Novel nucleases comprising enhanced performance properties

Abstract

The instant disclosure provides, inter alia, novel nucleases comprising enhanced performance properties. In certain embodiments, the disclosure is therefore related to nucleases having enhanced performance properties (benefits), nuclease compositions and protein preparations thereof, recombinant polynucleotides (expression construct) encoding mature nuclease of the disclosure, recombinant microbial host cells expressing / producing / secreting nucleases of the disclosure, recombinant microbial host cells co-expressing / co-producing nucleases and proteins of interest, and the like.

Description

NB42146-WO-PCT[2]NOVEL NUCLEASES COMPRISING ENHANCED PERFORMANCE PROPERTIES FIELD

[0001] The present disclosure is generally related to the fields of microbial cells, molecular biology, fermentation, protein production, protein recovery and the like. Certain embodiments are related to polynucleotides encoding novel nucleases having enhanced performance properties, such as therm tol erant nucleases capable of degrading DNA at high temperatures, protease tolerant nucleases, sodium phosphate tolerant nucleases, immobilized nucleases and the like.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to International Patent Application No. PCT / CN2025 / 078261, filed February 20, 2025, which is incorporated herein by referenced in its entirety.REFERENCE TO A SEQUENCE LISTING

[0003] The contents of the electronic submission of the text file Sequence Listing, named “NB42146-WO-PCT[2]_SequenceListing.xml” was created on February 09, 2026 and is 384,512 bytes in size, which is hereby incorporated by reference in its entirety.BACKGROUND

[0004] As generally known in the art, the presence of contaminating DNA (e.g., genomic DNA, recombinant DNA) in microbial cell fermentation broths and / or its presence in any down-stream protein recovery processes thereof, can lead to undesirable protein product qualities. DNA in microbial fermentations (e.g., present due to cell lysis) may also increase the broth viscosity in those fermentations, thereby having negative effects on oxygen (O₂) transfer with concomitant undesirable effects on microbial cell growth and / or protein production. Increased viscosity due to the presence of contaminating DNA also has undesirable (negative) effects on the recovery and / or purification of fermentation products (i.e., proteins of interest, e.g., enzymes) by for instance ultrafiltration.

[0005] Endonucleases can degrade DNA (and RNA) and are used in biotechnology for removal of nucleic acids and for viscosity reductions. However, nucleases are typically not thermotolerant, and are easily degraded (inactivated) due to the presence of protease in the microbial cell fermentation broths. Likewise, nucleases are not typically tolerant to high salt concentrations. These inadequacies render most nucleases useless when co-expressed with proteins of interest and / or when applied as additives, for instance, in recovery processes requiring high temperatures (e.g., recovering amylases produced via Bacillus sp. cells)NB42146-WO-PCT[2]or high salt concentrations, protease recovery processes (e.g., recovering proteases produced via Bacillus sp. cells) and the like.

[0006] As described herein, Applicant has identified novel nucleases comprising enhanced performance properties (benefits) addressing ongoing and unmet needs in the art, such as the need for thennotol erant nucleases capable of degrading DNA at high temperatures, the need for thermotolerant nucleases capable of degrading DNA at high temperatures and retaining (nuclease) activity during high temperature recovery processes, the need for nucleases that are tolerant to protease degradation, the need for nucleases that are tolerant to high salt concentrations and the like. In other aspects, the instant disclosure addresses unmet needs in the ait for novel nucleases which can be applied as additives before, during, or after one or more processes including, but not limited to, a fermentation process, a recovery process and / or a formulation process. Likewise, other embodiments address unmet needs in the art for microbial cells capable of co-expressing protein products (<?,#., enzymes of interest) and a novel nuclease of the disclosure, wherein the content or presence of DNA is reduced in the material recovered therefrom.SUMMARY

[0007] As briefly set forth above, certain one or more embodiments of the disclosure are related to novel nuclease proteins comprising enhanced performance properties, such as thermotolerant nucleases capable of degrading DNA at high temperatures, protease tolerant nucleases, sodium phosphate tolerant nucleases, immobilized nucleases and the like. Thus, certain one or more embodiments described herein are directed to nucleases comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30. In particular embodiments, nucleases of the disclosure comprise a protein family or domain selected from a DUF1524 domain, an Endonuclease_NS domain, a PLDc_2 domain and a DNase_NucA / NucB domain. In other embodiments, novel nucleases of the disclosure comprise enhanced performance properties (benefits) such as thermotolerant nucleases, protease resistant nucleases, sodium phosphate tolerant nucleases, immobilized nucleases and combined performance properties thereof. In certain one or more embodiments, nucleases of the disclosure retain substantial DNase activity following incubation at an elevated temperature for at least one (1) hour. In certain embodiments, the elevated temperature is 60°C. In yet other embodiments, nucleases of the disclosure retain substantial DNase activity when added to a fermentation broth comprising a protease (e.g., a subtilisin or metalloprotease) and / or when added to a downstream protein recovery process comprising a protease. In other embodiments, nucleases of the disclosure retain substantial DNase activity in presence of high sodium phosphate concentrations (e.g., at least about 20 mM sodium phosphate or higher). In some embodiments, nucleases of the disclosure are provided in soluble (free) form. In certain other embodiments, nucleases of the disclosure are provided in an immobilized form (e.g., immobilized on a matrix), wherein the immobilized nuclease retains activity.NB42146-WO-PCT[2]

[0008] In some embodiments, the nuclease (protein) may have any number of conservative amino acid substitutions, which are well recognized in the art. The present nucleases may be “precursor,” “immature,” or “full-length,” in which case they include a signal sequence, or “mature,” in which case they lack a signal sequence. Mature forms of the protein are generally the most useful. The present nucleases may also be truncated to remove the N or C-terminus or extended to include additional N or C-terminal residues, so long as the resulting nuclease retains activity.

[0009] In certain other embodiments, nucleases of the disclosure comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, or nuclease fragment (z.e., a truncated subsequence) thereof comprising a DUF1524 domain, an Endonuclease_NS domain, a PLDc_2 domain and a DNase_NucA / NucB domain. In certain embodiments, a truncated nuclease subsequence comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues deleted at the N and / or C-terminus and comprises at least about 70% to 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, wherein the truncated protein retains nuclease activity. In other embodiments, a truncated nuclease subsequence (fragment) comprises at least about 11 to 25 amino acid residues deleted at the N and / or C-terminus and comprises at least about 70% to 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, wherein the truncated protein retains nuclease activity. In certain preferred embodiments, a truncated nuclease fragment of the disclosure retains an intact protein family or domain sequence selected from a DUF1524 domain, an Endonuclease_NS domain, a PLDc_2 domain and a DNase_NucA / NucB domain.

[0010] Certain other embodiments are related to nucleic acids, polynucleotides, expression constructs (cassettes) and the like encoding a nuclease comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30.

[0011] Thus, certain other embodiments of the disclosure are related to polynucleotide expression constructs comprising an upstream promoter operably linked to a downstream nucleic acid encoding a signal (secretion) peptide sequence operably linked to a downstream nucleic acid encoding a mature nuclease comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30.

[0012] Other embodiments are related to recombinant microbial cells co-expressing a protein of interest (POI) and one or more nucleases comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30. For instance, certain embodiments provide Gram-positive bacterial cellsNB42146-WO-PCT[2](e.g., Bacillus sp.) co-expressing a POI and a nuclease of the disclosure. In another example, the disclosure provides filamentous fungal cells (e.g., Trichoderma sp.) co-expressing a POI and a nuclease of the disclosure.

[0013] Certain other embodiments are related to protease resistant nucleases comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature amino acid sequence of any one of SEQ ID NO: 27, 31, 33, 41, 43, 45, 47, 49, 142-176, 178, 182, 183, 184, 187, 188, 189, 190, 192, 193, 194, 196-199, 201, 210 and 220, wherein the nuclease retains substantial DNase activity in the presence of the at least one protease.

[0014] Other embodiments are related to thermotolerant nucleases comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature amino acid sequence of any one of SEQ ID NO: 27, 31, 33,, 43, 45, 47, 49, 133, 142, 143, 144, 145, 147, 148, 150, 151, 157, 158, 159, 160, 161, 162, 165, 168, 169, 171, 172, 174, 175, 176, 177-180, 183, 184, 188, 194, 195, 196, 197, 198, 199, 200, 201, 202, 219 and 220, wherein the nuclease retains substantial DNase activity following incubation at an elevated temperature (e.g., about 50°C, 51 °C, 52°C, 53°C, 54°C, 55 °C, 56°C, 57°C, 58°C, 59°C to about 60°C) for at least about one hour.

[0015] Certain other embodiments are directed to sodium phosphate tolerant nucleases comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature amino acid sequence of any one of SEQ ID NO: 31, 47, 133, 135, 145, 155, 177, 178, 180, 183, 189, 190, 192, 193, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 218 and 219, wherein the nuclease retains substantial DNase activity in presence of high sodium phosphate concentrations (e.g., at least about 20 mM sodium phosphate or higher).

[0016] Certain other embodiments are directed immobilized nucleases comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature amino acid sequence of any one of SEQ ID NO: 176, 213, 300 and 302.

[0017] In other embodiments, the disclosure is related to fermentation broths comprising a nuclease having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30. In yet other embodiments, the disclosure provides fermentation broths comprising a co-expressed protein of interest (POI) and nuclease having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30. For instance, in certain embodiments, the co-expressed POI is an enzyme is selected from the group consisting of acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypcptidascs, catalases, cellulases, chitinases, chymosins, cutinascs, deoxyribonucleases, epimerases, esterases, a-galactosidases, [3-galactosidases, a-glucanases, glucan lysases, endo-0-glucanases,NB42146-WO-PCT[2]glucoamylases, glucose oxidases, a-glucosidases, P-glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lactases, ligases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phosphatases, phytases, polygalacturonases, proteases, peptidases, rhamnogalacturonases, ribonucleases, transferases, transglutaminases, trehalases and xylanases.

[0018] Certain other embodiments of the disclosure provide methods for co-expressing a protein of interest (POI) and a nuclease in a Gram-positive bacterial cell comprising obtaining (or constructing) a Grampositive bacterial cell expressing a POI and introducing into the cell a polynucleotide expression construct (cassette) encoding a nuclease having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, and fermenting the modified cell under conditions for the expression of the POI, wherein the mature nuclease is secreted into the fermentation broth. In particular embodiments of the methods, the modified Gram-positive bacterial cell produces no less than 25% of the POI as compared to a control (isogenic) Gram-positive bacterial cell expressing the same POI, but not co-expressing a nuclease. In certain other embodiments, the disclosure provides methods for co-expressing a POI and a nuclease in a modified filamentous fungal cell comprising obtaining (or constructing) a filamentous fungal cell expressing a POI and introducing into the cell a polynucleotide expression construct encoding a mature nuclease having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, and fermenting the modified cell under conditions for the expression of the POI. In particular embodiments of the methods, the modified filamentous fungal cell produces no less than 25% of the POI as compared to a control (isogenic) filamentous fungal cell expressing the same POI, but not co-expressing a nuclease. In certain embodiments of the methods, the POI is an enzyme, wherein the enzyme is selected from the group consisting of acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, deoxyribonucleases, epimerases, esterases, a-galactosidases, [3-galactosidases, a-glucanases, glucan lysases, endo-0-glucanases, glucoamylases, glucose oxidases, a-glucosidases, p-glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lactases, ligases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phosphatases, phytases, polygalacturonases, proteases, peptidases, rhamnogalacturonases, ribonucleases, transferases, transport proteins, transglutaminases, trehalases, and xylanases.NB42146-WO-PCT[2]

[0019] In still other embodiments, the disclosure provides methods for producing a protein of interest (POI) essentially free of DNA, the method comprising obtaining or constructing a microbial cell expressing a POI and modifying the cell to express a nuclease having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, fermenting the modified cell under suitable conditions for the expression of the POI, wherein the POI in the broth is essentially free of DNA. In certain embodiments of the methods, the fermentation broth is harvesting at the end of fermentation. In other embodiments of the methods, the harvested fermentation broth is subjected to one or more downstream protein recovery processes, wherein the recovered protein is essentially free of DNA. In other embodiments, the modified cell produces no less than 25% of the POI as compared to a control (isogenic) cell expressing the same POI, but not expressing a nuclease.

[0020] In another embodiment, the disclosure provides methods for reducing the DNA content of a fermentation broth in which microbial (host) cells have been fermented, the methods comprising obtaining a microbial cell fermentation broth and treating the broth with a nuclease having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, wherein the treated broth is essentially free of DNA.100211 In yet other embodiments, the disclosure is related to methods for producing a protein of interest (POI) essentially free of DNA comprising obtaining (or constructing) a microbial cell expressing a POI and modifying the cell to express a nuclease having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, fermenting the modified cell under suitable conditions for the expression and secretion of the POI and nuclease into the broth, wherein the POI in the broth is essentially free of DNA. In certain embodiments of the methods, the broth is harvesting at the end of fermentation. In certain other embodiments of the methods, wherein the broth is harvesting at the end offermentation and held at a temperature of 60°C for at least 1 hour. In other embodiments of the methods, the broth is harvesting at the end of fermentation and subjected to one or more downstream protein recovery processes, wherein the recovered protein is essentially free of DNA. In other embodiments, the modified cell produces no less than 25% of the POI as compared to a control cell expressing the same POI, but not expressing a nuclease.

[0022] In other embodiments, the disclosure provides methods for producing a protein of interest (POI) essentially free of DNA, the method comprising fermenting a modified cell expressing a POI under suitable conditions for the expression of the POI, collecting / harvesting the fermentation broth and subjecting the harvested broth to one or more downstream protein recovery processes using an immobilized nucleaseNB42146-WO-PCT[2]composition, wherein the recovered protein of interest is essentially free of DNA. For example, a matrix having a nuclease immobilized thereon may be used in reactors, such as columns, vessels, or tank reactors,.BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 shows the mature amino acid sequences of nucleases co-expressed, as generally set forth in Examples 1-7. More particularly, FIG. 1A shows the mature amino acid sequence of a nuclease named “NsxNucl” (SEQ ID NO: 25), FIG. IB shows the mature amino acid sequence of a nuclease named “MetNuc70” (SEQ ID NO: 27), FIG. 1C shows the mature amino acid sequence of a nuclease named “BhoNuc2” (SEQ ID NO: 29), FIG. ID shows the mature amino acid sequence of a nuclease named “MetNuc71” (SEQ ID NO: 31), FIG. IE shows the mature amino acid sequence of a nuclease named “UstNuc9” (SEQ ID NO: 33), FIG. IF shows the mature amino acid sequence of a nuclease named “BhoNuc3” (SEQ ID NO: 35), FIG. 1G shows the mature amino acid sequence of a nuclease named “BhoNucl” (SEQ ID NO: 37), FIG. 1H shows the mature amino acid sequence of a nuclease named “UstNuc8” (SEQ ID NO: 39), FIG. II shows the mature amino acid sequence of a nuclease named “UstNucll” (SEQ ID NO: 41), FIG. 1J shows the mature amino acid sequence of a nuclease named “MetNucl9” (SEQ ID NO: 43), FIG. IK shows the mature amino acid sequence of a nuclease named “MetNuc72” (SEQ ID NO: 45), FIG. IL shows the mature amino acid sequence of a nuclease named “MetNuc73” (SEQ ID NO: 47), FIG. IM shows the mature amino acid sequence of a nuclease named “MetNuc36” (SEQ ID NO: 49), FIG. IN shows the mature amino acid sequence of a nuclease named “UstNuc7” (SEQ ID NO: 51), FIG. 10 shows the mature amino acid sequence of a nuclease named “TreNucl” (SEQ ID NO: 133) and FIG. IP shows the mature amino acid sequence of a nuclease named “AosNucl” (SEQ ID NO: 135).

[0024] Figure 2 shows the mature amino acid sequences of nucleases co-expressed, as generally described in Examples 8-16. More particularly, FIG. 2A shows the mature amino acid sequence of a nuclease named “PtaNucl” (SEQ ID NO: 142), FIG. 2B shows the mature amino acid sequence of a nuclease named “MabNucl” (SEQ ID NO: 143), FIG. 2C shows the mature amino acid sequence of a nuclease named “TspNuc5” (SEQ ID NO: 144), FIG. 2D shows the mature amino acid sequence of a nuclease named “TlaNucl” (SEQ ID NO: 145), FIG. 2E shows the mature amino acid sequence of a nuclease named “MetNuclO” (SEQ ID NO: 146), FIG. 2F shows the mature amino acid sequence of a nuclease named “MctNucll” (SEQ ID NO: 147), FIG. 2G shows the mature amino acid sequence of a nuclease named “MetNucl8” (SEQ ID NO: 148), FIG. 2H shows the mature amino acid sequence of a nuclease named “MetNuc26” (SEQ ID NO: 149), FIG. 21 shows the mature amino acid sequence of a nuclease named “MetNuc27” (SEQ ID NO: 150), FIG. 2J shows the mature amino acid sequence of a nuclease named “MetNuc35” (SEQ ID NO: 151), FIG. 2K shows the mature amino acid sequence of a nuclease named “MetNuc39” (SEQ ID NO: 152), FIG. 2L shows the mature amino acid sequence of a nuclease namedNB42146-WO-PCT[2]“MetNuc55” (SEQ ID NO: 153), FIG. 2M shows the mature amino acid sequence of a nuclease named “MetNuc66” (SEQ ID NO: 154), FIG. 2N shows the mature amino acid sequence of a nuclease named “MetNuc67” (SEQ ID NO: 155), FIG. 20 shows the mature amino acid sequence of a nuclease named “BspNucll” (SEQ ID NO: 156), FIG. 2P shows the mature amino acid sequence of a nuclease named “PspNuc6” (SEQ ID NO: 157), FIG. 2Q shows the mature amino acid sequence of a nuclease named “BspNucl4” (SEQ ID NO: 158), FIG. 2R shows the mature amino acid sequence of a nuclease named “PmuNuc4” (SEQ ID NO: 159), FIG. 2S shows the mature amino acid sequence of a nuclease named “MidNuc2” (SEQ ID NO: 160), FIG. 2T shows the mature amino acid sequence of a nuclease named “BspNucl6” (SEQ ID NO: 161), FIG. 2U shows the mature amino acid sequence of a nuclease named “PfrNucl” (SEQ ID NO: 162), FIG. 2V shows the mature amino acid sequence of a nuclease named “MidNuc3” (SEQ ID NO: 163), FIG. 2W shows the mature amino acid sequence of a nuclease named “BspNucl7” (SEQ ID NO: 164), FIG. 2X shows the mature amino acid sequence of a nuclease named “BspNucl8” (SEQ ID NO: 165), FIG. 2Y shows the mature amino acid sequence of a nuclease named “BvaNucl” (SEQ ID NO: 166), FIG. 2Z shows the mature amino acid sequence of a nuclease named “BspNuc35” (SEQ ID NO: 167), FIG. 2AA shows the mature amino acid sequence of a nuclease named “BspNucl9” (SEQ ID NO: 168), FIG. 2BB shows the mature amino acid sequence of a nuclease named “BspNuc21” (SEQ ID NO: 169), FIG. 2CC shows the mature amino acid sequence of a nuclease named “BvaNuc2” (SEQ ID NO: 170), FIG. 2DD shows the mature amino acid sequence of a nuclease named “BspNuc23” (SEQ ID NO: 171), FIG. 2EE shows the mature amino acid sequence of a nuclease named “BspNuc26” (SEQ ID NO: 172), FIG. 2FF shows the mature amino acid sequence of a nuclease named “BceNuc2” (SEQ ID NO: 173), FIG. 2GG shows the mature amino acid sequence of a nuclease named “BspNuc27” (SEQ ID NO: 174), FIG. 2HH shows the mature amino acid sequence of a nuclease named “CfiNucl” (SEQ ID NO: 175), FIG. 211 shows the mature amino acid sequence of a nuclease named “BspNuc28” (SEQ ID NO: 176), FIG. 2JJ shows the mature amino acid sequence of a nuclease named “TasNucl” (SEQ ID NO: 177), FIG. 2KK shows the mature amino acid sequence of a nuclease named “CpaNuc4” (SEQ ID NO: 178), FIG. 2LL shows the mature amino acid sequence of a nuclease named “TauNucl” (SEQ ID NO: 179), FIG. 2MM shows the mature amino acid sequence of a nuclease named “SspNuc5” (SEQ ID NO: 180), FIG. 2NN shows the mature amino acid sequence of a nuclease named “EsiNucl” (SEQ ID NO: 181), FIG. 200 shows the mature amino acid sequence of a nuclease named “EspNucl” (SEQ ID NO: 182), FIG. 2PP shows the mature amino acid sequence of a nuclease named “UstNucl” (SEQ ID NO: 183), FIG. 2QQ shows the mature amino acid sequence of a nuclease named “PfiNucl” (SEQ ID NO: 184), FIG. 2RR shows the mature amino acid sequence of a nuclease named “MctNuc42” (SEQ ID NO: 185), FIG. 2SS shows the mature amino acid sequence of a nuclease named “MetNuc44” (SEQ ID NO: 186), FIG. 2TT shows the mature amino acid sequence of a nuclease namedNB42146-WO-PCT[2]“MetNuc53” (SEQ ID NO: 187), FIG. 2UU shows the mature amino acid sequence of a nuclease named “MetNuc54” (SEQ ID NO: 188), FIG.2W shows the mature amino acid sequence of a nuclease named “MetNuc62” (SEQ ID NO: 189), FIG. 2WW shows the mature amino acid sequence of a nuclease named “MetNuc63” (SEQ ID NO: 190), FIG. 2XX shows the mature amino acid sequence of a nuclease named “MetNuc64” (SEQ ID NO: 191), FIG. 2YY shows the mature amino acid sequence of a nuclease named “MetNuc68” (SEQ ID NO: 192), FIG. 2ZZ shows the mature amino acid sequence of a nuclease named “MetNuc69” (SEQ ID NO: 193), FIG. 2AAA shows the mature amino acid sequence of a nuclease named “NspNuc3” (SEQ ID NO: 194), FIG. 2BBB shows the mature amino acid sequence of a nuclease named “JspNucl” (SEQ ID NO: 195), FIG. 2CCC shows the mature amino acid sequence of a nuclease named “RaqNuc2” (SEQ ID NO: 196), FIG. 2DDD shows the mature amino acid sequence of a nuclease named “BspNuc20” (SEQ ID NO: 197), FIG. 2EEE shows the mature amino acid sequence of a nuclease named “BspNuc22” (SEQ ID NO: 198), FIG. 2FFF shows the mature amino acid sequence of a nuclease named “BspNuc25” (SEQ ID NO: 199), FIG. 2GGG shows the mature amino acid sequence of a nuclease named “RarNuc5” (SEQ ID NO: 200), FIG. 2HHH shows the mature amino acid sequence of a nuclease named “BspNuc29” (SEQ ID NO: 201), FIG. 2III shows the mature amino acid sequence of a nuclease named “BspNuc32” (SEQ ID NO: 202), FIG. 2JJJ shows the mature amino acid sequence of a nuclease named “PcuNucl” (SEQ ID NO: 203), FIG. 2KKK shows the mature amino acid sequence of a nuclease named “CthNucl” (SEQ ID NO: 204), FIG. 2LLL shows the mature amino acid sequence of a nuclease named “TpuNucl” (SEQ ID NO: 205), FIG. 2MMM shows the mature amino acid sequence of a nuclease named “AspNuc7” (SEQ ID NO: 206), FIG. 2NNN shows the mature amino acid sequence of a nuclease named “RdeNucl” (SEQ ID NO: 207), FIG. 2000 shows the mature amino acid sequence of a nuclease named “CecNucl” (SEQ ID NO: 208), FIG. 2PPP shows the mature amino acid sequence of a nuclease named “TspNucl4” (SEQ ID NO: 209), FIG. 2QQQ shows the mature amino acid sequence of a nuclease named “SenNuc2” (SEQ ID NO: 210), FIG. 2RRR shows the mature amino acid sequence of a nuclease named “PsyNuc2” (SEQ ID NO: 211), FIG. 2SSS shows the mature amino acid sequence of a nuclease named “MplNucl” (SEQ ID NO: 212), FIG. 2TTT shows the mature amino acid sequence of a nuclease named “GbuNucl” (SEQ ID NO: 213), FIG. 2UUU shows the mature amino acid sequence of a nuclease named “BciNuc3” (SEQ ID NO: 214), FIG. 2VW shows the mature amino acid sequence of a nuclease named “PniNucl” (SEQ ID NO: 215), FIG. 2 WWW shows the mature amino acid sequence of a nuclease named “AglNuc2: (SEQ ID NO: 216), FIG. 2XXX shows the mature amino acid sequence of a nuclease named “GpeNucl” (SEQ ID NO: 217), FIG. 2YYY shows the mature amino acid sequence of a nuclease named “ZmeNucl” (SEQ ID NO: 218), FIG. 2ZZZ shows the mature amino acid sequence of a nuclease named “TspNucl” (SEQ ID NO: 219) and FIG. 2AAAA shows the mature amino acid sequence of a nuclease named “PmuNuc3” (SEQ ID NO: 220).NB42146-WO-PCT[2]

