

10. The transgene construct of any one of claims 1-9, wherein the distance between the 5' splice site and the branch site is between about 20 nt to about 950 nt; and / or wherein the distance between the branch site and the 3' splice site is between about 1 nt to about 50 nt.

11. The transgene construct of any one of claims 1-10, wherein the 5' splice site, the branch site, and the 3' splice site are derived from the same species or source; and / or wherein the at least one heterologous intron is derived from a natural fungal intron sequence, is synthetic, or is chimeric.

12. The transgene construct of any one of claims 1-11, wherein the at least one heterologous intron comprises an intron from the Saccharomyces cerevisiae TEF4 gene, from the Saccharomyces cerevisiae EFB 1 gene, or from the Saccharomyces cerevisiae UBC4 gene.

13. The transgene construct of any one of claims 1-12, wherein the location of the at least one heterologous intron within the bacterial polypeptide coding sequence is:(a) in the center of the open reading frame (ORF);(b) in the central portion that is 45% to 55% of the way through the ORF;(c) in the central portion that is 40% to 60% of the way through the ORF;(d) in the central portion that is 30% to 70% of the way through the ORF;(e) in the central portion that is 25% to 75% of the way through the ORF; or(f) in an active site codon of the ORF.

14. The transgene construct of any one of claims 1-13, wherein the at least one heterologous intron comprises at least one functional RNA element, optionally a small nucleolar RNA (snoRNA).

15. The transgene construct of any one of claims 1-14, which is comprised by a plasmid or vector.

16. The transgene construct of any one of claims 1-15, wherein the bacterial polypeptide encoded by the bacterial polypeptide coding sequence comprises an antibiotic resistance enzyme or an antibiotic-degrading enzyme; optionally wherein the antibiotic-degrading enzyme comprises a beta-lactamase enzyme; optionally wherein the beta-lactamase enzyme is a Temoneira-type (TEM) beta-lactamase (e.g., TEM1) or a cefotaximase-Munich-type (CTX-M) beta-lactamase (e.g., CTX-M-15).

17. The transgene construct of any one of claims 1-16, wherein the bacterial polypeptide encoded by the bacterial polypeptide coding sequence can confer an advantage if horizontally transmitted from the fungal host cell to a non-host bystander cell and expressed in the non-host bystander cell; optionally wherein the advantage comprises increased virulence and / or increased fitness; optionally wherein the increased fitness comprises increased nutritional capacity or increased adhesion to another cell; optionally wherein the nutritional capacity comprises utilization of alternative carbon sources and / or nitrogen sources.

18. The transgene construct of claim 17, wherein the non-host bystander cell is a bacterial cell.

19. The transgene construct of any one of claims 1-18, which produces a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a fungal host cell; and / or which does not produce a spliced mRNA encoding the full-length bacterial polypeptide when introduced to a bacterial cell.

20. A system comprising at least two transgene constructs, the system comprising:(a) a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and(b) a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence;wherein one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence.

21. The system of claim 20, wherein:(a) the first portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence;(b) the second portion of the bacterial polypeptide coding sequence is interrupted by at least one heterologous intron sequence; or(c) the first and second portions of the bacterial polypeptide coding sequence are each interrupted by at least one heterologous intron sequence.

22. The system of claim 20 or 21, wherein the first and second portions of the bacterial polypeptide coding sequence are spliced and translated into functional first and second portions of the bacterial polypeptide, optionally wherein the bacterial polypeptide is an antibiotic-degrading enzyme, optionally wherein the antibioticdegrading enzyme is a beta-lactamase enzyme; optionally wherein the beta-lactamase enzyme is a Temoneira-type (TEM) beta-lactamase (e.g., TEM1) or a cefotaximase- Muni ch-type (CTX-M) beta-lactamase (e.g., CTX-M-15).

23. At least one fungal host cell comprising the transgene construct of any one of claims 1-19 or the system of any one of claims 20-22.