[0025] Figure 3 shows the phylogenetic relationships of ninety-six (96) nucleases from different protein families. In particular, the Neighbor-joining phylogenetic tree (FIG.3) was built and analyzed by Mega11 based on the alignment generated by Muscle. As presented in FIG. 3, the scale bar represents 0.2 substitutions per site, and the clades of four (4) nuclease families are indicated by black arrows.

[0026] Figure 4 shows the mature amino acid sequences of the nucleases named “RarNuc3” (FIG. 4A;SEQ ID NO: 300) and “RdeNuc2” (FIG. 4B; SEQ ID NO: 302), as described in Example 17.NB42146-WO-PCT[2]BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0027] SEQ ID NO: 1 is the mature amino acid sequence of a Bacillus licheniformis a-amylase (amyL).

[0028] SEQ ID NO: 2 is a polynucleotide (DNA) sequence comprising a B. licheniformis amyL gene promoter region.

[0029] SEQ ID NO: 3 is a polynucleotide sequence comprising a Bacillus subtilis aprE 5 '-untranslated region (aprE 5'-UTR).

[0030] SEQ ID NO: 4 is a polynucleotide sequence comprising a B. subtilis spoVG terminator.

[0031] SEQ ID NO: 5 is the amino acid sequence of the NsxNucl signal peptide (NsxNucl_sp).

[0032] SEQ ID NO: 6 is a polynucleotide sequence encoding the NsxNucl_sp.

[0033] SEQ ID NO: 7 is the amino acid sequence of the MetNuc70 signal peptide (MetNuc70_sp).

[0034] SEQ ID NO: 8 is a polynucleotide sequence encoding the MetNuc70_sp.

[0035] SEQ ID NO: 9 is the amino acid sequence of the BhoNuc2 signal peptide (BhoNuc2_sp).

[0036] SEQ ID NO: 10 is a polynucleotide sequence encoding the BhoNuc2_sp.

[0037] SEQ ID NO: 11 is the amino acid sequence of the BhoNuc3 signal peptide (BhoNuc3_sp).

[0038] SEQ ID NO: 12 is a polynucleotide sequence encoding the BhoNuc3_sp.

[0039] SEQ ID NO: 13 is the amino acid sequence of the BhoNucl signal peptide (BhoNucl_sp).

[0040] SEQ ID NO: 14 is a polynucleotide sequence encoding the BhoNucl_sp.

[0041] SEQ ID NO: 15 is the amino acid sequence of the UstNuc8 signal peptide (UstNuc8_sp).

[0042] SEQ ID NO: 16 is a polynucleotide sequence encoding the UstNuc8_sp.

[0043] SEQ ID NO: 17 is the amino acid sequence of the UstNuc11 signal peptide (UstNuc11_sp).

[0044] SEQ ID NO: 18 is a polynucleotide sequence encoding the UstNuc11_sp.

[0045] SEQ ID NO: 19 is the amino acid sequence of the MetNuc36 signal peptide (MetNuc36_sp).

[0046] SEQ ID NO: 20 is a polynucleotide sequence encoding the MetNuc36_sp.

[0047] SEQ ID NO: 21 is the amino acid sequence of a B. licheniformis amyL signal peptide (amyL_sp).

[0048] SEQ ID NO: 22 is a polynucleotide sequence encoding the amyL_sp.

[0049] SEQ ID NO: 23 is the amino acid sequence of a B. licheniformis aprL signal peptide (aprL_sp).

[0050] SEQ ID NO: 24 is a polynucleotide sequence encoding the aprL_sp.

[0051] SEQ ID NO: 25 is the amino acid sequence of the mature nuclease named “NsxNucl”.

[0052] SEQ ID NO: 26 is a polynucleotide sequence encoding the mature NsxNucl nuclease.

[0053] SEQ ID NO: 27 is the amino acid sequence of the mature nuclease named “MetNuc70”.

[0054] SEQ ID NO: 28 is a polynucleotide sequence encoding the mature MetNuc70 nuclease.

[0055] SEQ ID NO: 29 is the amino acid sequence of the mature nuclease named “BhoNuc2”.

[0056] SEQ ID NO: 30 is a polynucleotide sequence encoding the mature BhoNuc2 nuclease.

[0057] SEQ ID NO: 31 is the amino acid sequence of the mature nuclease named “MetNuc71”.NB42146-WO-PCT[2]

[0058] SEQ ID NO: 32 is a polynucleotide sequence encoding the mature MetNuc71 nuclease.

[0059] SEQ ID NO: 33 is the amino acid sequence of the mature nuclease named “UstNuc9”.

[0060] SEQ ID NO: 34 is a polynucleotide sequence encoding the mature UstNuc9 nuclease.

[0061] SEQ ID NO: 35 is the amino acid sequence of the mature nuclease named “BhoNuc3”.

[0062] SEQ ID NO: 36 is a polynucleotide sequence encoding the mature BhoNuc3 nuclease.

[0063] SEQ ID NO: 37 is the amino acid sequence of the mature nuclease named “BhoNucl”.

[0064] SEQ ID NO: 38 is a polynucleotide sequence encoding the mature BhoNucl nuclease.

[0065] SEQ ID NO: 39 is the amino acid sequence of the mature nuclease named “UstNuc8”.

[0066] SEQ ID NO: 40 is a polynucleotide sequence encoding the mature UstNuc8 nuclease.

[0067] SEQ ID NO: 41 is the amino acid sequence of the mature nuclease named “UstNucll”.

[0068] SEQ ID NO: 42 is a polynucleotide sequence encoding the mature UstNuc11 nuclease.

[0069] SEQ ID NO: 43 is the amino acid sequence of the mature nuclease named “MetNucl9”.

[0070] SEQ ID NO: 44 is a polynucleotide sequence encoding the mature MetNucl9 nuclease.

[0071] SEQ ID NO: 45 is the amino acid sequence of the mature nuclease named “MetNuc72”.

[0072] SEQ ID NO: 46 is a polynucleotide sequence encoding the mature MetNuc72 nuclease.

[0073] SEQ ID NO: 47 is the amino acid sequence of the mature nuclease named “MetNuc73”.

[0074] SEQ ID NO: 48 is a polynucleotide sequence encoding the mature MetNuc73 nuclease.

[0075] SEQ ID NO: 49 is the amino acid sequence of the mature nuclease named “MetNuc36”.

[0076] SEQ ID NO: 50 is a polynucleotide sequence encoding the mature MetNuc36 nuclease.

[0077] SEQ ID NO: 51 is the amino acid sequence of the mature nuclease named “UstNuc7”.

[0078] SEQ ID NO: 52 is a polynucleotide sequence encoding the mature UstNuc7 nuclease.

[0079] SEQ ID NO: 53 is the amino acid sequence of the “signal sequence-nuclease construct 1” (“Construct 1”).

[0080] SEQ ID NO: 54 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 1 (Construct 1).

[0081] SEQ ID NO: 55 is the amino acid sequence of the “signal sequence-nuclease construct 2” (“Construct 2”).

[0082] SEQ ID NO: 56 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 2 (Construct 2).

[0083] SEQ ID NO: 57 is the amino acid sequence of the “signal sequence-nuclease construct 3” (“Construct 3”).

[0084] SEQ ID NO: 58 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 3 (Construct 3).NB42146-WO-PCT[2]

[0085] SEQ ID NO: 59 is the amino acid sequence of the “signal sequence-nuclease construct 4” (“Construct 4”).

[0086] SEQ ID NO: 60 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 4 (Construct 4).

[0087] SEQ ID NO: 61 is the amino acid sequence of the “signal sequence-nuclease construct 5” (“Construct 5”).

[0088] SEQ ID NO: 62 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 5 (Construct 5).

[0089] SEQ ID NO: 63 is the amino acid sequence of the “signal sequence-nuclease construct 6” (“Construct 6”).

[0090] SEQ ID NO: 64 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 6 (Construct 6).

[0091] SEQ ID NO: 65 is the amino acid sequence of a “signal sequence-nuclease construct 7” (“Construct 7”).

[0092] SEQ ID NO: 66 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 7 (Construct 7).

[0093] SEQ ID NO: 67 is the amino acid sequence of a “signal sequence-nuclease construct 8” (“Construct 8”).

[0094] SEQ ID NO: 68 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 8 (Construct 8).

[0095] SEQ ID NO: 69 is the amino acid sequence of a “signal sequence-nuclease construct 9” (“Construct 9”).

[0096] SEQ ID NO: 70 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 9 (Construct 9).

[0097] SEQ ID NO: 71 is the amino acid sequence of a “signal sequence-nuclease construct 10” (“Construct 10”).

[0098] SEQ ID NO: 72 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 10 (Construct 10).

[0099] SEQ ID NO: 73 is the amino acid sequence of a “signal sequence-nuclease construct 11” (“Construct 11”).

[0100] SEQ ID NO: 74 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 11 (Construct 11).

[0101] SEQ ID NO: 75 is the amino acid sequence of a “signal scqucncc-nuclcasc construct 12” (“Construct 12”).NB42146-WO-PCT[2]

[0102] SEQ ID NO: 76 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 12 (Construct 12).

[0103] SEQ ID NO: 77 is the amino acid sequence of a “signal sequence-nuclease construct 13” (“Construct 13”).

[0104] SEQ ID NO: 78 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 13 (Construct 13).

[0105] SEQ ID NO: 79 is the amino acid sequence of a “signal sequence-nuclease construct 14” (“Construct 14”).

[0106] SEQ ID NO: 80 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 14 (Construct 14).

[0107] SEQ ID NO: 81 is the amino acid sequence of a “signal sequence-nuclease construct 15” (“Construct 15”).

[0108] SEQ ID NO: 82 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 15 (Construct 15).

[0109] SEQ ID NO: 83 is the amino acid sequence of a “signal sequence-nuclease construct 16” (“Construct 16”).

[0110] SEQ ID NO: 84 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 16 (Construct 16).

[0111] SEQ ID NO: 85 is the amino acid sequence of a “signal sequence-nuclease construct 17” (“Construct 17”).

[0112] SEQ ID NO: 86 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 17 (Construct 17).

[0113] SEQ ID NO: 87 is the amino acid sequence of a “signal sequence-nuclease construct 18” (“Construct 18”).

[0114] SEQ ID NO: 88 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 18 (Construct 18).

[0115] SEQ ID NO: 89 is the amino acid sequence of a “signal sequence-nuclease construct 19” (“Construct 19”).

[0116] SEQ ID NO: 90 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 19 (Construct 19).

[0117] SEQ ID NO: 91 is the amino acid sequence of a “signal sequence-nuclease construct 20” (“Construct 20”).

[0118] SEQ ID NO: 92 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 20 (Construct 20).NB42146-WO-PCT[2]

[0119] SEQ ID NO: 93 is the amino acid sequence of a “signal sequence -nuclease construct 21” (“Construct 21”).

[0120] SEQ ID NO: 94 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 21 (Construct 21).

[0121] SEQ ID NO: 95 is the amino acid sequence of a “signal sequence-nuclease construct 22” (“Construct 22”).

[0122] SEQ ID NO: 96 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 22 (Construct 22).

[0123] SEQ ID NO: 97 is the amino acid sequence of a “signal sequence-nuclease construct 23” (“Construct 23”).

[0124] SEQ ID NO: 98 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 23 (Construct 23).

[0125] SEQ ID NO: 99 is the amino acid sequence of a “signal sequence-nuclease construct 24” (“Construct 24”).

[0126] SEQ ID NO: 100 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 24 (Construct 24).

[0127] SEQ ID NO: 101 is the amino acid sequence of a “signal sequence-nuclease construct 25” (“Construct 25”).

[0128] SEQ ID NO: 102 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 25 (Construct 25).

[0129] SEQ ID NO: 103 is the amino acid sequence of a “signal sequence-nuclease construct 26” (“Construct 26”).

[0130] SEQ ID NO: 104 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 26 (Construct 26).

[0131] SEQ ID NO: 105 is the amino acid sequence of a “signal sequence-nuclease construct 27” (“Construct 27”).

[0132] SEQ ID NO: 106 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 27 (Construct 27).

[0133] SEQ ID NO: 107 is the amino acid sequence of a “signal sequence-nuclease construct 28” (“Construct 28”).

[0134] SEQ ID NO: 108 is a synthetic polynucleotide sequence encoding the signal sequence-nuclease construct 28 (Construct 28).

[0135] SEQ ID NO: 109 is a polynucleotide sequence encoding a variant Bacillus amyloliquefaciens precursor subtilisin protease.NB42146-WO-PCT[2]

[0136] SEQ ID NO: 110 is the amino acid sequence of the variant B. amyloliquefaciens subtilisin (precursor) protease.

[0137] SEQ ID NO: 111 is a polynucleotide sequence encoding a variant Bacillus gibsonii precursor subtilisin protease.

[0138] SEQ ID NO: 112 is the amino acid sequence of the variant B. gibsonii subtilisin (precursor) protease.

[0139] SEQ ID NO: 113 is a polynucleotide sequence encoding a Thermobifida cellulosilytica precursor serine protease.

[0140] SEQ ID NO: 114 is the amino acid sequence of the T. cellulosilytica serine (precursor) protease.

[0141] SEQ ID NO: 115 is a polynucleotide sequence encoding a mature B. subtilis endo-beta-1,3-1,4 glucanase (BglS).

[0142] SEQ ID NO: 116 is the amino acid sequence of the mature BglS.

[0143] SEQ ID NO: 117 is a polynucleotide sequence comprising a B. subtilis 5' aprE gene flanking region (5' aprE-FR).

[0144] SEQ ID NO: 118 is a polynucleotide sequence comprising a B. subtilis P2 promoter region.

[0145] SEQ ID NO: 119 is a polynucleotide sequence encoding a B. subtilis aprE signal peptide (aprE_sp).

[0146] SEQ ID NO: 120 is the amino acid sequence of the aprE_sp.

[0147] SEQ ID NO: 121 is a polynucleotide sequence comprising a B. amyloliquefaciens BPN' terminator.

[0148] SEQ ID NO: 122 is a polynucleotide sequence comprising a MetNuc71 promoter region.

[0149] SEQ ID NO: 123 is a polynucleotide sequence encoding a MetNuc71 signal peptide (MetNuc71_sp).

[0150] SEQ ID NO: 124 the amino acid sequence of the MetNuc71_sp.

[0151] SEQ ID NO: 125 is a polynucleotide sequence comprising a B. subtilis 3' aprE gene flanking region (3' aprE-FR).

[0152] SEQ ID NO: 126 is a polynucleotide sequence encoding the BglS signal peptide (BglS_sp).

[0153] SEQ ID NO: 127 is the amino acid sequence of the BglS_sp.

[0154] SEQ ID NO: 128 is the amino acid sequence of a native B. subtilis aprE protease.

[0155] SEQ ID NO: 129 is the amino acid sequence of a native B. subtilis nprE protease.

[0156] SEQ ID NO: 130 is the amino acid sequence of a native B. subtilis bpr protease.

[0157] SEQ ID NO: 131 is the amino acid sequence of a native B. subtilis epr protease.

[0158] SEQ ID NO: 132 is a synthetic polynucleotide sequence encoding the mature nuclease named “TreNucl”.

[0159] SEQ ID NO: 133 is the amino acid sequence of the mature TreNucl nuclease.

[0160] SEQ ID NO: 134 is a polynucleotide sequence comprising a B. subtilis aprE gene promoter region.NB42146-WO-PCT[2]

[0161] SEQ ID NO: 135 is the amino acid sequence of the mature nuclease named “AosNucl”.

[0162] SEQ ID NO: 136 is a polynucleotide sequence encoding the mature AosNucl nuclease.

[0163] SEQ ID NO: 137 is a polynucleotide sequence comprising a Trichoderma reesei gpdl gene promoter region.

[0164] SEQ ID NO: 138 is a polynucleotide sequence encoding a T. reesei pepl signal peptide (pepl_sp).

[0165] SEQ ID NO: 139 is a polynucleotide sequence comprising a T. reesei CBHI terminator.

[0166] SEQ ID NO: 140 is the amino acid sequence of a T. reesei trehalase.

[0167] SEQ ID NO: 141 is a polynucleotide sequence encoding the T. reesei trehalase.

[0168] SEQ ID NO: 142 is the amino acid sequence of the mature nuclease named “PtaNucl”.

[0169] SEQ ID NO: 143 is the amino acid sequence of the mature nuclease named “MabNucl”.

[0170] SEQ ID NO: 144 is the amino acid sequence of the mature nuclease named “TspNuc5”.

[0171] SEQ ID NO: 145 is the amino acid sequence of the mature nuclease named “TlaNucl”.

[0172] SEQ ID NO: 146 is the amino acid sequence of the mature nuclease named “MetNuclO”.

[0173] SEQ ID NO: 147 is the amino acid sequence of the mature nuclease named “MetNucll”.

[0174] SEQ ID NO: 148 is the amino acid sequence of the mature nuclease named “MetNucl8”.

[0175] SEQ ID NO: 149 is the amino acid sequence of the mature nuclease named “MetNuc26”.

[0176] SEQ ID NO: 150 is the amino acid sequence of the mature nuclease named “MetNuc27”.

[0177] SEQ ID NO: 151 is the amino acid sequence of the mature nuclease named “MetNuc35”.

[0178] SEQ ID NO: 152 is the amino acid sequence of the mature nuclease named “MetNuc39”.

[0179] SEQ ID NO: 153 is the amino acid sequence of the mature nuclease named “MetNuc55”.

[0180] SEQ ID NO: 154 is the amino acid sequence of the mature nuclease named “MetNuc66”.

[0181] SEQ ID NO: 155 is the amino acid sequence of the mature nuclease named “MetNuc67”

[0182] SEQ ID NO: 156 is the amino acid sequence of the mature nuclease named “BspNucll”.

[0183] SEQ ID NO: 157 is the amino acid sequence of the mature nuclease named “PspNuc6”.

[0184] SEQ ID NO: 158 is the amino acid sequence of the mature nuclease named “BspNucl4”.

[0185] SEQ ID NO: 159 is the amino acid sequence of the mature nuclease named “PmuNuc4”.

[0186] SEQ ID NO: 160 is the amino acid sequence of the mature nuclease named “MidNuc2”.

[0187] SEQ ID NO: 161 is the amino acid sequence of the mature nuclease named “BspNucl6”.

[0188] SEQ ID NO: 162 is the amino acid sequence of the mature nuclease named “PfrNucl”.

[0189] SEQ ID NO: 163 is the amino acid sequence of the mature nuclease named “MidNuc3”.

[0190] SEQ ID NO: 164 is the amino acid sequence of the mature nuclease named “BspNucl7”.

[0191] SEQ ID NO: 165 is the amino acid sequence of the mature nuclease named “BspNucl8”.

[0192] SEQ ID NO: 166 is the amino acid sequence of the mature nuclease named “BvaNucl”.

[0193] SEQ ID NO: 167 is the amino acid sequence of the mature nuclease named “BspNuc35”.NB42146-WO-PCT[2]

[0194] SEQ ID NO: 168 is the amino acid sequence of the mature nuclease named“BspNucl9”.

[0195] SEQ ID NO: 169 is the amino acid sequence of the mature nuclease named “BspNuc21”.

[0196] SEQ ID NO: 170 is the amino acid sequence of the mature nuclease named “BvaNuc2”.

[0197] SEQ ID NO: 171 is the amino acid sequence of the mature nuclease named “BspNuc23”.

[0198] SEQ ID NO: 172 is the amino acid sequence of the mature nuclease named “BspNuc26”.

[0199] SEQ ID NO: 173 is the amino acid sequence of the mature nuclease named “BceNuc2”.

[0200] SEQ ID NO: 174 is the amino acid sequence of the mature nuclease named “BspNuc27”.

[0201] SEQ ID NO: 175 is the amino acid sequence of the mature nuclease named “CfiNucl”.

[0202] SEQ ID NO: 176 is the amino acid sequence of the mature nuclease named “BspNuc28”.

[0203] SEQ ID NO: 177 is the amino acid sequence of the mature nuclease named “TasNucl”.

[0204] SEQ ID NO: 178 is the amino acid sequence of the mature nuclease named “CpaNuc4”.

[0205] SEQ ID NO: 179 is the amino acid sequence of the mature nuclease named “TauNucl”.

[0206] SEQ ID NO: 180 is the amino acid sequence of the mature nuclease named “SspNuc5”.

[0207] SEQ ID NO: 181 is the amino acid sequence of the mature nuclease named “EsiNucl”.

[0208] SEQ ID NO: 182 is the amino acid sequence of the mature nuclease named “EspNucl”.

[0209] SEQ ID NO: 183 is the amino acid sequence of the mature nuclease named “UstNucl”.

[0210] SEQ ID NO: 184 is the amino acid sequence of the mature nuclease named “PfiNucl”. |0211| SEQ ID NO: 185 is the amino acid sequence of the mature nuclease named “MetNuc42”.

[0212] SEQ ID NO: 186 is the amino acid sequence of the mature nuclease named “MetNuc44”.

[0213] SEQ ID NO: 187 is the amino acid sequence of the mature nuclease named “MetNuc53”.

[0214] SEQ ID NO: 188 is the amino acid sequence of the mature nuclease named “MetNuc54”.

[0215] SEQ ID NO: 189 is the amino acid sequence of the mature nuclease named “MetNuc62”.

[0216] SEQ ID NO: 190 is the amino acid sequence of the mature nuclease named “MetNuc63”.

[0217] SEQ ID NO: 191 is the amino acid sequence of the mature nuclease named “MetNuc64”.

[0218] SEQ ID NO: 192 is the amino acid sequence of the mature nuclease named “MetNuc68”.

[0219] SEQ ID NO: 193 is the amino acid sequence of the mature nuclease named “MetNuc69”.

[0220] SEQ ID NO: 194 is the amino acid sequence of the mature nuclease named “NspNuc3”.

[0221] SEQ ID NO: 195 is the amino acid sequence of the mature nuclease named “JspNucl”.

[0222] SEQ ID NO: 196 is the amino acid sequence of the mature nuclease named “RaqNuc2”.

[0223] SEQ ID NO: 197 is the amino acid sequence of the mature nuclease named “BspNuc20”.

[0224] SEQ ID NO: 198 is the amino acid sequence of the mature nuclease named “BspNuc22”.

[0225] SEQ ID NO: 199 is the amino acid sequence of the mature nuclease named “BspNuc25”.

[0226] SEQ ID NO: 200 is the amino acid sequence of the mature nuclease named “RarNuc5”.

[0227] SEQ ID NO: 201 is the amino acid sequence of the mature nuclease named “BspNuc29”.NB42146-WO-PCT[2]

[0228] SEQ ID NO: 202 is the amino acid sequence of the mature nuclease named “BspNuc32”.

[0229] SEQ ID NO: 203 is the amino acid sequence of the mature nuclease named “PcuNucl”.

[0230] SEQ ID NO: 204 is the amino acid sequence of the mature nuclease named “CthNucl”.

[0231] SEQ ID NO: 205 is the amino acid sequence of the mature nuclease named “TpuNucl”.

[0232] SEQ ID NO: 206 is the amino acid sequence of the mature nuclease named “AspNuc7”.

[0233] SEQ ID NO: 207 is the amino acid sequence of the mature nuclease named “RdeNucl”.

[0234] SEQ ID NO: 208 is the amino acid sequence of the mature nuclease named “CecNucl”.

[0235] SEQ ID NO: 209 is the amino acid sequence of the mature nuclease named “TspNucl4”.

[0236] SEQ ID NO: 210 is the amino acid sequence of the mature nuclease named “SenNuc2”.

[0237] SEQ ID NO: 211 is the amino acid sequence of the mature nuclease named “PsyNuc2”.

[0238] SEQ ID NO: 212 is the amino acid sequence of the mature nuclease named “MplNucl”.

[0239] SEQ ID NO: 213 is the amino acid sequence of the mature nuclease named “GbuNucl”.

[0240] SEQ ID NO: 214 is the amino acid sequence of the mature nuclease named “BciNuc3”.

[0241] SEQ ID NO: 215 is the amino acid sequence of the mature nuclease named “PniNucl”.

[0242] SEQ ID NO: 216 is the amino acid sequence of the mature nuclease named “AglNuc2”.

[0243] SEQ ID NO: 217 is the amino acid sequence of the mature nuclease named “GpeNucl”.

[0244] SEQ ID NO: 218 is the amino acid sequence of the mature nuclease named “ZmeNucl”.

[0245] SEQ ID NO: 219 is the amino acid sequence of the mature nuclease named “TspNucl”.

[0246] SEQ ID NO: 220 is the amino acid sequence of the mature nuclease named “PmuNuc3”.

[0247] SEQ ID NO: 221 is a codon optimized DNA sequence encoding the mature nuclease PtaNucl.

[0248] SEQ ID NO: 222 is a codon optimized DNA sequence encoding the mature nuclease MabNucl.

[0249] SEQ ID NO: 223 is a codon optimized DNA sequence encoding the mature nuclease TspNuc5.

[0250] SEQ ID NO: 224 is a codon optimized DNA sequence encoding the mature nuclease TlaNucl.

[0251] SEQ ID NO: 225 is a codon optimized DNA sequence encoding the mature nuclease MetNuclO.

[0252] SEQ ID NO: 226 is a codon optimized DNA sequence encoding the mature nuclease MetNucl 1.

[0253] SEQ ID NO: 227 is a codon optimized DNA sequence encoding the mature nuclease MetNucl 8.

[0254] SEQ ID NO: 228 is a codon optimized DNA sequence encoding the mature nuclease MetNuc26.

[0255] SEQ ID NO: 229 is a codon optimized DNA sequence encoding the mature nuclease MetNuc27.

[0256] SEQ ID NO: 230 is a codon optimized DNA sequence encoding the mature nuclease MetNuc35.

[0257] SEQ ID NO: 231 is a codon optimized DNA sequence encoding the mature nuclease MetNuc39.

[0258] SEQ ID NO: 232 is a codon optimized DNA sequence encoding the mature nuclease MetNuc55.

[0259] SEQ ID NO: 233 is a codon optimized DNA sequence encoding the mature nuclease MetNuc66.

[0260] SEQ ID NO: 234 is a codon optimized DNA sequence encoding the mature nuclease MetNuc67.

[0261] SEQ ID NO: 235 is a codon optimized DNA sequence encoding the mature nuclease BspNucl 1.NB42146-WO-PCT[2]

[0262] SEQ ID NO: 236 is a codon optimized DNA sequence encoding the mature nuclease PspNuc6.

[0263] SEQ ID NO: 237 is a codon optimized DNA sequence encoding the mature nuclease BspNucl4.

[0264] SEQ ID NO: 238 is a codon optimized DNA sequence encoding the mature nuclease PmuNuc4.

[0265] SEQ ID NO: 239 is a codon optimized DNA sequence encoding the mature nuclease MidNuc2.

[0266] SEQ ID NO: 240 is a codon optimized DNA sequence encoding the mature nuclease BspNucl6.

[0267] SEQ ID NO: 241 is a codon optimized DNA sequence encoding the mature nuclease PfrNucl.

[0268] SEQ ID NO: 242 is a codon optimized DNA sequence encoding the mature nuclease MidNuc3.

[0269] SEQ ID NO: 243 is a codon optimized DNA sequence encoding the mature nuclease BspNuc17.

[0270] SEQ ID NO: 244 is a codon optimized DNA sequence encoding the mature nuclease BspNucl8.

[0271] SEQ ID NO: 245 is a codon optimized DNA sequence encoding the mature nuclease BvaNucl.

[0272] SEQ ID NO: 246 is a codon optimized DNA sequence encoding the mature nuclease BspNuc35.

[0273] SEQ ID NO: 247 is a codon optimized DNA sequence encoding the mature nuclease BspNucl9.

[0274] SEQ ID NO: 248 is a codon optimized DNA sequence encoding the mature nuclease BspNuc21.

[0275] SEQ ID NO: 249 is a codon optimized DNA sequence encoding the mature nuclease BvaNuc2.

[0276] SEQ ID NO: 250 is a codon optimized DNA sequence encoding the mature nuclease BspNuc23.

[0277] SEQ ID NO: 251 is a codon optimized DNA sequence encoding the mature nuclease BspNuc26.

[0278] SEQ ID NO: 252 is a codon optimized DNA sequence encoding the mature nuclease BceNuc2.

[0279] SEQ ID NO: 253 is a codon optimized DNA sequence encoding the mature nuclease BspNuc27.

[0280] SEQ ID NO: 254 is a codon optimized DNA sequence encoding the mature nuclease CfiNucl.

[0281] SEQ ID NO: 255 is a codon optimized DNA sequence encoding the mature nuclease BspNuc28.

[0282] SEQ ID NO: 256 is a sequence encoding the mature nuclease TasNucl.

[0283] SEQ ID NO: 257 is a codon optimized DNA sequence encoding the mature nuclease CpaNuc4.

[0284] SEQ ID NO: 258 is a codon optimized DNA sequence encoding the mature nuclease TauNucl.

[0285] SEQ ID NO: 259 is a codon optimized DNA sequence encoding the mature nuclease SspNuc5.

[0286] SEQ ID NO: 260 is a codon optimized DNA sequence encoding the mature nuclease EsiNucl.

[0287] SEQ ID NO: 261 is a codon optimized DNA sequence encoding the mature nuclease EspNucl.

[0288] SEQ ID NO: 262 is a codon optimized DNA sequence encoding the mature nuclease UstNuc 1.

[0289] SEQ ID NO: 263 is a codon optimized DNA sequence encoding the mature nuclease PfiNucl.

[0290] SEQ ID NO: 264 is a codon optimized DNA sequence encoding the mature nuclease MetNuc42.

[0291] SEQ ID NO: 265 is a codon optimized DNA sequence encoding the mature nuclease MetNuc44.

[0292] SEQ ID NO: 266 is a codon optimized DNA sequence encoding the mature nuclease MetNuc53.

[0293] SEQ ID NO: 267 is a codon optimized DNA sequence encoding the mature nuclease MetNuc54.

[0294] SEQ ID NO: 268 is a codon optimized DNA sequence encoding the mature nuclease MetNuc62.

[0295] SEQ ID NO: 269 is a codon optimized DNA sequence encoding the mature nuclease MetNuc63.NB42146-WO-PCT[2]

[0296] SEQ ID NO: 270 is a codon optimized DNA sequence encoding the mature nuclease MetNuc64

[0297] SEQ ID NO: 271 is a codon optimized DNA sequence encoding the mature nuclease MetNuc68

[0298] SEQ ID NO: 272 is a codon optimized DNA sequence encoding the mature nuclease MetNuc69

[0299] SEQ ID NO: 273 is a codon optimized DNA sequence encoding the mature nuclease NspNuc3.

[0300] SEQ ID NO: 274 is a codon optimized DNA sequence encoding the mature nuclease JspNucl.

[0301] SEQ ID NO: 275 is a codon optimized DNA sequence encoding the mature nuclease RaqNuc2.

[0302] SEQ ID NO: 276 is a codon optimized DNA sequence encoding the mature nuclease BspNuc20.

[0303] SEQ ID NO: 277 is a codon optimized DNA sequence encoding the mature nuclease BspNuc22.

[0304] SEQ ID NO: 278 is a codon optimized DNA sequence encoding the mature nuclease BspNuc25.

[0305] SEQ ID NO: 279 is a codon optimized DNA sequence encoding the mature nuclease RarNuc5.

[0306] SEQ ID NO: 280 is a codon optimized DNA sequence encoding the mature nuclease BspNuc29.

[0307] SEQ ID NO: 281 is a codon optimized DNA sequence encoding the mature nuclease BspNuc32.

[0308] SEQ ID NO: 282 is a DNA sequence encoding the mature nuclease PcuNucl.

[0309] SEQ ID NO: 283 is a codon optimized DNA sequence encoding the mature nuclease CthNucl.

[0310] SEQ ID NO: 284 is a codon optimized DNA sequence encoding the mature nuclease TpuNucl.

[0311] SEQ ID NO: 285 is a codon optimized DNA sequence encoding the mature nuclease AspNuc7.

[0312] SEQ ID NO: 286 is a codon optimized DNA sequence encoding the mature nuclease RdeNucl.

[0313] SEQ ID NO: 287 is a codon optimized DNA sequence encoding the mature nuclease CecNuc1.

[0314] SEQ ID NO: 288 is a codon optimized DNA sequence encoding the mature nuclease TspNucl4.

[0315] SEQ ID NO: 289 is a codon optimized DNA sequence encoding the mature nuclease SenNuc2.

[0316] SEQ ID NO: 290 is a codon optimized DNA sequence encoding the mature nuclease PsyNuc2.

[0317] SEQ ID NO: 291 is a codon optimized DNA sequence encoding the mature nuclease MplNucl.

[0318] SEQ ID NO: 292 is a codon optimized DNA sequence encoding the mature nuclease GbuNuc1.

[0319] SEQ ID NO: 293 is a codon optimized DNA sequence encoding the mature nuclease BciNuc3.

[0320] SEQ ID NO: 294 is a codon optimized DNA sequence encoding the mature nuclease PniNucl.

[0321] SEQ ID NO: 295 is a codon optimized DNA sequence encoding the mature nuclease AglNuc2.

[0322] SEQ ID NO: 296 is a codon optimized DNA sequence encoding the mature nuclease GpeNucl.

[0323] SEQ ID NO: 297 is a codon optimized DNA sequence encoding the mature nuclease ZmeNucl.

[0324] SEQ ID NO: 298 is a codon optimized DNA sequence encoding the mature nuclease TspNucl.

[0325] SEQ ID NO: 299 is a codon optimized DNA sequence encoding the mature nuclease PmuNuc3.

[0326] SEQ ID NO: 300 is the amino acid sequence of the mature nuclease named “RarNuc3”.

[0327] SEQ ID NO: 301 is a polynucleotide sequence encoding the mature RarNuc3 nuclease.

[0328] SEQ ID NO: 302 is the amino acid sequence of the mature nuclease named “RdcNuc2”.

[0329] SEQ ID NO: 303 is a polynucleotide sequence encoding the mature RdeNuc2 nuclease.NB42146-WO-PCT[2]DETAILED DESCRIPTIONI. OVERVIEW

[0330] As generally set forth above, the presence of contaminating DNA (e.g., genomic DNA, recombinant DNA) in microbial cell fermentation broths and / or its presence in down-stream (protein) recovery processes and / or (protein) formulation processes, can lead to undesirable or negative (protein) product qualities / properties. More particularly, as described herein and presented in the Examples below, Applicant has identified novel nucleases having enhanced performance properties (enhanced benefits) that address ongoing and unmet needs in the art, including, but not limited to, the need for nucleases capable of degrading DNA at high temperatures (i.e., thermotolerant nucleases), the need for nucleases capable of retaining nuclease activity during high temperature protein recovery processes, the need for nucleases that are tolerant (resistant) to protease degradation, the need for nucleases that are tolerant (resistant) to phosphate stress conditions, the need for nucleases that are tolerant (resistant) to salt stress conditions, and the like. Other embodiments of the disclosure address the needs need for novel nucleases which can be applied as additives before, during, or after one or more processes including, but not limited to, fermentation processes, recovery processes and / or formulation processes, and / or the need for microbial cells capable of co-expressing protein products (e.g., enzymes of interest) and a novel nuclease (or several nucleases) of the disclosure, wherein the content, size or presence of DNA is reduced in the material recovered therefrom, and / or the viscosity of the fermentation broth is reduced, and / or the need for microbial cells capable of co-expressing a protein of interest (PO1) and a novel nuclease (or several nucleases), wherein microbial cells retain the ability to express / produce a significant amount of the POI relative to microbial control cells that only express the POI, and the like.II. DEFINITIONS

[0331] Terms not defined herein should be accorded their ordinary meaning as used in the art.

[0332] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present compositions and methods apply. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present compositions and methods, representative illustrative methods and materials are now described. All publications and patents cited herein are incorporated by reference in their entirety.

[0333] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only”, “excluding”, “not including” and the like, in connection with the recitation of claim elements, or use of a “negative” limitation or proviso thereof.NB42146-WO-PCT[2]

[0334] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present compositions and methods described herein. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0335] As used herein, a “microbial cell”, a “microbial host cell”, a “microbial strain” and the like refer to microbial cells that have the capacity to act as a host or expression vehicle for a newly introduced (heterologous) DNA sequence. In certain embodiments, a microbial host cell is selected from the group consisting of a Gram negative bacterial cell, a Gram positive bacterial cell, a filamentous fungal cell and a yeast cell (e.g., recombinant Bacilli cells, recombinant E. coli cells, recombinant fungal cells, recombinant yeast cells).

[0336] As used herein, the terms “Gram positive bacteria”, Gram positive bacterial strains” and “Gram positive bacterial cells” are the same meaning of the terms used in the art. For example, Gram positive bacteria include all strains of Actinobacteria and Firmicutes. In certain embodiments, such Gram positive bacteria are of the classes Bacilli, Clostridia and Mollicutes.

[0337] As used herein, the term “Ascomycete fungal cell” refers to any organism in the Division Ascomycota in the Kingdom Fungi. Examples of Ascomycetes fungal cells include, but are not limited to, filamentous fungi in the subphylum Pezizomycotina, such as Trichoderma sp., Aspergillus sp., Myceliophthora sp., Penicillium sp., and the like.

[0338] As used herein, the terms “filamentous fungus”, “filamentous fungal strains” and “filamentous fungal cells” refers to all filamentous forms of the subdivision Eumycota and Oomycota. For example, filamentous fungi include, without limitation, Acremonium, Aspergillus, Emericella, Fusarium, Elumicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and Trichoderma species.

[0339] As used herein, the terms “recombinant” or “non-natural” refer to an organism, microorganism, cell, nucleic acid molecule, or vector that has at least one engineered genetic alteration, or has been modified by the introduction of a heterologous nucleic acid molecule, or refer to a cell (e.g., a microbial cell) that has been altered such that the expression of a heterologous or endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from a non-natural cell or is progeny of a non-natural cell having one or more such modifications. Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, or other nucleic acid molecule additions, deletions, substitutions or other functional alteration of a cell’s genetic material. For example, recombinant cells may express genes or other nucleic acid molecules that are not found in identical or homologous form within a native (wild-type) cell (e.g., a fusion or chimeric protein), or may provide anNB42146-WO-PCT[2]altered expression pattern of endogenous genes, such as being over-expressed, under-expressed, minimally expressed, or not expressed at all.

[0340] The term “derived” encompasses the terms “originated”, “obtained,” “obtainable,” and “created,” and generally indicates that one specified material or composition finds its origin in another specified material or composition, or has features that can be described with reference to another specified material or composition.

[0341] As used herein, the phrases “heterologous a-amylase” and “amylase reporter protein” may be used interchangeably, wherein the mature amino acid sequence of the amylase reporter protein is set forth as SEQ ID NO: 1.

[0342] As used herein, the phrases “B. amyloliquefaciens subtilisin’’ and “B. amyloliquefaciens reporter protease” may be used interchangeably, wherein the mature amino acid sequence of the B. amyloliquefaciens reporter protease is set forth as SEQ ID NO: 110.

[0343] As used herein, the terms “B. gibsonii subtilisin’’ and “B. gibsonii reporter protease” may be used interchangeably, wherein the amino acid sequence of the B. gibsonii reporter protease is set forth as SEQ ID NO: 112.

[0344] As used herein, the terms “T. cellulosilytica subtilisin’’ and “T. cellulosilytica reporter protease” may be used interchangeably, wherein the amino acid sequence of the T. cellulosilytica reporter protease is set forth as SEQ ID NO: 114.

[0345] As used herein, the term “f>-glucanase” and “[ -glucanase reporter protein” may be used interchangeably, wherein the amino acid sequence -glucanase reporter protein is set forth as SEQ ID NO: 116.

[0346] As used herein, the terms “Irehalase”, “trehalase enzyme” and “trehalase reporter protein” may be used interchangeably, and refer to the trehalase reporter protein set forth as SEQ ID NO: 140.

[0347] As used herein, the terms “deoxyribonuclease” (abbreviated “DNase”) and “nuclease” are used interchangeably, and refer to enzymes capable of degrading DNA by non-specifically cleaving (hydrolyzing) a phosphodiester bond in the DNA backbone. DNase proteins (enzymes) of the disclosure are thus defined as non-specific DNases, in contrast to nucleotide sequence-specific DNases, such as restriction enzymes.