24. The at least one fungal host cell of claim 23, wherein the fungal host cell is a probiotic cell.

25. The at least one fungal host cell of claim 23 or 24, wherein the fungal host cell is a hemiascomycetous yeast; or wherein the fungal host cell belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Debaryomyces, Candida, Yarrowia, Kazachslania, I.achancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyer ozyma, Hanseniaspora, Rhizophagus, and Ogataea, or wherein the fungal host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces unisporus, Saccharomyces servazzii, Saccharomyces kluyveri, Kluyveromyces marxianus, Pichia angusta, Debaryomyces hansenii, Candida tropicalis, Yarrowia lipolytica, Kluyveromyces lactis, Candida albicans, Candida glabrata, Candida krusei, Candidaauris, Lachancea thermotolerans, Malassezia furfur, Malassezia globosa, Malassezia pachyder malis. Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis, Schizosaccharomyces pombe, Komagataella pastoris, Pichia guilliermondii, Meyer ozyma gruessi, Hanseniaspora osmophila, and Rhizophagus irregularis.

26. The at least one fungal host cell of any one of claims 23-25, wherein the at least one fungal host cell is dried and viable.

27. The at least one fungal host cell of any one of claims 23-26, wherein a first fungal host cell comprises a first transgene construct comprising a first portion of a bacterial polypeptide coding sequence; and wherein a second fungal host cell comprises a second transgene construct comprising a second portion of the bacterial polypeptide coding sequence; wherein one or both of the first and second portions of the bacterial polypeptide coding sequence are interrupted by at least one heterologous intron sequence.

28. The at least one fungal host cell of claim 27, wherein the first and second portions of the bacterial polypeptide coding sequence are spliced and translated into functional first and second portions of the bacterial polypeptide, optionally wherein the bacterial polypeptide is an antibiotic-degrading enzyme, optionally wherein the antibioticdegrading enzyme is a beta-lactamase enzyme; optionally wherein the beta-lactamase enzyme is a Temoneira-type (TEM) beta-lactamase (e.g., TEM1) or a cefotaximase- Muni ch-type (CTX-M) beta-lactamase (e.g., CTX-M-15).

29. A composition comprising the at least one fungal host cell of any one of claims 23-28, formulated for delivery to a subject.

30. A food composition comprising the at least one fungal host cell of any one of claims 23-28.

31. A medical food comprising the at least one fungal host cell of any one of claims 23- 28.

32. A supplement comprising the at least one fungal host cell of any one of claims 23-28.

33. A probiotic composition comprising the at least one fungal host cell of any one of claims 23-28.

34. A pharmaceutical composition comprising the at least one fungal host cell of any one of claims 23-28 and a pharmaceutically acceptable carrier.

35. A preparation comprising the at least one fungal host cell of any one of claims 23-28 and a carrier permitting application to a plant or to soil.

36. A method of limiting or eliminating potential for horizontal transmission of a bacterial transgene from a fungal host cell to a bacterial cell, the method comprising introducing at least one heterologous intron into the bacterial polypeptide coding sequence of the bacterial transgene.

37. The method of claim 36, wherein the method limits or eliminates horizontal transmission of the bacterial transgene nucleic acid and / or the bacterial polypeptide coding sequence to bacterial cells.

38. A method of preventing or treating a disease or infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the at least one fungal host cell of any one of claims 23-28, the composition of claim 29, the food composition of claim 30, the medical food of claim 31, the supplement of claim 32, the probiotic composition of claim 33, or the pharmaceutical composition of claim 34.

39. A method of promoting heath in a subject, the method comprising administering to the subject an effective amount of the at least one fungal host cell of any one of claims 23-28, the composition of claim 29, the food composition of claim 30, the medical food of claim 31, the supplement of claim 32, the probiotic composition of claim 33, or the pharmaceutical composition of claim 34.

40. The method of claims 38 or 39, wherein the at least one fungal host cell is an agent for bio-control, bio-stimulation, and / or bio-nutrition in the subject.

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