[0348] The Pfam database is a widely used resource for classifying protein sequences into families and domains (Mistry et al., 2021). As used herein, a protein “domain” is a region of a protein’s polypeptide chain that is self-stabilizing and that folds independently from the rest. For instance, each domain forms a compact folded three-dimensional structure.NB42146-WO-PCT[2]

[0349] In certain embodiments, the term nuclease particularly refers to four groups of polypeptides (i.e., domains) herein referred to as “DUF1524”, “DNase NucA / NucB”, “PLDc_2” and “Endonuclease_NS” polypeptides (e.g., see FIG. 3).

[0350] Thus, as used herein, terms and phrases such as “DUF1524” domain (PF07510), “Deoxyribonuclease (DNase) NucA / NucB” family (PF14040), “PLDc_2” domain (PF13091), and / or “Endonuclease_NS” domain (PF01223) polypeptide refer to such protein domains as understood by one skilled in the relevant art. For instance, phrases such as “protein of unknown function DUF1524” and “conserved protein domain family DUF1524”, are used interchangeably (abbreviated, “DUF1524” domain) and refer to a family of uncharacterized proteins having a conserved “HXXP” motif, in which H is the amino acid histidine, P is the amino acid proline and X is any amino acid.

[0351] Phrases such as “Endonuclease_NS” and “conserved protein domain family Endonuclease_NS”, are used interchangeably (abbreviated, “Endonuclease_NS” domain) and refer to a family of bacterial and eukaryotic endonucleases sharing certain characteristics, e.g., acting on both DNA and RNA, cleaving double-stranded and single-stranded nucleic acids and requiring a divalent ion such for activity, wherein a histidine (H) has been shown to be essential for the activity of the S. marcescens nuclease, which H residue is located in a conserved region which also contains an aspartic acid residue that could be implicated in the binding of the divalent ion (e.g., Mg2+).|0352| The term “PLDc_2” domain refers to a family of proteins containing a phospholipase D-like domain. “PLDc_2” domain belongs to PLD superfamily which is defined by a common sequence motif HxK(x)4D(x)6GSxN, and includes enzymes in signal transduction, lipid biosynthesis and endonucleases. The endonuclease from Salmonella typhimurium was the first enzymes whose structure was solved in this family (Stuckey et al., 1999).

[0353] The term “Deoxyribonuclease (DNase) NucA / NucB” refers to a family of endonucleases, which can non-specifically hydrolyze both single- and double-stranded DNA substrates.

[0354] For the purposes of the instant disclosure, nuclease (DNase) activity / function is determined according to the procedures described below in the Examples. For instance, in certain embodiments, DNase activity / function may correlate with DNA concentration and / or DNA size according to one or more procedures described herein. In certain embodiments, an increase in DNase activity / function is stated as a nuclease capable of reducing the concentration of DNA in a sample by at least 1.4x to about 100x. In other embodiments, an increase in DNase activity / function is stated as a nuclease capable of reducing the size of DNA in a sample by at least 1.4x to about 20x. Likewise, in other embodiments, DNase activity / function may be determined via fluorescence detection according to procedures described herein. Other methods and techniques to assay nuclease activity / function are available to one of skill in the art.NB42146-WO-PCT[2]

[0355] As used herein, phrases such as “thermal stress”, “heat stress” and “thermally stressed nuclease” refer to subjecting a nuclease to an elevated temperature for a specified period of time. In certain embodiments, an elevated temperature is a temperature of at least about 40°C, at least about 50°C, or at least 60°C. For instance, in certain embodiments, a nuclease is subjected to an elevated temperature of at least about 40°C, 41°C, 42°C, 43°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C for a specified period of time. In related embodiments, the specified period of time is at least about thirty (30) minutes to about sixty (60) minutes. In other embodiments, the specified period of time is at least about one (1) hour to about twenty four (24) hours. For instance, in certain embodiments, a thermally stressed nuclease is subjected to an elevated temperature of at least about 40°C to about 60°C for at least about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

[0356] The term “thermotolerant” nuclease refers to nucleases capable of withstanding one or more heat stress conditions described herein. In certain embodiments, thermotolerant nucleases retain at least about 55% or higher residual activity of the purified nuclease after 60 minutes pre -incubation at 60°C in 50 mM Tris-HCl buffer (pH 7). For instance, in certain embodiments, a nuclease retaining “substantial” DNase activity following a heat stress condition retain at least about 55% or higher activity. In certain embodiments, the nuclease is purified and in others it is crude, unpurified enzyme.

[0357] As used herein, phrases such as “sodium phosphate stressed nucleases”, “sodium phosphate stress conditions”, “sodium phosphate resistant nucleases”, “sodium phosphate tolerant nucleases”, and the like refer to nucleases capable of withstanding one or more sodium phosphate stress conditions described herein. In certain embodiments, sodium phosphate resistant nucleases retain at least about 40% or higher residual activity of purified nuclease in the presence of at least 100 mM sodium phosphate stress condition (50°C, pH 7, 10 minutes). For instance, as used herein, a nuclease retaining “substantial” DNase activity following a sodium phosphate stress condition retains at least 40% or higher activity. In certain embodiments, the nuclease is purified and in others it is crude, unpurified enzyme. In other embodiments, sodium phosphate resistant nucleases retain substantial activity in the presence of at least about 20 mM to 200 mM sodium phosphate.

[0358] As used herein, phrases such as “protease stressed nucleases”, “protease stress conditions”, “protease resistant nuclease”, “protease tolerant nucleases, “protease stable nucleases”, and the like refer to nucleases capable of retaining at least 10% residual activity after incubation of a purified nuclease enzyme with 5 ppm B. amyloliquefaciens subtilisin protease in 50 mM HEPES (pH 8.0) at 50°C for 60 minutes. For instance, as used herein, a nuclease retaining “substantial” DNase activity following aNB42146-WO-PCT[2]protease stress condition retains at least 10% or higher activity. In certain embodiments, the nuclease is purified and in others it is crude, unpurified enzyme.

[0359] As herein, the term “immobilized nuclease” refers to a nuclease that is physically or chemically confined to a solid phase, including but not limited to nucleases bound by covalent attachment, adsorption, ionic interaction, cross -linking, or entrapment, wherein the nuclease retains substantial DNase activity. Immobilization of the nuclease on a matrix is advantageous for increasing the usage life of the enzyme. In some cases, immobilization allows the nuclease to be used in industrial scale processes for commercial production of proteins of interest essentially free of contaminating DNA. For example, a matrix having nuclease protein immobilized thereon may be used in reactors, such as columns, vessels, or tank reactors, to isolate and / or purify a protein of interest present in a microbial cell fermentation broth.

[0360] In contrast, a “free nuclease” is a nuclease in solution that is not immobilized on a solid support and is capable of diffusing freely within a reaction medium. In some embodiments, a soluble (free) nuclease may be used in reactors, such as columns, vessels, or tank reactors, to isolate and / or purify a protein of interest present in a microbial cell fermentation broth.

[0361] As used herein, the term “essentially free of DNA”, when used in phrases such as “producing a protein of interest (POI) essentially free of DNA”, a “fermentation broth essentially free of DNA”, a “recovered protein essentially free of DNA”, and the like generally refer to DNA concentrations lower than assay detection limits and / or DNA sizes smaller (or larger) than the assay detection limits. In other embodiments, essentially free of DNA refers to nuclease activity assays described in the Examples.

[0362] As used herein, “nucleic acid” refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, as well as to DNA, cDNA, and RNA of genomic or synthetic origin, which may be doublestranded or single-stranded, whether representing the sense or antisense strand. It will be understood that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences may encode a given protein. It is understood that the polynucleotides (or nucleic acid molecules) described herein include “genes”, “vectors” and “plasmids”.

[0363] Accordingly, the term “gene”, refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all, or part of a protein coding sequence, and may include regulatory (nontranscribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions (UTRs), including introns, 5 '-untranslated regions (UTRs), and 3'-UTRs, as well as the coding sequence.

[0364] As used herein, the term “coding sequence” (“CDS”) refers to a nucleotide sequence, which directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of the coding sequence (CDS) arc generally determined by an open reading frame (“ORF”), which usually begins with an ATG start codon. The coding sequence typically includes DNA, cDNA, and recombinant nucleotide sequences.NB42146-WO-PCT[2]

[0365] As defined herein, an “endogenous gene” refers to a gene in its natural location in the genome of an organism.

[0366] As used herein, a “heterologous” gene, a “non-endogenous” gene, or a “foreign” gene refer to a gene (or ORF) not normally found in the host organism, but that is introduced into the host organism by gene transfer. As used herein, the term “foreign” gene(s) comprise native genes (or ORFs) inserted into a non-native organism and / or chimeric genes inserted into a native or non-native organism.

[0367] As defined herein, a “heterologous control sequence”, refers to a gene expression control sequence (e.g., a promoter or enhancer) which does not function in nature to regulate (control) the expression of the gene of interest. Generally, heterologous nucleic acid sequences are not endogenous (native) to the cell, or a part of the genome in which they are present, and have been added to the cell, by infection, transfection, transformation, microinjection, electroporation, and the like. A “heterologous” nucleic acid construct may contain a control sequence / DNA coding (ORF) sequence combination that is the same as, or different, from a control sequence / DNA coding sequence combination found in the native host cell.

[0368] As used herein, the terms “signal sequence” and “signal peptide” refer to a sequence of amino acid residues that may participate in the secretion or direct transport of a mature protein or precursor form of a protein. The signal sequence is typically located N-terminal to the precursor or mature protein sequence. The signal sequence may be endogenous or exogenous. A signal (pre) sequence is normally absent from the mature protein. A signal (pre) sequence is typically cleaved from the protein by a signal peptidase after the protein is transported.

[0369] As used herein, the term “expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA, derived from a nucleic acid molecule of the disclosure. Expression may also refer to translation of mRNA into a polypeptide. Thus, the term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, secretion and the like.

[0370] The term “promoter” as used herein refers to a nucleic acid sequence capable of controlling the expression of a coding sequence (CDS) or functional RNA. In general, a coding sequence is located 3’ (downstream) to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleic acid segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.NB42146-WO-PCT[2]

[0371] The term “operably linked” as used herein refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence (e.g., an ORF) when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. In another example, DNA encoding a secretory leader sequence (i.e., a signal peptide) is operably linked to DNA encoding a polypeptide if it is expressed as a pre -protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0372] As used herein, “suitable regulatory sequences” refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, RNA processing site, effector binding site and stem-loop structure.

[0373] As used herein, the term “introducing”, as used in phrases such as “introducing into a cell” or “introducing into a (microbial) cell at least one polynucleotide, or a gene thereof, or a vector thereof, includes methods known in the art for introducing polynucleotides into a cell, including, but not limited to protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation and the like.

[0374] As used herein, “transformed” or “transformation” mean a cell has been transformed by use of recombinant DNA techniques. Transformation typically occurs by insertion of one or more nucleotide sequences (e.g., a polynucleotide, an ORF or gene) into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e., a sequence that is not naturally occurring in cell that is to be transformed). Transformation therefore generally refers to introducing an exogenous DNA into a host cell so that the DNA is maintained as a chromosomal integrant or a self-replicating extra-chromosomal vector.

[0375] As used herein, the terms “plasmid”, “vector” and “cassette” refer to extrachromosomal elements, often carrying genes which are typically not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single -NB42146-WO-PCT[2]stranded or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3’ untranslated sequence into a cell.

[0376] As used herein, the term “plasmid” refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes. In some embodiments, plasmids become incorporated into the genome of the host cell, in some embodiments plasmids exist in a parental cell and are lost in the daughter cell.

[0377] A used herein, a “transformation cassette” refers to a specific vector comprising a gene (or ORF thereof), and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.

[0378] As used herein, the term “vector” refers to any nucleic acid that can be replicated (propagated) in cells and can carry new genes or DNA segments into cells. Thus, the term refers to a nucleic acid construct designed for transfer between different host cells. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), PLACs (plant artificial chromosomes), and the like, that are “episomes” (i.e., replicate autonomously or can integrate into a chromosome of a host organism).

[0379] An “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA in a cell. Many prokaryotic and eukaryotic expression vectors are commercially available and known to one skilled in the art. Selection of appropriate expression vectors is within the knowledge of one skilled in the art.

[0380] As used herein, the terms “expression cassette” and “expression vector” refer to a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell i.e., these are vectors or vector elements, as described above). The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments, DNA constructs also include a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. In certain embodiments, a DNA construct of the disclosure comprises a selective marker and an inactivating chromosomal or gene or DNA segment as defined herein.

[0381] As used herein, a “targeting vector” is a vector that includes polynucleotide sequences that are homologous to a region in the chromosome of a host cell into which the targeting vector is transformed andNB42146-WO-PCT[2]that can drive homologous recombination at that region. For example, targeting vectors find use in introducing mutations into the chromosome of a host cell through homologous recombination. In some embodiments, the targeting vector comprises other non-homologous sequences, e.g., added to the ends (z.e., staffer sequences or flanking sequences). The ends can be closed such that the targeting vector forms a closed circle, such as, for example, insertion into a vector.

[0382] As used herein, the term “protein of interest” or “POI” refers to a polypeptide of interest that is desired to be expressed in a recombinant microbial cell of the disclosure. Thus, as used herein, a POI may be an enzyme, a substrate-binding protein, a surface-active protein, a structural protein, a receptor protein, and the like.

[0383] As used herein, a “gene of interest” or “GOI” refers a nucleic acid sequence (e.g., a polynucleotide) which encodes a POI. A “gene of interest” encoding a “protein of interest” may be a naturally occurring gene, a mutated gene or a synthetic gene.

[0384] As used herein, the terms “polypeptide” and “protein” are used interchangeably, and refer to polymers of any length comprising amino acid residues linked by peptide bonds. The conventional one (1) letter or three (3) letter codes for amino acid residues are used herein. The polypeptide may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term polypeptide also encompasses an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0385] In certain embodiments, a gene encodes a commercially relevant industrial protein of interest, such as an enzyme, e.g., a acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, deoxyribonucleases, epimerases, esterases, a-galactosidases, -galactosidases, a-glucanases, glucan lysases, endo-P-glucanases, glucoamylases, glucose oxidases, a-glucosidases, P-glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases, lysozymes, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phytases, poly esterases, polygalacturonases, proteases, peptidases, rhamno-galacturonases, ribonucleases, transferases, transport proteins, transglutaminases, xylanases, hexose oxidases, and combinations thereof.

[0386] As used herein, the terms “modification” and “genetic modification” are used interchangeably and include: (a) the introduction, substitution, or removal of one or more nucleotides in a gene (or an ORFNB42146-WO-PCT[2]thereof), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the gene (or ORF thereof), (b) a gene disruption, (c) a gene conversion, (d) a gene deletion, (e) the down-regulation of a gene, (f) specific mutagenesis and / or (g) random mutagenesis of any one or more the genes disclosed herein.

[0387] As used herein, a “mutation” refers to any change or alteration in a nucleic acid sequence. Several types of mutations exist, including point mutations, deletion mutations, silent mutations, frame shift mutations, splicing mutations and the like. Mutations may be performed specifically (e.g., via site directed mutagenesis) or randomly (e.g., via chemical agents, passage through repair minus bacterial strains).

[0388] As used herein, in the context of a polypeptide or a sequence thereof, the term “substitution” means the replacement (i.e., substitution) of one amino acid with another amino acid.

[0389] A “variant” of an enzyme, protein, polypeptide, nucleic acid, or polynucleotide, as used herein means that the variant is derived from a parent (or reference or control) polypeptide, or a parent (or reference or control) nucleic acid, that includes at least one genetic modification as compared to the parent (or reference or control) sequence. In certain embodiments, variant proteins differ from a parental (or control or reference) protein e.g., a wild-type protein) by substitutions, deletions, and / or insertions at a small number of amino acid residue positions. The number of differing amino acid residues between the variant and parental (or control or reference) protein can be one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more amino acid residues. Variant proteins can share at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more amino acid sequence identity with a reference protein. A variant protein can also differ from a reference protein in selected motifs, domains, epitopes, conserved regions, and the like.

[0390] As used herein, the phrases “substantially similar” and “substantially identical”, in the context of at least two nucleic acids or polypeptides, typically means that a polynucleotide or polypeptide comprises a sequence that has at least about 60% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85 % identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or even at least about 99% identity, or more, compared to the reference sequence. Thus, substantially similar sequence identity can be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters.

[0391] As used herein, the term “homologous” protein refers to a protein that has similar activity, function and / or structure to a reference protein. It is not intended that homologs necessarily be evolutionarily related. Thus, it is intended that the term encompass the same, similar, or corresponding protein(s) (i.e., in terms ofNB42146-WO-PCT[2]structure and function) obtained from different organisms. In some embodiments, it is desirable to identify a homolog that has a quaternary, tertiary and / or primary structure similar to the reference protein.

[0392] The degree of homology between sequences can be determined using any suitable method known in the art (see, e.g., Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., 1984). For purposes of the present disclosure, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970), as implemented in the Needle program of the EMBOSS package (Rice et al., 2000), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the nobrief option) is used as the percent identity and is calculated as follows:(Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0393] Likewise, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), as implemented in the Needle program of the EMBOSS package (Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0394] As used herein “an incoming sequence” refers to a DNA sequence that is introduced into a microbial cell. In some embodiments, the incoming sequence is part of a DNA construct. In other embodiments, the incoming sequence encodes one or more proteins of interest. In some embodiments, the incoming sequence comprises a sequence that may or may not already be present in the genome of the cell to be transformed (i.e., it may be either a homologous or heterologous sequence). In some embodiments, the incoming sequence encodes one or more proteins of interest, a gene, and / or a mutated or modified gene. In alternative embodiments, the incoming sequence encodes a functional wild-type gene or operon, a functional mutant gene or operon, or a nonfunctional gene or operon. In some embodiments, the nonfunctional sequence may be inserted into a gene to disrupt function of the gene. In another embodiment, the incoming sequence includes a selective marker. In a further embodiment the incoming sequence includes two homology boxes.

[0395] As used herein, “homology box” refers to a nucleic acid sequence, which is homologous to a sequence in the microbial cell. More specifically, a homology box is an upstream or downstream regionNB42146-WO-PCT[2]having between about 80 and 100% sequence identity, between about 90 and 100% sequence identity, or between about 95 and 100% sequence identity with the immediate flanking coding region of a gene or part of a gene to be deleted, disrupted, inactivated, down-regulated and the like, according to the invention. These sequences direct where in the chromosome a DNA construct is integrated and directs what part of the chromosome is replaced by the incoming sequence. While not meant to limit the present disclosure, a homology box may include about between 1 base pair (bp) to 200 kilobases (kb). Preferably, a homology box includes about between 1 bp and 10.0 kb; between 1 bp and 5.0 kb; between 1 bp and 2.5 kb; between 1 bp and 1.0 kb, and between 0.25 kb and 2.5 kb. A homology box may also include about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb and 0.1 kb. In some embodiments, the 5’ and 3’ ends of a selective marker are flanked by a homology box wherein the homology box comprises nucleic acid sequences immediately flanking the coding region of the gene.[0396J As used herein, a “flanking sequence” refers to any sequence that is either upstream or downstream of the sequence being discussed (e.g., for genes A-B-C, gene B is flanked by the A and C gene sequences). In certain embodiments, the incoming sequence is flanked by a homology box on each side. In another embodiment, the incoming sequence and the homology boxes comprise a unit that is flanked by stuffer sequence on each side. In some embodiments, a flanking sequence is present on only a single side (either 3’ or 5'), but in preferred embodiments, it is on each side of the sequence being flanked. The sequence of each homology box is homologous to a sequence in the chromosome. These sequences direct where in the chromosome the new construct gets integrated and what part of the chromosome will be replaced by the incoming sequence. In other embodiments, the 5' and 3’ ends of a selective marker are flanked by a polynucleotide sequence comprising a section of the inactivating chromosomal segment. In some embodiments, a flanking sequence is present on only a single side (either 3' or 5'), while in other embodiments, it is present on each side of the sequence being flanked.

[0397] As used herein, the terms “selectable marker” and “selective marker” refer to a nucleic acid (e.g., a gene) capable of expression in host cell which allows for ease of selection of those hosts containing the vector. Examples of such selectable markers include, but are not limited to, antimicrobials. Thus, the term “selectable marker” refers to genes that provide an indication that a host cell has taken up an incoming DNA of interest or some other reaction has occurred. Typically, selectable markers are genes that confer antimicrobial resistance or a metabolic advantage on the host cell to allow cells containing the exogenous DNA to be distinguished from cells that have not received any exogenous sequence during the transformation.

[0398] As used herein, the terms “purified”, “isolated” or “enriched” are meant that a biomolecule (e.g., a polypeptide or polynucleotide) is altered from its natural state by virtue of separating it from some, or all of, the naturally occurring constituents with which it is associated in nature. Such isolation or purificationNB42146-WO-PCT[2]may be accomplished by art-recognized separation techniques such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulphate precipitation or other protein salt precipitation, centrifugation, size exclusion chromatography, filtration, microfiltration, ultrafiltration, gel electrophoresis or separation on a gradient to remove whole cells, cell debris, impurities, extraneous proteins, or enzymes undesired in the final composition. It is further possible to then add constituents to a purified or isolated biomolecule composition which provide additional benefits, for example, activating agents, anti-inhibition agents, desirable ions, compounds to control pH or other enzymes or chemicals.

[0399] As used herein, a “protein preparation” is any material, typically a solution, generally aqueous, comprising one or more proteins.

[0400] As used herein, the terms “broth”, “cultivation broth” and “fermentation broth” may be used interchangeably, and particularly refer to a whole fermentation broth.

[0401] The term “whole fermentation broth” as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and / or purification. For example, whole fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of proteins by host cells) and secretion into cell culture medium. Typically, the whole fermentation broth is unfractionated and comprises spent cell culture medium, extracellular polypeptides, and microbial cells.

[0402] “Cell debris” refers to cell walls and other insoluble cellular components that are released after disruption of the cell membrane, e.g., after lysis of microbial cells.

[0403] “Cell kill” means a process rendering the host organisms inactive and no longer able to replicate.

[0404] “Broth conditioning” refers to pretreatment of a microbial fermentation broth designed to improve subsequent broth handling properties. Broth conditioning changes the chemical composition and / or physical and / or rheological properties of the broth to facilitate its use in downstream recovery and / or formulation processes. Broth conditioning may include one or more treatments such as pH modification, heat treatment, cooling, addition of additives (e.g., calcium, salt(s), flocculant(s), reducing agent(s), enzyme activator(s), enzyme inhibitor(s), and / or surfactant(s)), mixing, and / or timed hold (e.g., 0.5 to 200 hours) of the broth without further treatment. As set forth and further described below, certain aspects of the disclosure provide novel DNase compositions (e.g., DNase protein preparations) suitable for degrading contaminating DNA present in microbial cell fermentation broths. In related embodiments, a DNase preparation of the disclosure is added during a broth conditioning / treatment process, including but not limited to, broth stabilization processes, broth pH and / or temperature optimization processes, broth conditioning with additives, broth holding times, and the like.NB42146-WO-PCT[2]

[0405] The terms “recovery”, “recovered” and “recovering” as used herein refer to treatment or stabilization of broth, or at least partial separation of a protein from one or more soluble components of a microbial broth and / or at least partial separation from one or more solvents in the broth (e.g., water or ethanol). A recovered protein is often of higher purity than prior to the recovery process. However, in some embodiments, a recovered protein may be of the same or lower purity than prior to the recovery process.III. POLYNUCLEOTIDES ENCODING NOVEL NUCLEASES|0406| As set forth above, the presence of contaminating DNA in microbial cell fermentation broths and / or its presence in any down-stream protein recovery processes thereof can lead to undesirable protein product qualities. DNA in microbial fermentations, e.g., present due to cell lysis, can increase the broth viscosity in those fermentations, thereby having negative effects on oxygen (O2) transfer with concomitant undesirable effects on microbial cell growth and / or protein production. Increased viscosity due to the presence of contaminating D A also has undesirable (negative) effects on the recovery and / or purification of fermentation products (i.e., proteins of interest, e.g., enzymes) by for instance ultrafiltration. Endonucleases can degrade DNA and are used in biotechnology for removal of nucleic acids and viscosity reductions, e.g., see PCT Publication Nos: WO1999 / 50389, W02008 / 065200, WO2011 / 015327, WO2018 / 210794, WO2021 / 254832, WO2022 / 272155, WO2023 / 023644, WO2023 / 118565

[0407] However, nuclease polypeptides are typically not thermotol erant, and are readily degraded (inactivated) due to the presence of protease in the microbial cell fermentation broths. Likewise, nucleases are not typically tolerant to high sodium phosphate concentrations. Such inadequacies render most nucleases useless when co-expressed with proteins of interest and / or when applied as additives, for instance, in recovery processes requiring high temperatures and / or high sodium phosphate concentrations, protease recovery processes, and the like.

[0408] As described herein and set forth in the Examples below, Applicant has identified novel nucleases having enhanced performance properties that address various ongoing and unmet needs in the art. Certain embodiments of the disclosure therefore provide inter alia, novel nucleases having enhanced performance properties, such as thermotolerant nucleases which are capable of degrading DNA at high temperatures, and / or capable of degrading DNA during one or more high temperature protein recovery and / or formulation processes, novel nucleases that are tolerant or resistant to proteases and / or tolerant to high sodium phosphate stress and capable of degrading DNA during fermentation processes and'or protein recovery process and / or protein formulation process and the like.

[0409] More specifically, as presented in Example I. various natural isolates from Applicant’s microbial strain collection were selected as potential sources for nuclease enzymes useful in industrial applications. For example, to identify the enzymes and genes that encode these enzymes, the entire genomes of theNB42146-WO-PCT[2]microbial strains were sequenced using Illumina sequencing by synthesis (SBS) technology. In particular, genes encoding enzymes with homology to nucleases of various other bacteria and with a predicted signal peptide were selected for further assessment. The amino acid sequences of the mature chains of these selected nucleases (z.<?., nuclease enzymes without a signal peptide sequence) are shown in FIG. 1, wherein the molecular weight (Da), sequence identification number (SEQ ID), total number of amino acid residues and domain family for each mature nuclease are summarized below in TABLE 1.TABLE 1MATURE NUCLEASE SEQUENCE DETAILSName SEQ ID Molecular Weight (Da) Residues Domain Family NsxNucl 25 20,148 184 DUF1524 MetNuc70 27 20,105 182 DUF1524 BhoNuc2 29 20,265 182 DUF1524 MetNuc71 31 20,104 182 DUF1524 UstNuc9 33 20,188 182 DUF1524 BhoNuc3 35 18,880 171 DUF1524 BhoNucl 37 20,211 182 DUF1524 UstNuc8 39 20,241 182 DUF1524 UstNucll 41 20,062 182 DUF1524 MetNucl9 43 20,167 182 DUF1524 MetNuc72 45 19,999 182 DUF1524 MetNuc73 47 20,199 181 DUF1524 MetNuc36 49 20,135 182 DUF1524 UstNuc7 51 19,989 182 DUF1524 TreNucl 133 20,090 186 DUF1524AosNucl 135 29,047 258 Endonuclease_NS Domain

[0410] Example 2 generally describes the construction of recombinant Bacillus sp. strains co-expressing a heterologous a-amylase reporter and a heterologous nuclease. More particularly, the recombinant strains constructed comprised introduced nuclease cassettes having an upstream heterologous promoter and 5'-UTR region operably linked to DNA encoding a heterologous signal (pre) sequence set forth in TABLE 2 operably linked to DNA encoding a mature nuclease sequence set forth in TABLE 3 operably linked to a downstream transcriptional terminator, to build a series of modified Bacillus sp. strains overexpressing both the a-amylase reporter and heterologous nuclease set forth in TABLE 3. In particular, for each mature nuclease presented in TABLE 3, several combinations were constructed with the various signal sequences (TABLE 2), where a list of all nuclease expression cassettes constructed are shown in TABLE 4.

[0411] As described below in Example 3, the modified strains co-expressing the amylase reporter and introduced nuclease cassette (Example 2) were assessed for amylase production, nuclease activity andNB42146-WO-PCT[2]nuclease thermotolerance. More specifically, as presented in TABLE 5, all constructs produced at least 82% of the amylase reporter as compared to the parental (control) strain, with a number of constructs producing slightly more amylase than the control.

[0412] Additionally, a portion of the broth was then held at 60°C for one (1) hour to test the thermal stability of the nuclease. After the hold at 60°C, genomic DNA (gDNA) from the parent strain was added to thermally-stressed broth or unstressed broth, and held at 37°C for one (1) hour, and the integrity of the added gDNA was determined by running the samples on a ZAG fragment analyzer. For example, both the remaining DNA concentration and the average DNA size are presented in TABLE 6 and TABLE 7, respectively. Thus, as described in Example 3, the results of this analysis demonstrate that the coexpression of nucleases with amylases can be optimized by (mature) nuclease and signal sequence selection to minimize impact on production of the reporter protein of interest. As shown in TABLE 6, there are several nucleases capable of reducing the concentration of DNA in a fermentation broth by at least 1.4x to about 100x, both with and without heat stress. Likewise, as presented in TABLE 7, these nucleases also reduce the size of the remaining DNA by at least 1.4x to about 20x, both with and without heat stress as compared to the parent strain not expressing a heterologous nuclease.

[0413] Example 4 particularly describes the construction of recombinant Bacillus sp. strains co-expressing a heterologous subtilisin reporter or a heterologous 0-glucanase reporter, and a heterologous nuclease. For instance, the modified Bacillus sp. strains expressing the subtilisin protease in absence or presence of a coexpressed nuclease are listed in TABLE 8. Likewise, the modified Bacillus sp. strains expressing the 0-glucanase in absence or presence of a co-expressed nuclease are presented in TABLE 9.

[0414] As described in Example 5, the strains constructed in Example 4 were assessed for production of the reporter proteins. More specifically, B. subtilis strains expressing the subtilisin (protease) or 0-glucanase and co-expressing a nuclease were compared to the parental strain not expressing a nuclease. In particular, as shown in TABLE 10 (Example 5), supernatants of B. subtilis strains CB455 (control, no nuclease co-expression) and AP514 (nuclease co-expression) were compared for production of the B. amyloliquefaciens subtilisin variant, wherein similar to slightly higher subtilisin production was observed in strain AP514 co-expressing the Bacillus sp. nuclease and strain CB472 co-expressing the T. reesei nuclease. Additionally, as presented in TABLE 11, supernatants of B. subtilis strains CZ508 (control, no nuclease co-expression) and CZ507 (nuclease co-expression) were compared for production of the B. gibsonii subtilisin variant, wherein similar to higher subtilisin production in strain CZ507 (co-expressing a nuclease was observed compared to the parent (control) strain CZ508.

[0415] As further described in Example 5 (TABLE 12), supernatants of B. subtilis strains CZ509 (control, no nuclease co-expression) and CZ510 (nuclease co-expression) were compared for production of the T.NB42146-WO-PCT[2]cellulosilytica subtilisin protease. As shown in TABLE 12, similar subtilisin production in strain CZ509 (control) and strain CZ510 that co-expresses the nuclease was observed.

[0416] Likewise, as described in Example 5 (TABLE 13), the parent B. subtilis strain was compared to the strains expressing p-glucanase (CZ483, CZ493 and CZ502), wherein the nuclease co-expression in strains CZ493 and CZ502 does not negatively affect expression of the p-glucanase. Furthermore, as presented in TABLE 14, reduction of DNA concentration and / or DNA size was observed in the strains co-expressing a nuclease (CZ493, CZ502, CB472, AP514, CZ507, CZ510) compared to their respective parent (control) strains (CZ483, CB455, CZ5O8, CZ509) not expressing the nuclease.

[0417] In other embodiments, Applicant constructed recombinant Trichoderma strains co-expressing a protein of interest (POI) and a nuclease. In particular, Examples 6 (TABLE 15) describes the construction of Trichoderma strains co-expressing a trehalase reporter protein (enzyme) and a nuclease (SEQ ID NO: 136). As described in Example 7, production of the trehalase reporter protein in the strain coexpressing the nuclease (TABLE 16; FS65) was compared to the parental (control) strain (TABLE 16; FS00) not expressing the nuclease. As shown in TABLE 16, similar or slightly higher trehalase production was observed in strain FS65 compared to the parent (control) strain FS00. As further described in Example 16, nuclease activity was assessed (TABLE 17; Relative DNA Concentration and Relative DNA Size), wherein a reduction of both DNA concentration and DNA size were observed in the FS65 strain co-expressing the nuclease as compared to the parent (control) FS00 strain.

[0418] Examples 8 and 9 describe certain other natural isolates from Applicant’s microbial strain collection which were selected as a potential source for nuclease enzymes useful in industrial applications. In particular’, DNA sequences encoding the mature nucleases (TABLE 18) were codon-optimized for the expression in B. subtilis (TABLE 19), wherein the amino acid sequences of the mature chains of these selected nucleases are shown in FIG. 2. For instance, the mature nuclease sequences (TABLE 18) were screened for enhanced properties (e.g., enhanced protease stability, enhanced thermostability, enhanced phosphate tolerance, etc.). Example 10 describes the construction, expression, fermentation and purification of mature nucleases of the disclosure.

[0419] Thus, Example 11 describes the identification of nucleases having enhanced protease stability. For instance, as presented in TABLE 20 (Example 11), among the sixty-seven (67) nuclease candidates, forty-one (41) demonstrated enhanced protease stability with over 60% residual activities remaining after stressing. In other embodiments, the activity of crude nuclease samples were assayed before and after preincubation with a protease. In particular, as described in Example 12, forty -two (42) crude nuclease samples were harvested for protease stability measurements, and the residual activity was determined. As shown in TABLE 21 (Example 12), among the 42 crude nuclease samples, twenty-three (23) samples retained over 80% residual activity after stressing.NB42146-WO-PCT[2]

[0420] Example 13 further describes the identification of nucleases having enhanced thermostability, determined by assaying nuclease activity before and after pre-incubation at 60°C for one (1) hour. More particularly, as described in this example, the nucleases were sampled for residual activity (TABLE 22), wherein approximately 40% of the nuclease candidates retained over 60% of the activity after thermal (heat) stress. In other embodiments, the activity of crude nuclease samples were assayed for activity before and after pre-incubation at 60°C for twenty-four (24) hours (Example 14). For instance, as presented in TABLE 23 (Examplel4), after heat stressing at 60°C for 24 hours, nineteen (19) out of the forty-two (42) crude nuclease samples retained over 60% residual activity.

[0421] Example 15 describes the identification nucleases having enhanced salt stability / tolerance. In particular, the phosphate (salt) tolerance of nucleases was identified by the residual nuclease activities in different concentrations of sodium phosphate buffer. For instance, as set forth in TABLE 24 (Example 15), the activity of nucleases are significantly inhibited in the presence of phosphates. More particularly, eighteen ( 18) out of forty-four (44) candidate nucleases retained over 50% activity in the presence of 20 mM phosphates (TABLE 24), and seven (7) of the forty-four (44) candidate nucleases continued to retain over 50% of their activities. As further described in Example 15, certain nucleases having enhanced salt tolerance / stability were selected for further characterization in higher phosphate stress conditions. For instance, to better distinguish the performance of these candidate nuclease molecules, a series of nuclease reaction dosages (0-0.5 ppm) were set with phosphate concentrations of 100 mM (TABLE 25), 150 mM (TABLE 26), and 200 mM (TABLE 27), wherein activity of nuclease was indicated by the corrected (A) fluorescence signal, before and after reactions. In certain other embodiments, forty-two (42) nuclease crude samples were harvested for phosphate tolerance measurements, as generally set forth in Example 16. More specifically, the detected fluorescence signal and calculated residual activities are shown in TABLE 28 (Example 16). As presented in TABLE 28, among the 42 nuclease crude samples, thirteen (13) samples retained over 50% of their activities in the presence of 20 mM and ten (10) samples retained over 50% of their activities in the presence of 100 mM phosphates.

[0422] As further described in Example 17, Applicant identified and characterized additional novel nuclease polypeptides encoded by newly disclosed polynucleotides, thereby expanding the repertoire of nucleases having enhanced performance properties. In particular, Example 17 describes two newly identified nucleases, designated RarNuc3 and RdeNuc2 (members of the endonuclease_NS domain family), wherein the mature amino acid sequences are set forth in SEQ ID NO: 300 and SEQ ID NO: 302, respectively, with the corresponding polynucleotide sequences set forth in SEQ ID NOs: 301 and 303.

[0423] More particularly, Example 17 evaluated the suitability of selected nucleases of the disclosure for immobilized enzyme applications, an important consideration for industrial DNA degradation processes requiring enzyme reuse, fixed-bed operation, prolonged exposure to harsh processing conditions and / or toNB42146-WO-PCT[2]prevent the nuclease from being present in the final product. To this end, purified nuclease preparations were subjected to an immobilization assay using solid supports and chemical binding strategies, and immobilization efficiency was quantified by measuring the amount of residual enzyme remaining in solution after immobilization and wash steps, as summarized in TABLE 29 (Example 17). For instance, several nucleases evaluated in TABLE 29 exhibited exceptionally high immobilization efficiencies, indicating strong and stable association with the support material and minimal loss during washing. In particular, the nucleases RarNuc3 (SEQ ID NO: 300), RdeNuc2 (SEQ ID NO: 302), BspNuc28 (SEQ ID NO: 176) and GbuNucl (SEQ ID NO: 213) were identified as top performers, wherein each of these enzymes demonstrated immobilization efficiencies exceeding approximately 95%. This performance indicates that the vast majority of the applied enzyme was successfully captured on the solid support, with negligible enzyme detected in the filtrate or subsequent wash fractions.104241 The strong immobilization behavior observed for these nucleases suggests that their surface properties and structural features are particularly well suited for interaction with immobilization matrices and linker chemistries. Without being bound by theory, such properties may include favorable surface charge distribution, accessible amino acid side chains for covalent or electrostatic attachment, and structural robustness that prevents denaturation during immobilization and washing. Importantly, the best-performing immobilized nucleases belong to different nuclease families described in the disclosure, demonstrating that high immobilization efficiency is not restricted to a single domain class (e.g., DLJE1524 or DNase_NucA / NucB), but rather represents a broadly exploitable feature of select novel nucleases disclosed herein.

[0425] Thus, in certain one or more embodiments, the disclosure provides, inter alia, isolated polynucleotides encoding novel nucleases having enhanced performance properties (benefits), polynucleotides encoding mature nucleases, polynucleotides encoding signal (pre) peptide sequences, expression constructs comprising a DNA sequence encoding a signal peptide sequence operably linked to a DNA sequence encoding a mature nuclease, recombinant microbial cells expressing novel nucleases having enhanced performance properties, recombinant microbial cells co-expressing a novel nuclease and a protein of interest (POI), novel nucleases immobilized on a matrix and the like.

[0426] In some embodiments, a novel nuclease of the disclosure is immobilized on a matrix as described in, for example, U. S. Patent Nos. 3,796,634, 4,355,105, 4,713,333, 5,177,005, 5,437,993, 5,811,280, 5,916,789, and 7,297,510. In some embodiments, immobilization of a nuclease may include a colloidal silica, activated charcoal, hydroxyapatite, alumina C gamma, bentonite, diatomaceous earth or a combination thereof. In some embodiments, the immobilized nuclease contains polyethylenimine and glutaraldehyde. In some embodiments, the nuclease is immobilized on a bead. In some embodiments, the nuclease is immobilized on a resin. In some embodiments, the nuclease is immobilized on an ion exchangeNB42146-WO-PCT[2]resin. In some embodiments, the nuclease is immobilized on a matrix by weakly basic ion exchange (i.e., electrostatic interaction based on the charge of the protein and the charge of a matrix such as a resin). In some embodiments, the nuclease is immobilized by non-specific binding to porous regions of a matrix, such as a resin.IV. MOLECULAR BIOLOGY

[0427] As briefly set forth above, certain embodiments of the disclosure are related to polynucleotides (expression constructs) encoding novel nucleases (DNases) having enhanced performance properties (benefits). Certain other embodiments are related to polynucleotides (expression constructs) encoding proteins of interest (e.g., enzymes). Thus, certain other embodiments provide, inter alia, nucleic acids, polynucleotides, plasmids, vectors, expression constructs, gene cassettes, recombinant microbial host cells and methods thereof suitable for expressing / producing novel nucleases, and / or co-expressing / co-producing novel nucleases and proteins of interest.

[0428] Certain embodiments are therefore directed to one or more novel nucleases described herein. In certain embodiments, a nuclease of the disclosure comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 133, 135, 142-220, 300 and 302. In other embodiments, a nuclease of the disclosure comprises at least about 70%, 75%, 80%, 85%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 133, 135 and 142-220 and has an enhanced performance property (benefit) selected from enhanced thermotolerance, enhanced sodium phosphate tolerance and / or enhanced protease tolerance.

[0429] In certain embodiments, a gene, nucleic acid, polynucleotide, open reading frame (ORF) and the like (e.g., encoding nucleases, proteins of interest) is genetically modified. For instance, genetic modifications include, but are not limited to, the introduction, substitution, or removal of one or more nucleotides in a gene (or an ORF thereof), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the gene (or ORF thereof), a gene disruption, a gene conversion, a gene deletion, the down-regulation of a gene, specific mutagenesis and / or random mutagenesis of any one or more the genes disclosed herein.

[0430] Those of skill in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli, Bacillus sp., etc.), filamentous fungal cells (e.g., Aspergillus sp., Trichoderma sp., etc.), yeast cells (e.g., Saccharomyces sp.) and the like (i.e., microbial cells).

[0431] As generally specified above, certain embodiments of the disclosure are directed to expressing, producing and / or secreting one or more nucleases and / or proteins of interest which are heterologous to the to the microbial host cell. Therefore, the instant disclosure generally relies on routine techniques in theNB42146-WO-PCT[2]field of recombinant genetics. Basic texts disclosing the general methods of use in present disclosure include Sambrook et al., (1989; 2011; 2012) and Ausubel et al., (1994).

[0432] In particular embodiments, the disclosure relates to recombinant (modified) nucleic acids comprising a gene or ORF encoding a nuclease (DNase) protein. For example, in certain embodiments, a recombinant nucleic acid is a polynucleotide expression construct (gene cassette) for expression / production of a novel nuclease of the disclosure. In certain other embodiments, the recombinant nucleic acid (polynucleotide) further comprises one or more selectable markers. Selectable markers for use in Gram negative bacteria, Gram positive bacteria filamentous fungi and yeast are generally known in the art. Thus, in certain embodiments, a polynucleotide construct encoding a nuclease and / or a protein of interest, comprises a nucleic acid sequence encoding a selectable marker operably linked thereto.

[0433] In other embodiments, nucleic acids comprising a gene (or ORF) encoding a nuclease further comprise operably linked regulatory or control sequences. An example of regulatory or control sequences may be a promoter sequence or a functional part thereof, (i.e., a part which is sufficient for affecting expression of the nucleic acid sequence). Other control sequences include, but are not limited to, a leader sequence, a pro-peptide sequence, a signal sequence, a transcription terminator, a transcriptional activator and the like. Thus, in certain embodiments, a recombinant (modified) polynucleotide comprises an upstream (5') promoter (P) sequence driving the expression of a gene (or ORF) encoding a nuclease, or a POI of the disclosure. More particularly, in certain embodiments, the promoter is a constitutive or an inducible promoter active (functional) in the microbial host cell. For example, one of skill in the art can use any suitable promoter capable of driving the expression of a gene of interest in a microbial expression host cell. Thus, in certain aspects, a recombinant nucleic acid of the disclosure comprises a promoter (P) sequence which is 5' (upstream) and operably linked to a nucleic acid (gene) sequence encoding a nuclease (e.g., 5'-[P]-[gene]-3').

[0434] In certain other aspects, a recombinant nucleic acid (e.g., an expression cassette) comprises an upstream (5') promoter (P) sequence operably linked to a downstream (3') nucleic acid encoding a nuclease (or encoding a POI), further comprises a terminator (T) sequence downstream and operably linked thereto. For example, in certain embodiments, a recombinant nucleic acid of the disclosure comprises a promoter (P) sequence which is 5' (upstream) and operably linked to a nucleic acid (gene) sequence encoding a nuclease (or POI) which is operably linked to a downstream terminator (T) sequence (e.g., 5'-[P]-[gene]-[71-3').

[0435] Suitable promoters for driving the expression of genes of interest in a microbial host cell of the disclosure are generally known in the art. For example, exemplary Bacillus sp. promoters include, but are not limited to, tac promoter sequences, 0-lactamasc promoter sequences, aprE promoter sequences, groES promoter sequences, ftsEI promoter sequences, titfA promoter sequences, secDF promoter sequences, rninCNB42146-WO-PCT[2]promoter sequences, spoVG promoter sequences, veg promoter sequences, hbs promoter sequences, amylases promoter sequences, P43 promoter sequence and the like, exemplary filamentous fungal promoters include, but are not limited to, Trichoderma sp. promoters (e.g., cellobiohydrolase promoters, endoglucanase promoters, P-glucosidase promoters, xylanases promoters, glucoamylase promoters), Aspergillus sp. promoters (e.g., trpC promoters, glucoamylase promoters), and the like. However, it is not intended that the present disclosure be limited to any particular promoter, as any suitable promoter known to those in the art finds use with the present invention.

[0436] Thus, certain other embodiments are related to cultivating (fermenting) microbial host cells expressing a nuclease and / or a POI, wherein the expressed nuclease and / or POI are secreted into the culture (fermentation) broth. For example, in certain other embodiments, a recombinant nucleic acid comprises an upstream (5') heterologous promoter (P) sequence operably linked to a downstream (3') nucleic acid sequence (ss) encoding a protein signal sequence operably linked to a downstream (3') nucleic acid (gene') encoding a nuclease (e.g., 5'-[2’]-[5'5']-[^ne]-3').

[0437] Any suitable (protein) signal sequence (signal peptide) functional in the microbial cell of choice may be used for the secretion (transport) of a mature nuclease protein (e.g., SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 133, 135,142-220, 300 and 302) and / or other proteins of interest. The signal sequence is typically located N-terminal to the precursor or mature protein sequence. For example, suitable signal sequences for use include, but are not limited to, signal sequences from secreted proteases, peptidases, amylases, glucoamylases, cellulases, lipases, esterases, arabinases, glucanases, chitosanases, lyases, xylanases, nucleases, phosphatases, transport and binding proteins, etc. In certain embodiments, a signal sequence is selected from an aprE signal sequence, a nprE signal sequence, a vpr signal sequence, a bglC signal sequence, a bglS signal sequence, a sacB signal sequence and amylase signal sequence, a heterologous signal sequence and / or a synthetic signal sequence,

[0438] Thus, in certain embodiments, standard techniques for transformation of microbial cells (which are well known to one skilled in the art) are used to transform a microbial host cell of the disclosure. Thus, the introduction of a DNA construct or vector into a host cell includes techniques such as transformation, electroporation, nuclear microinj ection, transduction, transfection (e.g., lipofection mediated and DEAE-Dextrin mediated transfection), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, gene gun or biolistic transformation, protoplast fusion and the like. General transformation techniques are known in the art (see, e.g., Ausubel et al., 1987, Sambrook et al., 2001 and 2012, and Campbell et al., 1989).

[0439] In certain embodiments, a heterologous gene, polynucleotide or ORF is cloned into an intermediate vector, before being transformed into a the microbial (host) cells for replication and / or expression. These intermediate vectors can be prokaryotic vectors, such as, e.g., plasmids, or shuttle vectors. Thus, theNB42146-WO-PCT[2]expression vector / construct typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the heterologous sequence. For example, a typical expression cassette contains a 5' promoter operably linked to the heterologous nucleic acid sequence encoding a protein of interest and may further comprise sequence signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

[0440] The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include bacteriophages and M13, as well as plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc. The elements that can be included in expression vectors may also be a replicon, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, or unique restriction sites in nonessential regions of the plasmid to allow insertion of heterologous sequences.

[0441] The methods of transformation of the present invention may result in the stable integration of all or part of the transformation vector into the genome of the microbial cell. However, transformation resulting in the maintenance of a self-replicating extra-chromosomal transformation vector is also contemplated. Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). Also of use is the Agrobacterium-mediated transfection method such as the one described in U. S. Patent No. 6,255,115. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the heterologous gene.

[0442] After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the genes of interest. Large batches of transformed cells can be cultured as described herein. Finally, the broth and / or product(s) are recovered from the culture using standard techniques. Thus, the disclosure herein provides for the expression and secretion of desired proteins (e.g., DNase proteins, enzymes and the like).

[0443] Microbial cells of the disclosure may comprise genetic modifications of one or more endogenous genes and / or one or more introduced (heterologous) genes described herein. For example, microbial cells may be constructed to reduce or eliminate the expression of endogenous genes (e.g., reduce or eliminateNB42146-WO-PCT[2]genes encoding proteases), using methods well known in the art, e.g., insertions, disruptions, replacements, or deletions. The portion of the gene to be modified or inactivated may be, for example, the coding region or a regulatory element required for expression of the coding region.

[0444] In certain embodiments, a modified cell of the disclosure is constructed by introducing, substituting, or removing one or more nucleotides in the gene or a regulatory element required for the transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a frame-shift of the open reading frame. Such a modification may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art.

[0445] In another embodiment, a modified cell is constructed by the process of gene conversion. For example, in the gene conversion method, a nucleic acid sequence corresponding to the gene(s) is mutagenized in vitro to produce a defective nucleic acid sequence, which is then transformed into the parental cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. It may be desirable that the defective gene or gene fragment also encodes a marker which may be used for selection of transformants containing the defective gene. For example, the defective gene may be introduced on a non-replicating or temperature-sensitive plasmid in association with a selectable marker. Selection for integration of the plasmid is affected by selection for the marker under conditions not permitting plasmid replication. Selection for a second recombination event leading to gene replacement is affected by examination of colonies for loss of the selectable marker and acquisition of the mutated gene. Alternatively, the defective nucleic acid sequence may contain an insertion, substitution, or deletion of one or more nucleotides of the gene, as described below.

[0446] In other embodiments, a modified cell is constructed by established anti-sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the gene. More specifically, expression of the gene by a cell may be reduced (down-regulated) or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated. Such anti-sense methods include, but are not limited to RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, and the like, all of which are well known to the skilled artisan.

[0447] In other embodiments, a modified cell is produced / constructed via CRISPR-Cas9 editing. For example, a gene of interest can be disrupted (or deleted or down-regulated) by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g., Cas9) and Cpfl or a guide DNA e.g., NgAgo), which recruits the endonuclease to the target sequence on the DNA, wherein theNB42146-WO-PCT[2]endonuclease can generate a single or double stranded break in the DNA. This targeted DNA break becomes a substrate for DNA repair, and can recombine with a provided editing template to disrupt or delete the gene. For example, the gene encoding the nucleic acid guided endonuclease (for this purpose Cas9 from S. pyogenes) or a codon optimized gene encoding the Cas9 nuclease is operably linked to a promoter active in the microbial cell and a terminator active in the microbial cell, thereby creating a microbial cell Cas9 expression cassette. Likewise, one or more target sites unique to the gene of interest are readily identified by a person skilled in the art. For example, to build a DNA construct encoding a gRNA -directed to a target site within the gene of interest, the variable targeting domain (VT) will comprise nucleotides of the target site which are 5' of the (PAM) protospacer adjacent motif (TGG), which nucleotides are fused to DNA encoding the Cas9 endonuclease recognition domain for S. pyogenes Cas9 (GER). The combination of the DNA encoding a VT domain and the DNA encoding the CER domain thereby generate a DNA encoding a gRNA. Thus, a microbial cell expression cassette for the gRNA is created by operably linking the DNA encoding the gRNA to a promoter active in the microbial cells and a terminator active in the microbial cells. The Cas9 expression cassette, the gRNA expression cassette and the editing template can be co-delivered to cells using many different methods (e.g., protoplast fusion, electroporation, natural competence, or induced competence). The transformed cells are screened by PCR amplifying the target gene locus, by amplifying the locus with a forward and reverse primer. These primers can amplify the wild-type locus or the modified locus that has been edited by the RGEN.

[0448] In yet other embodiments, a modified cell is constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis and transposition. Modification of the gene may be performed by subjecting the parental cell to mutagenesis and screening for mutant cells in which expression of the gene has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods. Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), N-methyl-N’-nitrosoguanidine (NTG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the parental cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutant cells exhibiting reduced or no expression of the gene.NB42146-WO-PCT[2]V. MICROBIAL HOST CELLS

[0449] Certain embodiments are related to recombinant microbial (host) cells comprising and expressing heterologous polynucleotides which encode one or more novel nuclease (DNase) proteins of the disclosure. Certain other embodiments are directed to such microbial cells co-expressing a novel nuclease and a protein of interest. Microbial cells of the disclosure include Gram negative bacterial cells, Gram positive bacterial cells, filamentous fungal cells and yeast cell.

[0450] In certain embodiments, a Gram negative bacterial cell includes all bacteria of the classes of proteobacteria, such as alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria, delta-proteobacteria, epsilon-proteobacteria (e.g., including, but not limited to, proteobacteria such as Acidithiobacillal.es, Aeromonadales, Alteromonadales, Cardiobacteriales, Chromatiales, Enterbacteriales, Legionellales, Methylococcales, Oceanospirillales, Pasteurellales, Pseudomonadales, Thiotrichales, Vibrionales, Xanthomonadales. Enter obacteriales are Arsenophonus, Brenneria, Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Dickeya, Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella, Moellerella, Marganella, Obesumbacterium, Pantoea, Pectobacterium, Photorhabdus, Plesiomonas, Pragia, Proteus, Providencia, Rahnella, Raoultella, Saccharobacter, Salmonella, Samsonia, Serratia, Shigella, Sodalis, Tatumella, Thorsellia, Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia, Yokenella and the like).|04511 In certain embodiments, a Gram positive bacterial cell includes the classes Bacilli, Clostridia and Mollicutes (e.g., including Lactobacillales with the families Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Oscillospiraceae, Streptococcaceae and the Bacillales with the families Alicyclobacellaceae, Bacillaceae, Caryophanaceae, Listeriaceae, Paenibacillaceae, Planococcaceae, Sporolactobacillaceae, Staphylococcaceae, Thermoactinomycetaceae, Turicibacteraceae). In other embodiments, species of the family Bacillaceae include Alkalibacillus, Amphibacillus, Anoxybacillus, Bacillus, Caldalkali bacillus, Cerasilbacillus, Exiguobacterium, Filobacillus, Geobacillus, Gracili bacillus, Halobacillus, Halolactibacillus, Jeotgalibacillus, I.entibaciUus, Marinibacillus, Oce anobacillus, Ornithinibacillus, Paraliobacillus, Paucisalibacillus, Pontibacillus, Pontibacillus, Saccharococcus, Salibacillus, Salinibacillus, Tenuibacillus, Thalassobacillus, Ureibacillus, Virgibacillus.

[0452] In other embodiments, Bacillus sp. cells include, but are not limited to, B. acidiceler, B. acidicola, B. acidocaldarius, B. acidoterrestris, B. aeolius, B. aerius, B. aerophilus, B. agaradhaerens. B. agri, B. aidingensis, B. akibai, B. alcalophilus, B. algicola, B. alginolyticus, B. alkalidiaw-trophicus, B. alkalinitrilicus, B. alkalitelluris, B. altitudinis, B. alveayuensis, B. alvei, B. amylolyticus, B. aneurinilyticus, B. aneurinolyticus, B. anthracia, B. aquimaris, B. arenosi, B. arseniciselenatis, B. arsenicoselenatis, B. arsenicus, B. arvi, B. asahii, B. atrophaeus, B. aurantiacus, B. axarquiensis, B. awtofixans, B.NB42146-WO-PCT[2]azotoformans, B. badius, B. barbaricus, B. bataviensis, B. beijingensis, B. benzoevorans, B. bogoriensis, B. boroniphilus, B. borstelenis, B. butanolivorans, B. carboniphilus, B. cecembensis, B. cellulosilyticus, B. centrosporus, B. chagannorensis, B. chitinolyticus, B. chondroitinus, B. choshinensis, B. cibi, B. circulans, B. clarkii, B. clausii, B. coagulans, B. coahuilensis, B. cohnii, B. curdianoly cus, B. cycloheptanicus, B. decisifrondis, B. decolor adonis, B. dipsosauri, B. drentensis, B. edaphicus, B. ehimensis, B. endophyticus, B. farraginis, B.fasddiosus, B.firmus, B. plexus, B.foraminis, B. fordii, B.formosus, B. fords, B.fumarioli, B. funiculus, B. fusiformis, B. galactophilus, B. galactosidilydcus, B. geladni, B. gibsonii, B. ginsengi, B. ginsengihumi, B. globisporus, B. globisporus subsp. globisporus, B. globisporus subsp. marinus, B. glucanolyticus, B. gordonae, B. halmapalus, B. haloalkaliphilus, B. halodenitrificans, B. halodurans, B. halophilus, B. hemicellulosilyticus, B. herbersteinensis, B. horikoshii, B. horn, B. hemi, B. hwajinpoensis, B. idriensis, B. indicus, B. infantis, B. infernus, B. insolitus, B. isabeliae, B. jeotgali, B. kaustophilus, B. kobensis, B. koreensis, B. kribbensis, B krulwichiae, B. laevolacdcus, B. larvae, B. laterosporus, B. lautus, B. lehensis, B. lentimorbus, B. lentus, B. litoralis, B. luciferensis, B. macauensis, B. macerans, B. macquariensis, B. macyae, B. malacitensis, B. mannanilyticus, B. marinus, B. marisflavi, B. marismortui, B. massiliensis, B. methanolicus, B. migulanus, B. mojavensis, B. mucilaginosus, B. muralis, B. murimardni, B. mycoides, B. naganoensis, B. nealsonii, B. neidei. B, niabensis, B. niacini, B. novalis, B. odysseyi, B. okhensis, B. okuhidensis, B. oleronius, B. oshimensis, B. pabuli, B. pallidus, B. pallidus (illeg.), B. panaciterrae, B. pantothenticus, B. parabrevis, B. pasteurii, B. patagoniensis, B. peoriae, B. plakortidis, B. pocheonensis, B. polygoni, B. polymyxa, B. popilliae, B. pseudalcaliphilus, B. pseudofirmus, B. pseudomycoides, B. psychrodurans, B. psyc.hr ophilus, B. psychrosaccarolydcus, B. psychrotolerans, B. pulvifaciens, B. pycnus, B. qingdaonensis, B. reuszeri, B. runs, B. safensis, B. salarius, B. salexigens, B. saliphilus, B. schlegelii, B. selenatar senatis, B. selenitrireducens, B. seohaeanensis, B. shackletonii, B. silvestris, B. simplex, B. siralis, B. smithii, B. soli, B. sonorensis, B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. stratosphericus, B. subterraneus, B. subtilis subsp. spizizenii, B. subtilis subsp. subtilis, B. taeanensis, B. tequilensis, B. thermantarcticus, B. thermoaerophilus, B. thermoamylovorans, B. thermoantarcticus, B. thermocatenulatus, B. thermocloacae, B. thermodenitrificans, B. thermoglucosidasius, B. thermoleovorans, B. thermoruber, B. thermosphaericus, B. thiaminolyticus, B. thioparans, B. thuringiensis, B. tusciae, B. validus, B. vallisrnords, B. vedderi, B. velezensis, B. vietnamensis, B. vire, B. vulcani, B. wakoensis and B. weihenstephanensis. In one preferred embodiment, the Bacillus sp. cell is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have beenNB42146-WO-PCT[2]reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus”.

[0453] Exemplary yeast (or yeast cells) includes budding yeast from the order Saccharomycetales. Particular examples of yeast are Saccharomyces sp., including but not limited to S. cerevisiae.

[0454] Filamentous fungal cells include, without limitation, Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and Trichoderma species. In some embodiments, a filamentous fungus is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride. In other embodiments, a filamentous fungus may be an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae.VI. FERMENTING MICROBIAL CELLS FOR THE PRODUCTION OF PROTEINS

[0455] In certain embodiments, the present disclosure provides recombinant microbial cells capable of producing proteins of interest. More particularly, certain embodiments are related genetically modified (recombinant) microbial cells expressing heterologous polynucleotides encoding novel nucleases. In other embodiments, the disclosure provides modified microbial cells co-expressing a nuclease and a protein of interest. Thus, particular embodiments are related to cultivating (fermenting) microbial cells for the production of proteins.

[0456] In general, fermentation methods well known in the art are used to ferment the microbial cells. In some embodiments, the cells are grown under batch or continuous fermentation conditions. A classical batch fermentation is a closed system, where the composition of the medium is set at the beginning of the fermentation and is not altered during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism(s). In this method, fermentation is permitted to occur without the addition of any components to the system. Typically, a batch fermentation qualifies as a “batch” with respect to the addition of the carbon source, and attempts are often made to control factors such as pH and oxygen concentration. The metabolite and biomass compositions of the batch system change constantly up to the time the fermentation is stopped. Within batch cultures, cells progress through a static lag phase to a high growth log phase and finally to a stationary phase, where growth rate is diminished or halted. If untreated, cells in the stationary phase eventually die. In general, cells in log phase are responsible for the bulk of production of product.

[0457] A suitable variation on the standard batch system is the “fed-batch fermentation” system. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression likely inhibits the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of theNB42146-WO-PCT[2]changes of measurable factors, such as pH, dissolved oxygen and the partial pressure of waste gases, such as CO2. Batch and fed-batch fermentations are common and well known in the art.

[0458] Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous lermenialion generally maintains the cultures at a constant high density, where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, a limiting nutrient, such as the carbon source or nitrogen source, is maintained at a fixed rate and all other parameters are allowed to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidily, is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

[0459] Culturing / fermenting is generally accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, a carbon and energy source material, molecular oxygen, and, of course, a starting inoculum of the microbial host to be employed.|0460| In addition to the carbon and energy source, oxygen, assimilable nitrogen, and an inoculum of the microorganism, it is necessary to supply suitable amounts in proper proportions of mineral nutrients to assure proper microorganism growth, maximize the assimilation of the carbon and energy source by the cells in the microbial conversion process, and achieve maximum cellular yields with maximum cell density in the fermentation media.

[0461] The composition of the aqueous mineral medium can vary over a wide range, depending in part on the microorganism and substrate employed, as is known in the art. The mineral media should include, in addition to nitrogen, suitable amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium, in suitable soluble assimilable ionic and combined forms, and also present preferably should be certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, and others, again in suitable soluble assimilable form, all as known in the art.

[0462] The fermentation reaction is an aerobic process in which the molecular oxygen needed is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion.

[0463] The fermentation temperature can vary somewhat, but for most microbial cells the temperature generally will be within the range of about 20°C to 40°C.NB42146-WO-PCT[2]

[0464] The microorganisms also require a source of assimilable nitrogen. The source of assimilable nitrogen can be any nitrogen-containing compound or compounds capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganism. While a variety of organic nitrogen source compounds, such as protein hydrolysates, can be employed, usually cheap nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds can be utilized. Ammonia gas itself is convenient for large scale operations, and can be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia can also be employed to assist in pH control.

[0465] The pH range in the aqueous microbial ferment (fermentation admixture) should be in the exemplary range of about 2.0 to 8.0. Preferences for pH range of microorganisms are dependent on the media employed to some extent, as well as the particular microorganism, and thus change somewhat with change in media as can be readily determined by those skilled in the art.

[0466] Preferably, the fermentation is conducted in such a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing good conversion of the carbon-containing substrate to cells and avoiding contamination of the cells with a substantial amount of unconverted substrate. The latter is not a problem with water-soluble substrates, since any remaining traces are readily washed off. It may be a problem, however, in the case of non-water-soluble substrates, and require added product-treatment steps such as suitable washing steps.

[0467] As described above, the time to reach this level is not critical and may vary with the particular microorganism and fermentation process being conducted. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether or not the desired level of carbon source has been achieved.

[0468] If desired, part or all of the carbon and energy source material and / or part of the assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium to the fermenter.

[0469] Each of the streams introduced into the reactor preferably is controlled at a predetermined rate, or in response to a need determinable by monitoring such as concentration of the carbon and energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gases from the fermenter, cell density measurable by dry cell weights, light transmittancy, or the like. The feed rates of the various materials can be varied so as to obtain as rapid a cell growth rate as possible, consistent with efficient utilization of the carbon and energy source, to obtain as high a yield of microorganism cells relative to substrate charge as possible.NB42146-WO-PCT[2]

[0470] In either a batch, or the preferred fed batch operation, all equipment, reactor, or fermentation means, vessel or container, piping, attendant circulating or cooling devices, and the like, are initially sterilized, usually by employing steam such as at about 121°C for at least about 15 minutes. The sterilized reactor then is inoculated with a culture of the selected microorganism in the presence of all the required nutrients, including oxygen, and the carbon-containing substrate. The type of fermenter employed is not critical. VII. PROTEINS OF INTEREST

[0471] A protein of interest (POI) of the instant disclosure can be any endogenous or heterologous protein, and it may be a variant of such a POI. The protein can contain one or more disulfide bridges or is a protein whose functional form is a monomer or a multimer, i.e., the protein has a quaternary structure and is composed of a plurality of identical (homologous) or non-identical (heterologous) subunits, wherein the POI or a variant POI thereof is preferably one with properties of interest. Thus, in certain embodiments, a modified cell of the disclosure expresses an endogenous POI, a heterologous POI, or a combination of one or more of such POIs.104721 In certain embodiments, a modified cell produces one or more proteins of interest. In other embodiments, a modified cell may produce an increased amount of a POI relative to a parental (control or reference) cell, wherein the increased amount of the POI is at least about a 0.01% increase, at least about a 0.10% increase, at least about a 0.50% increase, at least about a 1.0% increase, at least about a 2.0% increase, at least about a 3.0% increase, at least about a 4.0% increase, at least about a 5.0% increase, or an increase greater than 5.0%. In certain embodiments, the POI is detected by assaying enzymatic activity and / or by assaying / quantifying the specific productivity (Qp) of the protein thereof.

[0473] In certain embodiments, a POI or a variant POI thereof is selected from the group consisting of acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, arylesterases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, deoxyribonucleases, epimerases, esterases, a-galactosidases, [3-galactosidases, a-glucanases, glucan lysases, endo-0-glucanases, glucoamylases, glucose oxidases, a-glucosidases, p-glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, ligases, lipases, lyases, lysozymes, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phcnoloxidascs, phosphodiesterases, phytases, polycstcrascs, polygalacturonases, proteases, peptidases, rhamno-galacturonases, ribonucleases, trehalases, transferases, transport proteins, transglutaminases, xylanases, hexose oxidases, and combinations thereof.

[0474] Thus, in certain embodiments, a POI or a variant POI thereof is an enzyme selected from Enzyme Commission (EC) Number EC 1, EC 2, EC 3, EC 4, EC 5 or EC 6.NB42146-WO-PCT[2]

[0475] One skilled in the art may utilize other routine methods and techniques known in the art for detecring, assaying, measuring, etc. the expression, production or secretion of one or more proteins of interest. In particular, for proteases, there are assays based on the release of acid-soluble peptides from casein or hemoglobin measured as absorbance at 280 nm or colorimetrically, using the Folin method. Other exemplary assays include succinyl-Ala-Ala-Pro-Phe-para-nitroanilide assay (SAAPFpNA) and the 2,4,6-trinitrobenzene sulfonate sodium salt assay (TNBS assay). International PCT Publication No. WO2014 / 164777 discloses Ceralpha a-amylase activity assays useful for amylase activities described herein. Means for determining the levels of secretion of a protein of interest in a host cell and detecting expressed proteins include the use of immunoassays with either polyclonal or monoclonal antibodies specific for the protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and fluorescent activated cell sorting (FACS).VIII. BROTH CONDITIONING, PROTEIN RECOVERY PROCESSES AND USES THEREOF

[0476] As generally described above, certain aspects of the disclosure are related to cultivating (fermenting) microbial cells for the production of nucleases and / or other proteins of interest. Certain embodiments are therefore related to fermentation broths obtained by fermenting microbial cells expressing nuclease proteins of the disclosure. Certain other embodiments are related to fermentation broths obtained by fermenting microbial cells co-expressing protein(s) of interest and a novel nuclease of the disclosure.

[0477] More particularly, such microbial cell fermentation broths comprising certain novel nucleases of the disclosure are particularly useful in degrading DNA according to many aspects of the disclosure. In related embodiments, such microbial cell fermentation broths are further subjected to a protein recovery process. Certain other embodiments are therefore directed to compositions and methods for producing a protein of interest (POI) essentially free from DNA. For example, certain embodiments provide protein preparations essentially free from DNA produced according to the compositions and methods of the disclosure.

[0478] Thus, in other embodiments, the disclosure relates to methods for recovering proteins of interest from microbial cell fermentation broths, such as (a) obtaining a microbial cell fermentation broth comprising a POI, (b) treating the broth with an exogenously introduced protein preparation comprising one or more nucleases (z.e., free nucleases) and / or treating the broth with one or more immobilized nucleases, or a combination thereof and (c) recovering the POI from the broth, wherein the recovered protein(s) is / are essentially free from contaminating DNA. For example, in the case of immobilized nuclease, the recovered POI is free from contaminating DNA, with no recombinant nuclease present in the recovered POI composition.

[0479] Certain other embodiments therefore provide protein preparations (or proteins isolated therefrom) essentially free from DNA, recovered according to the disclosed methods and compositions herein.NB42146-WO-PCT[2]

[0480] Thus, certain other embodiments relate to methods for reducing the DNA content of a fermentation broth in which microbial host cells have been fermented comprising introducing into the fermentation broth an exogenous protein preparation comprising one or more novel nucleases of the disclosure and / or treating the broth with one or more immobilized nuclease matrices. In other embodiments, one or more (multiple) microbial cell fermentation broths (e.g., comprising one or more proteins of interest) may be combined (mixed) in the presence of exogenous nuclease (DNase) protein preparations and / or treated with one or more immobilized nucleases, or a combination thereof. In related embodiments, the combined fermentation broth(s) is / are further subjected to one or more protein recovery steps performed in the presence of at least one exogenously introduced protein preparation comprising one or more nucleases of the disclosure and / or in the presence of one or more immobilized nucleases of the disclosure.

[0481] More particularly, as described herein, one or more nuclease (DNase) proteins, and / or one or more proteins of interest can be secreted into the fermentation broth and / or retained intracellularly. Therefore, in certain aspects, the fermentation broth is subjected to at least one the protein recovery process selected from a cell lysis process, a cell separation process, a protein concentration process and / or a protein purification process. More particularly, in certain aspects, the fermentation broth is subjected to at least one protein recovery process performed in the presence of an exogenously introduced nuclease protein preparation and / or performed in the presence of an immobilized nuclease. The recovery of proteins from a fermentation broth can be done by procedures known to one of skill in the art to obtain a desired protein preparation.

[0482] The fermentation broth will generally contain cellular debris, including cells, various suspended solids and other biomass contaminants, as well as the desired protein (e.g., enzyme) products and / or DNase proteins. For example, suitable protein recovery processes include, but are not limited to, conventional solid-liquid separation techniques such as, e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes, to produce a cell-free filtrate.

[0483] The terms “cell separation” or “cell separation process” are not meant to be limiting, and include methods of cell separation and broth clarification know to those skilled in the art, such as centrifugation, rotary vacuum drum filtration, filter pressing, microfiltration and the like. Likewise, the terms “concentration” or “concentration process” are not meant to be limiting, and include concentration methods know to those skilled in the art, such as ultrafiltration, evaporation, centrifugation and the like. In certain aspects, a microbial cell fermentation broth comprising a protein of interest is treated for a sufficient amount of time with an introduced (exogenous) DNase protein preparation, wherein the protein of interest is recovered from the broth following such DNase treatment, and the protein of interest recovered therefrom is essentially free from contaminating DNA. For example, in certain embodiments, the broth is treated with a DNase protein preparation for a sufficient amount of time to render the broth essentially free fromNB42146-WO-PCT[2]contaminating DNA. One skilled in the art may monitor DNA content of the broth using known techniques and adjust the amount of time accordingly and / or the total activity of a particular DNase preparation. In certain embodiments, a sufficient amount of time is about one (1) second to about forty-eight (48) hours.

[0484] It may be preferable to further concentrate the fermentation broth or the cell-free filtrate prior to crystallization using techniques such as ultrafiltration, evaporation or precipitation. Precipitating the proteinaceous components of the supernatant or filtrate may be accomplished by means of a salt, e.g., ammonium sulfate, followed by purification by a variety of chromatographic procedures, e.g., ion exchange chromatography, affinity chromatography or similar art recognized procedures.

[0485] Thus, as generally set forth above, protein preparations according to the instant disclosure may be recovered, purified, enriched and the like using methods known to one skilled the art (e.g., art-recognized separation techniques such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulphate precipitation (or other protein salt precipitation), centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis or separation on a gradient to remove whole cells, cell debris, impurities, extraneous proteins, or enzymes undesired in the final composition. It is further possible to then add constituents to a purified or isolated biomolecule composition which provide additional benefits, for example, activating agents, anti-inhibition agents, desirable ions, compounds to control pH or other enzymes or chemicals.|0486| With regard to Gram negative bacterial cells e.g., E. coll), purification steps for separating endotoxins (lipopolysaccharides; LPS) which are known in the art may can be used (e.g., anion exchange chromatography; affinity chromatography; ion exchange chromatography, in particular ion exchange chromatography using alkanediol; ultrafiltration; purification using affinity adsorbents such as, e.g., L-histidine, poly-L-histidine, poly(gamma-methyl L-glutamate), polymyxin B; gel filtration; gel filtration chromatography; sucrose gradient centrifugation; purification using dual-phase micelle systems; triton X-114-based phase separation; temperature -induced phase separation; purification by a non-selective adsorption with hydrophobic adsorbents or anion exchangers; polyacrylamide gel electrophoresis, in particular slab polyacrylamide gel electrophoresis; SDS gel electrophoresis; membrane-based chromatography; agarose gel electrophoresis; caesium chloride gradient centrifugation; affinity purification using beads).

[0487] Thus, in certain embodiments, a protein preparation comprises at least one nuclease protein of the disclosure. In certain other embodiments, a protein preparation comprises at least one nuclease protein of the disclosure and a protein of interest (e.g., an enzyme) or multiple proteins of interest (e.g., enzyme combinations / blends). The protein preparations of the disclosure (e.g., a nuclease preparation, an enzyme (POI) preparation, a combined nuclcasc / cnzymc (POI) preparation, etc.) can be solid (e.g., a lyophilized powder), paste -like or liquid (e.g., an aqueous solution or dispersion).NB42146-WO-PCT[2]

[0488] Therefore, certain embodiments of the disclosure provide novel nuclease compositions suitable for degrading DNA. In certain embodiments, novel nucleases are co-expressed with a protein of interest (POI), wherein the expressed nucleases degrade DNA present in the broth. In other aspects, novel nucleases are expressed in a microbial cell of the disclosure, wherein nuclease (protein) preparations are obtained, derived or recovered from the broth. In certain aspects, a nuclease preparation is a liquid preparation, such as concentrated broth (e.g., a nuclease UFC). In related embodiments, a liquid nuclease preparation is used to treat a protein preparation comprising a protein of interest. For example, in certain aspects, liquid nuclease preparations are used to treat a protein of interest (z.e., a protein preparation comprising the POI) during a POI purification process and / or a POI formulation process, thereby removing DNA from the purified and / or formulated POI.

[0489] In some embodiments, nucleases of the disclosure are immobilized on a matrix. For example, such immobilized nuclease compositions offer multiple advantages in industrial bioprocessing, including, but not limited to, reusability, enabling multiple cycles of DNA degradation without replenishing enzyme, improved process economics, due to reduced enzyme consumption, enhanced operational stability, particularly under elevated temperature, protease, or phosphate stress conditions, compatibility with continuous processing systems, such as fixed-bed or packed-column reactors and the like.

[0490] Thus, the nucleases of the disclosure are particularly useful for degrading DNA present in microbial cell fermentation broths. In certain aspects, a nuclease preparation is added to a microbial cell fermentation broth comprising a protein of interest (POI). In other embodiments, the broth comprising the POI is collected and subjected to immobilized nuclease treatment. In related embodiments, the collected broth is harvested and / or the collected broth is treated with a soluble and / or immobilized nuclease. In any of these aspects or embodiments of the disclosure, a nuclease preparation (soluble or immobilized) may be used to remove / degrade DNA therein. For example, broth treatments include, but are not limited to, broth stabilization processes, broth pH optimization, broth temperature optimization, broth additives, broth holding times, and the like.IX. EXEMPLARY EMBODIMENTS

[0491] Non-limiting embodiments of the disclosure include, but are not limited to:

[0492] 1. A nuclease (DNase) polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, wherein the polypeptide has nuclease activity.

[0493] 2. The nuclease of embodiment 1, wherein the nuclease has an enhanced performance property selected from thermotolerance, protease tolerance, salt tolerance, phosphate tolerance, immobilization compatibility, or combinations thereof.NB42146-WO-PCT[2]

[0494] 3. The nuclease of embodiment 1, wherein the nuclease comprises a protein family or domain selected from a DUF1524 domain, an Endonuclease_NS domain, a PLDc_2 domain, and a DNase_NucA / NucB domain.

[0495] 4. The nuclease of any one of embodiments 1-3, wherein the nuclease is a mature nuclease lacking a signal peptide.

[0496] 5. The nuclease of any one of embodiments 1-3, wherein the nuclease is a precursor (immature / full-length) nuclease comprising a signal peptide.

[0497] 6. The nuclease of any one of embodiments 1 -5, wherein the nuclease comprises one or more amino acid substitutions, insertions, and / or deletions relative to a reference mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, and retains nuclease activity.

[0498] 7. The nuclease of any one of embodiments 1-6, wherein the nuclease comprises one or more conservative amino acid substitutions and retains nuclease activity.

[0499] 8. The nuclease of any one of embodiments 1-7, wherein the nuclease is truncated at the N-terminus and / or C-terminus relative to a reference mature nuclease sequence set forth in TABLE 1, TABLE 18 and / or TABLE 30, and retains nuclease activity.

[0500] 9. The nuclease of embodiment 8, wherein the truncation comprises deletion of about 1-10 amino acids, about 11-25 amino acids, or more, at the N-terminus and / or C-terminus.105011 10. The nuclease of any one of embodiments 1-9, wherein the nuclease retains an intact domain selected from DUF1524, Endonuclease_NS, PLDc_2, and DNase_NucA / NucB.

[0502] 11. The nuclease of any one of embodiments 1-10, wherein the nuclease is provided as a soluble (free) nuclease.

[0503] 12. The nuclease of any one of embodiments 1-10, wherein the nuclease is provided in an immobilized form on a solid support and retains nuclease activity.

[0504] 13. The nuclease of any one of embodiments 1-12, wherein the nuclease reduces DNA concentration in a sample and / or reduces DNA size in a sample.

[0505] 14. The nuclease of any one of embodiments 1-13, wherein the nuclease is thermotolerant and retains substantial nuclease activity following incubation at an elevated temperature for at least about 30 minutes, 1 hour, 2 hours, or up to about 24 hours.

[0506] 15. The nuclease of embodiment 14, wherein the elevated temperature is at least about 40°C, 50°C, or about 60°C.

[0507] 16. The nuclease of any one of embodiments 1-13, wherein the nuclease is protease tolerant and retains substantial nuclease activity in the presence of at least one protease.

[0508] 17. The nuclease of embodiment 16, wherein the protease comprises a subtilisin and / or a metalloprotease present in a fermentation broth and / or in a downstream recovery process.NB42146-WO-PCT[2]

[0509] 18. The nuclease of any one of embodiments 1-13, wherein the nuclease is tolerant to phosphate stress and retains substantial nuclease activity in the presence of sodium phosphate.

[0510] 19. The nuclease of embodiment 18, wherein the sodium phosphate concentration is at least about 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

[0511] 20. The nuclease of any one of embodiments 1-19, wherein the nuclease exhibits one or more combined enhanced properties selected from thermotolerance and protease tolerance; thermotolerance and phosphate tolerance; protease tolerance and phosphate tolerance; or thermotolerance, protease tolerance, and phosphate tolerance.

[0512] 21. An isolated polynucleotide encoding the nuclease of any one of embodiments 1-20.

[0513] 22. The polynucleotide of embodiment 21, wherein the polynucleotide is codon-optimized for expression in a microbial host cell.

[0514] 23. An expression construct comprising a promoter operably linked to the polynucleotide of embodiment 21 or embodiment 22.

[0515] 24. The expression construct of embodiment 23, further comprising a nucleic acid encoding a signal peptide operably linked to a nucleic acid encoding the nuclease, such that the nuclease is secreted.

[0516] 25. The expression construct of embodiment 23 or embodiment 24, further comprising a transcription terminator.|0517| 26. The expression construct of any one of embodiments 23-25, wherein the promoter is constitutive or inducible and is functional in a Gram-positive bacterial host cell, a Gram-negative bacterial host cell, a filamentous fungal host cell, and / or a yeast host cell.

[0518] 27. A vector comprising the expression construct of any one of embodiments 23-26.

[0519] 28. The vector of embodiment 27, wherein the vector is a plasmid, an integrating vector, a targeting vector, or a shuttle vector.

[0520] 29. A recombinant microbial host cell comprising the polynucleotide of embodiment 21 or 22, the expression construct of any one of embodiments 23-26, and / or the vector of embodiment 27 or 28.

[0521] 30. The recombinant microbial host cell of embodiment 29, wherein the host cell is a Gram-positive bacterial cell.

[0522] 31. The recombinant microbial host cell of embodiment 29 or 30, wherein the host cell is a Bacillus cell.

[0523] 32. The recombinant microbial host cell of embodiment 29, wherein the host cell is a Gramnegative bacterial cell.

[0524] 33. The recombinant microbial host cell of embodiment 29, wherein the host cell is a filamentous fungal cell.NB42146-WO-PCT[2]

[0525] 34. The recombinant microbial host cell of embodiment 33, wherein the filamentous fungal cell is a Trichoderma cell.

[0526] 35. The recombinant microbial host cell of embodiment 29, wherein the host cell is a yeast cell.

[0527] 36. The recombinant microbial host cell of any one of embodiments 29-35, further comprising a nucleic acid encoding a protein of interest (POI).

[0528] 37. The recombinant microbial host cell of embodiment 36, wherein the POI is an enzyme, a substrate-binding protein, a transport protein, a structural protein, or an antibody.

[0529] 38. The recombinant microbial host cell of embodiment 36 or 37, wherein the POI is an enzyme selected from acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, epimerases, esterases, a-galactosidases, p-galactosidases, a-glucanases, glucan lysases, endo-β-glucanases, glucoamylases, glucose oxidases, a-glucosidases, P-glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lactases, ligases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phosphatases, phytases, polygalacturonases, proteases, peptidases, rhamnogalacturonases, transferases, transglutaminases, trehalases, xylanases, and combinations thereof.|0530| 39. The recombinant microbial host cell of any one of embodiments 29-38, wherein co-expression of the nuclease reduces DNA concentration and / or DNA size in a fermentation broth relative to a control cell lacking the nuclease.

[0531] 40. A fermentation broth comprising the nuclease of any one of embodiments 1-20.

[0532] 41. A fermentation broth comprising the recombinant microbial host cell of any one of embodiments 29-39, wherein the broth comprises the nuclease and optionally a co-expressed POI.

[0533] 42. The fermentation broth of embodiment 40 or 41, wherein the broth has reduced DNA concentration and / or reduced DNA size compared to a control broth lacking the nuclease.

[0534] 43. A protein preparation comprising (i) a POI and (ii) the nuclease of any one of embodiments 1-20, wherein the preparation is essentially free of DNA.

[0535] 44. A method for co-expressing a POI and a nuclease comprising: providing a microbial host cell expressing the POI and introducing into the host cell a polynucleotide encoding the nuclease of embodiment 21 or 22 or an expression construct of any one of embodiments 23-86 and cultivating the host cell under conditions suitable for expression of the POI and the nuclease.

[0536] 45. The method of embodiment 44, wherein the nuclease is secreted into a fermentation broth.

[0537] 46. A method for producing a POI essentially free of DNA, comprising cultivating the recombinant microbial host cell of any one of embodiments 36-39 under conditions suitable for expression of the POINB42146-WO-PCT[2]and the nuclease and recovering the POI from the cultivation medium, wherein DNA in the cultivation medium and / or recovered POI is reduced by action of the nuclease.

[0538] 47. The method of embodiment 46, further comprising holding the cultivation medium at an elevated temperature prior to recovery of the POI.

[0539] 48. The method of embodiment 47, wherein the elevated temperature is about 60°C and the holding time is at least about 1 hour.

[0540] 49. A method for reducing DNA content of a fermentation broth, comprising contacting the broth with a nuclease of any one of embodiments 1-20 provided as an exogenous nuclease preparation.

[0541] 50. The method of embodiment 49, wherein contacting occurs before, during, or after a broth conditioning step, a recovery step, and / or a formulation step.

[0542] 51. The method of any one of embodiments 46-50, further comprising one or more downstream processing steps selected from centrifugation, filtration, microfiltration, ultrafiltration, precipitation, chromatography, and combinations thereof.

[0543] 52. An immobilized nuclease composition comprising the nuclease of any one of embodiments 1-20 immobilized on a solid support, wherein the immobilized nuclease retains nuclease activity.

[0544] 53. The immobilized nuclease composition of embodiment 52, wherein the solid support comprises a bead, resin, ion exchange resin, membrane, porous matrix, colloidal silica, activated charcoal, hydroxyapatite, alumina, bentonite, diatomaceous earth, or combinations thereof.

[0545] 54. The immobilized nuclease composition of embodiment 52 or 53, wherein immobilization is by covalent attachment, adsorption, ionic interaction, cross-linking, entrapment, or combinations thereof.

[0546] 55. The immobilized nuclease composition of any one of embodiments 52-54, wherein the immobilized nuclease is compatible with repeated use and / or continuous processing.

[0547] 56. A method for reducing DNA content of a fermentation broth or a protein preparation, comprising contacting the fermentation broth or protein preparation with the immobilized nuclease composition of any one of embodiments 52-55 under conditions suitable for DNA degradation.

[0548] 57. The method of claim 56, wherein suitable conditions comprise a temperature of about 20-60 °C, a pH of about 6-9, and a contacting time of about 1 second to 48 hours.

[0549] 58. The method of embodiment 56, wherein contacting is performed in a packed-bed reactor, fixed-bed reactor, column, vessel, tank reactor, or combinations thereof.

[0550] 59. The method of any one of embodiments 56-58, wherein the immobilized nuclease composition is reused for multiple cycles of DNA degradation.

[0551] 60. The immobilized nuclease composition of any one of embodiments 52-55 or the methods of any one of embodiments 56-59, wherein the nuclease is selected from SEQ ID NO: 176, 213, 300 and 302.NB42146-WO-PCT[2]

[0552] 61. Any one of the preceding embodiments, wherein the nuclease is provided alone or in combination with one or more additional nucleases.

[0553] 62. Any one of the preceding embodiments, wherein the nuclease is used alone or in combination with one or more additives selected from salts, calcium, surfactants, activators, inhibitors, reducing agents, or combinations thereof.

[0554] 63. Any one of the preceding embodiments, wherein the nuclease is used in a process for reducing viscosity and / or improving downstream filtration of a fermentation broth.

[0555] 64. Any one of the preceding embodiments, wherein the nuclease, expression construct, microbial cell, broth, immobilized nuclease composition, and / or method is used for degrading DNA in an industrial biotechnology process.

[0556] 65. A protease resistant nuclease comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature amino acid sequence of any one of SEQ ID NO: 27, 31, 33, 41, 43, 45, 47, 49, 142-176, 178, 182, 183, 184, 187, 188, 189, 190, 192, 193, 194, 196-199, 201, 210 and 220, wherein the nuclease retains activity in the presence of the at least one protease.

[0557] 67. A thermotolerant nuclease comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature amino acid sequence of any one of SEQ ID NO: 27, 31, 33, 43, 45, 47, 49, 133, 142, 143, 144, 145, 147, 148, 150, 151, 157, 158, 159, 160, 161, 162, 165, 168, 169, 171, 172, 174, 175, 176, 177-180, 183, 184, 188, 194, 195, 196, 197, 198, 199, 200, 201, 202, 219 and 220, wherein the nuclease retains activity following incubation at an elevated temperature for at least one hour.

[0558] 68. A sodium phosphate tolerant nuclease comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature amino acid sequence of any one of SEQ ID NO: 31, 47, 133, 135, 145, 155, 177, 178, 180, 183, 189, 190, 192, 193, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 218 and 219, wherein the nuclease retains activity in presence of high sodium phosphate concentrations.

[0559] 69. The broth of any one of embodiments 40-42, wherein the nuclease comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NO: 31, 53, 55, 57, 61, 63, 65, 67, 71, 73, 77, 79, 81, 83, 87, 89, 95, 97, 99, 101, 103 and 105, and reduces the DNA concentration in the broth by at least 1.4x.

[0560] 70. The broth of any one of embodiments 40-42, wherein the nuclease comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NO: 31, 53, 55, 61, 65, 67, 71, 73, 77, 79, 85, 87, 89, 93, 97, 103, 105, 107 and 133, and reduces the DNA size in the broth at least 1.4x.NB42146-WO-PCT[2]

[0561] 71. The broth of any one of embodiments 40-42, wherein the nuclease comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NO: 55, 67, 73, 79, 81, 83, 87, 89, 97, 101, 103, and 105 and reduces the DNA concentration in the broth at least 1.4x following incubation at an elevated temperature for at least one hour.

[0562] 72. The broth of any one of embodiments 40-42, wherein the nuclease comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NO: 55, 57, 61, 65, 67, 71, 77, 79, 81, 87, 93, 95, 99, 101 and 103, and reduces the DNA size in the broth by at least 1.4x following incubation at an elevated temperature for at least 1 hour.

[0563] 73. The method of embodiment 44 or 45, wherein the cell produces no less than 25% of the POI as compared to a control cell expressing the same POI, but not co-expressing the nuclease.

[0564] 74. The method of embodiment 44 or 45, wherein the cell produces no less than 50% of the POI as compared to a control cell expressing the same POI, but not co-expressing the nuclease.

[0565] 75. The method of embodiment 44 or 45, wherein the cell produces no less than 75% of the POI as compared to a control cell expressing the same POI, but not co-expressing the nuclease.NB42146-WO-PCT[2]EXAMPLES

[0567] Certain aspects of the present disclosure may be further understood in light of the following examples, which should not be construed as limiting. Modifications to materials and methods will be apparent to those skilled in the art. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989).EXAMPLE 1DISCOVERY AND IDENTIFICATION OF NOVEL NUCLEASES

[0568] The instant example describes the discovery, identification and selection of novel nucleases. More particularly, various natural isolates from Applicant’s microbial strain collection were selected as a potential source for enzymes useful in industrial applications. For example, to identify these enzymes and the genes that encode these enzymes, the entire genomes of these strains were sequenced using Illumina sequencing by synthesis (SBS) technology. Genome sequencing, assembly and annotation of the sequence data was performed by BaseClear (Leiden, The Netherlands). In particular, genes encoding enzymes with homology to nucleases of various other bacteria and with a predicted signal peptide (Teufel et al., 2022) were selected for further assessment. The amino acid sequences of the mature chains of these selected nucleases (i.e., nuclease enzymes without a signal peptide sequence) are shown in FIG.1.EXAMPLE 2CONSTRUCTION OF BACILLUS STRAINS CO-EXPRESSINGAN AMYLASE AND NUCLEASE

[0569] In the present example, a parent B. licheniformis strain overexpressing a heterologous a-amylase (SEQ ID NO: 1) reporter protein was modified with the introduction of nuclease expression cassettes in the format of an upstream (5') amyL promoter region (SEQ ID NO: 2) operably linked to the aprE 5'-UTR (SEQ ID NO: 3) operably linked to DNA encoding a signal (peptide) sequence set forth in TABLE 2 operably linked to DNA encoding a mature nuclease sequence set forth in TABLE 3 operably linked to a spoVG transcriptional terminator (SEQ ID NO: 4) operably linked to a selectable marker to build a series of modified B. licheniformis strains overexpressing both the native alpha amylase reporter (SEQ ID NO: 1) and a heterologous nuclease set forth in TABLE 3.NB42146-WO-PCT[2]TABLE 2LIST OF SIGNAL SEQUENCES USED IN THE NUCLEASE CONSTRUCTSSignal Sequence PRT SEQ ID NO DNA SEQ ID NONsxNucl_sp 5 6MetNuc70_sp 7 8BhoNuc2_sp 9 10BhoNuc3_sp 11 12BhoNucl_sp 13 14UstNuc8_sp 15 16UstNuc11_sp 17 18MetNuc36_sp 19 20amyL_sp 21 22aprL_sp 23 24TABLE 3LIST OF MATURE NUCLEASE SEQUENCES USED IN THE NUCLEASE CONSTRUCTSMature Nuclease PRT SEQ ID NO DNA SEQ ID NO SequenceNsxNucl 25 26MetNuc70 27 28BhoNuc2 29 30MetNuc71 31 32UstNuc9 33 34BhoNuc3 35 36BhoNucl 37 38UstNuc8 39 40UstNuc11 41 42MetNuc19 43 44MetNuc72 45 46MetNuc73 47 48MetNuc36 49 50UstNuc7 51 52

[0570] In particular, all nuclease expression cassettes were transformed into the parental strain using the methods described PCT Publication No. W02019 / 040412 (incorporated herein by referenced in its entirety). More specifically, for each mature nuclease presented above in TABLE 3, several combinations were constructed with the various signal sequences shown above in TABLE 2. For instance, a list of all nuclease expression cassettes constructed in this example are set forth below in TABLE 4.NB42146-WO-PCT[2]TABLE 4LIST OF CONSTRUCTS ENCODING A SIGNAL SEQUENCE OPERABLY LINKED TO A MATURE NUCLEASE SEQUENCENuclease Construct Signal Sequence Mature Nuclease PRT SEQ DNA SEQ Number ID NO ID NO1 NsxNucl_sp NsxNucl 53 542 MetNuc70_sp MetNuc70 55 563 BhoNuc2_sp BhoNuc2 57 584 BhoNuc3_sp BhoNuc3 59 605 BhoNucl_sp BhoNucl 61 626 UstNuc8_sp UstNuc8 63 647 UstNuc11_sp UstNuc11 65 668 MetNuc36_sp MetNuc36 67 689 amyL_sp NsxNucl 69 7010 amyL_sp MetNuc70 71 7211 amyL_sp MetNuc71 73 7412 amyL_sp BhoNuc3 75 7613 amyL_sp UstNuc11 77 7814 amyL_sp MetNuc19 79 8015 amyL_sp MetNuc72 81 8216 amyL_sp MetNuc73 83 8417 amyL_sp NsxNucl 85 8618 amyL_sp BhoNuc2 87 8819 amyL_sp MetNuc71 89 9020 amyL_sp UstNuc9 91 9221 amyL_sp BhoNuc3 93 9422 amyL_sp BhoNucl 95 9623 amyL_sp UstNuc8 97 9824 amyL_sp UstNuc11 99 10025 amyL_sp MetNuc19 101 10226 amyL_sp MetNuc72 103 10427 amyL_sp MetNuc36 105 10628 amyL_sp UstNuc7 107 108EXAMPLE 3ASSAYING STRAINS FOR PRODUCTION OF AMYLASE AND NUCLEASE ACTIVITY AND THERMOTOLERANCE

[0571] In the instant example, the parent B. licheniformis strain without a nuclease co-expression cassette and the twenty-eight (28) constructed strains comprising an introduced nuclease expression cassette described in Example 1 (TABLE 4) were grown under small scale assay conditions as described in PCTNB42146-WO-PCT[2]Publication Nos. WO2018 / 156705 and WO2019 / 055261. For instance, total a-amylase reporter production was determined by measuring total protein in the cell-free broth using the method of Bradford, as presented below in TABLE 5.TABLE 5AMYLASE PRODUCTION IN STRAINS CO-EXPRESSING A MATURE NUCLEASE Strain Number Nuclease Construct Nuclease Normalized Amylase Number SEQ ID NO ProductionParent (control) N / A N / A 1.01 1 53 0.96 2 2 55 0.85 3 3 57 1.04 4 4 59 0.99 5 5 61 1.00 6 6 63 1.02 7 7 65 1.01 8 8 67 0.82 9 9 69 0.98 10 10 71 0.86 11 11 73 1.00 12 12 75 0.99 13 13 77 1.02 14 14 79 1.02 15 15 81 0.99 16 16 83 0.95 17 17 85 1.04 18 18 87 1.03 19 19 89 1.08 20 20 91 1.0421 21 93 1.0422 22 95 0.9223 23 97 1.0424 24 99 1.0725 25 101 1.0726 26 103 0.9927 27 105 0.9928 28 107 1.05

[0572] As shown above in TABLE 5, all constructs produced at least 82% of the a-amylase reporter as the parental (control) strain, with a number of constructs producing slightly more a-amylase than the control.

[0573] A portion of the broth was then held at 60°C for one (1) hour to test the thermal stability of the nuclease. After the hold at 60°C, five-hundred (500) ng of genomic DNA (gDNA) from the parent strain was added to ten (10) pl of thermally-stressed broth or unstressed broth, and held at 37°C for one (1) hour. Integrity of the added (spiked) gDNA was determined by running the samples on a ZAG DNA AnalyzerNB42146-WO-PCT[2]System (Agilent) per manufacturer’s instructions. More specifically, both the remaining DNA concentration and the average DNA size were determined from this analysis, and are presented below in TABLE 6 and TABLE 7, respectively.TABLE 6RELATIVE DNA CONCENTRATION IN THERMAL-STRESSED AND UNSTRESSED BROTHS (100-18000 BP+)Strain Nuclease Nuclease Relative DNA Relative DNA Number Construct Number SEQ ID Concentration Unstressed Concentration Stressed Parent N / A N / A 1.000 1.000 1 1 53 0.215 2.496 2 2 55 0.346 0.589 3 3 57 0.012 2.466 4 4 59 1.344 1.302 5 5 61 0.000 1.329 6 6 63 0.006 1.353 7 7 65 0.196 1.606 8 8 67 0.383 0.537 9 9 69 1.166 1.198 10 10 71 0.238 1.130 11 11 73 0.000 0.005 12 12 75 4.305 1.875 13 13 77 0.250 1.292 14 14 79 0.407 0.524 15 15 81 0.040 0.000 16 16 83 0.004 0.017 17 17 85 1.513 1.257 18 18 87 0.045 0.512 19 19 89 0.205 0.013 20 20 91 0.983 1.148 21 21 93 2.804 1.785 22 22 95 0.015 ND* 23 23 97 0.057 0.586 24 24 99 0.018 ND* 25 25 101 0.080 0.035 26 26 103 0.015 0.000 27 27 105 0.000 0.00428 28 107 0.846 1.371ND*: Not determined due to overlap with analytical marker100-18000 BP+: Under the conditions of the analysis on the ZAG DNA Analyzer, fragments less than (<) 100 base pairs or greater than (>)18,000 base pairs would not be detected.NB42146-WO-PCT[2]TABLE 7RELATIVE AVERAGE DNA SIZE IN THERMAL-STRESSED AND UNSTRESSED BROTHS (100-18000 BP+)Strain Nuclease Nuclease Relative DNA Size Relative DNA Number Construct SEQ ID Unstressed Size Stressed NumberParent N / A N / A 1.000 1.0001 1 53 0.100 0.668 2 2 55 0.067 0.057 3 3 57 0.726 0.064 4 4 59 0.992 0.983 5 5 61 0.000 0.043 6 6 63 0.850 0.913 7 7 65 0.100 0.503 8 8 67 0.074 0.056 9 9 69 0.720 1.000 10 10 71 0.181 0.056 11 11 73 0.000 0.618 12 12 75 0.902 0.954 13 13 77 0.066 0.565 14 14 79 0.051 0.064 15 15 81 0.632 0.591 16 16 83 0.959 0.940 17 17 85 0.047 0.976 18 18 87 0.313 0.069 19 19 89 0.241 0.741 20 20 91 0.911 0.959 21 21 93 0.161 0.261 22 22 95 0.673 0.175 23 23 97 0.544 1.089 24 24 99 0.933 0.119 25 25 101 0.760 0.389 26 26 103 0.514 0.541 27 27 105 0.000 0.94428 28 107 0.460 0.966100-18000 BP+: Under the conditions of the analysis on the ZAG DNA Analyzer, fragments less than (<) 100 base pairs or greater than (>) 18,000 base pairs would not be detected.

[0574] As generally shown above in TABLES 6 and 7, some nucleases might decrease both the relative concentration (Lower relative values in TABLE 6) and the relative size of DNA (Lower relative values TABLE 7) after thermal stress. Additionally, one might find nucleases that decrease the overall concentration of DNA (low relative DNA values in TABLE 6) after thermal stress while leaving some larger DNA fragments (Higher relative values TABLE 7) or nucleases that significantly decrease the average size of the DNA after thermal stress (low relative values TABLE 7) while leaving a higherNB42146-WO-PCT[2]concentration of DNA (higher relative values TABLE 6). To someone skilled in the art, all three of these would be recognized as having thermal stable nuclease activity by virtue of decreasing the DNA concentration (TABLE 6), the DNA size (TABLE 7), or both.

[0575] As presented above, the results of this analysis indicate that co-expression of nucleases with a-amylases can be optimized by nuclease and signal sequence selection to minimize impact on production of the protein of interest. Additionally, as shown in TABLE 6, there are several nucleases capable of reducing the concentration of DNA in a fermentation broth by at least 1.4 x to about lOOx, both with and without heat stress. Likewise, as presented above in TABLE 7, these nucleases also reduce the size of the remaining DNA by at least 1.4x to about 20x, both with and without heat stress as compared to the parent strain not expressing a heterologous nuclease.EXAMPLE 4CONSTRUCTION OF BACILLUS STRAINS CO-EXPRESSING AN ENZYME OF INTEREST AND NUCLEASE

[0576] In the instant example, B. subtilis strains expressing a subtilisin protease (i.e., either SEQ ID NO: 110, SEQ ID NO: 112 or SEQ ID NO: 114), or a [3-glucanase (SEQ ID NO: 116) were modified to include the co-expression of a heterologous nuclease (i.e., SEQ ID NO: 31 or SEQ ID NO: 133).

[0577] Co-expression of a Protease with a Heterologous Nuclease

[0578] B. subtilis strains expressing a B. amyloliquefaciens subtilisin variant (SEQ ID NO: 110), a B. gibsonii subtilisin variant (SEQ ID NO: 112), or a T. cellulosilytica subtilisin (SEQ ID NO: 114) were constructed as described hereafter. A first DNA fragment comprising a 5' aprE gene flanking region (5' rzpi'E-FR; SEQ ID NO: 117) was operably linked to a polynucleotide construct (e.g. expression cassette) comprising the nucleotide sequence of a B. subtilis P2 (SEQ ID NO: 118) promoter region operably linked to a downstream nucleotide sequence comprising a B. subtilis aprE 5'-UTR (SEQ ID NO: 3) operably linked to a downstream nucleotide sequence encoding B. subtilis aprE signal sequence (aprE_sp; SEQ ID NO: 119) operably linked to a nucleotide sequence encoding the pro and mature (precursor) sequence of the selected protease (SEQ ID NO: 109, SEQ ID NO: 111 or SEQ ID NO: 113) gene operably linked to a downstream Bacillus amyloliquefaciens BPN' terminator (SEQ ID NO: 121).

[0579] Likewise, a first nuclease expression cassette was constructed, wherein the cassette comprises an upstream heterologous aprE gene promoter sequence (SEQ ID NO: 134) operably linked to downstream DNA encoding a heterologous signal peptide (MetNuc71_sp; SEQ ID NO: 123) operably linked to downstream DNA (SEQ ID NO: 32) encoding a heterologous nuclease (SEQ ID NO: 31) operably linked to a downstream DNA comprising a spoVG terminator sequence (SEQ ID NO: 4) operably linked to a 3' aprE gene flanking region (3' oprE-FR; SEQ ID NO: 125).NB42146-WO-PCT[2]

[0580] In another instance, such as the B. subtilis strain CB472 shown below in TABLE 8 (expressing subtilisin variant of SEQ ID NO: 109), a second nuclease expression cassette was constructed. In particular, this cassette comprises an upstream heterologous aprE gene promoter (SEQ ID NO: 134) operably linked to downstream DNA (SEQ ID NO: 119) encoding an aprE signal sequence (aprE_sp; SEQ ID NO: 120) operably linked to a downstream DNA sequence (SEQ ID NO: 132) encoding a heterologous nuclease (SEQ ID NO: 133) operably linked to a downstream DNA sequence comprising a spoVG terminator sequence (SEQ ID NO: 4), and integrated into B. subtilis by double-crossing-over at the ppsC locus.

[0581] More specifically, the B. subtilis strains expressing a subtilisin protease (SEQ ID NO: 110, SEQ ID NO: 112 or SEQ ID NO: 114) in absence or presence of a co-expressed nuclease (SEQ ID NO: 31, or SEQ ID NO: 133) are listed below in TABLE 8.TABLE 8BACILLUS STRAINS EXPRESSING A PROTEASE IN ABSENCE AND PRESENCE OF A CO-EXPRESSED NUCLEASEStrain Name Protease MatureSEQ ID NO NucleaseSEQ ID NO CB455 110 N / ACB472 110 133AP514 110 31CZ508 112 N / ACZ507 112 31CZ509 114 N / ACZ510 114 31

[0582] Co-expression of a p-glucanase with a Heterologous Nuclease

[0583] In the present example, a B. subtilis strain deleted for the native exoproteases aprE (SEQ ID NO: 128), nprE (SEQ ID NO: 129), bpr (SEQ ID NO:130) and epr (SEQ ID NO:131) and over-expressing a P-glucanase (SEQ ID NO: 116) was constructed as described hereafter. A first DNA fragment comprising a 5' aprE gene flanking region (5' aprE-PR SEQ ID NO: 117) was operably linked to a polynucleotide construct (e.g., expression cassette) comprising an upstream DNA sequence comprising a B. subtilis P2 (SEQ ID NO: 118) promoter region operably linked to a downstream DNA sequence comprising a B. subtilis aprE 5'-UTR (SEQ ID NO: 3) operably linked to a downstream DNA sequence (SEQ ID NO: 126) encoding a B. subtilis -glucanase signal sequence (BglS_sp; SEQ ID NO: 127) operably linked to a downstream DNA sequence (SEQ ID NO: 115) encoding the endo-beta-glucanase (BglS; SEQ ID NO: 116) operably linked to a downstream DNA sequence comprising a Bacillus amyloliquefaciens BPN terminatorNB42146-WO-PCT[2](SEQ ID NO: 121). This linear DNA fragment was used to transform B. subtilis cells. The resulting strain was called CZ483.

[0584] Likewise, in the instant example, the first cassette with the reporter 0-glucanase gene was operably linked to a second expression cassette containing a nuclease gene. More particularly, the second nuclease cassette comprises an upstream heterologous promoter sequence (SEQ ID NO: 122) operably linked to a downstream DNA sequence (SEQ ID NO: 123) encoding a heterologous signal peptide (MetNuc71_sp; SEQ ID NO: 124) operably linked to a downstream DNA sequence (SEQ ID NO: 32) encoding a heterologous nuclease (SEQ ID NO: 31) operably linked to a downstream DNA sequence comprising a spoVG terminator (SEQ ID NO: 4) operably linked to a 3' aprE gene flanking region (3' aprE-FR SEQ ID NO: 125). Bacillus subtilis cells were transformed with this linear DNA cassette to create strain CZ502.

[0585] In a second example, the first cassette with the reporter -glucanase gene was operably linked to a to a different downstream expression cassette containing a nuclease gene. In this instance, the nuclease expression cassette comprises an upstream aprE promoter (SEQ ID NO: 134) region operably linked to a downstream DNA sequence (SEQ ID NO: 119) encoding an aprE signal sequence (aprE_sp; SEQ ID NO: 120) operably linked to a downstream DNA sequence (SEQ ID NO: 132) encoding a heterologous nuclease (SEQ ID NO: 133) operably linked to a downstream DNA sequence comprising a spoVG terminator sequence (SEQ ID NO: 4) and by double-crossing-over in Bacillus subtilis ppsC locus to create strain CZ493. Thus, these DNA fragments were assembled using standard molecular biology techniques and were used as templates to develop linear DNA expression cassettes for integration in B. subtilis genome. More particularly, B. subtilis strains expressing the 0-glucanase (SEQ ID NO: 116) in absence or presence of a co-expressed nuclease (SEQ ID NO: 31 or SEQ ID NO: 133) are listed below in TABLE 9.TABLE 9BACILLUS STRAINS EXPRESSING A GLUCANASE IN ABSENCE AND PRESENCE OF A CO-EXPRESSED NUCLEASEStrain P-glucanase Mature NucleaseName SEQ ID NO SEQ ID NOCZ483 116 N / ACZ493 116 133CZ502 116 31NB42146-WO-PCT[2]EXAMPLE 5NUCLEASE CO-EXPRESSION IN BACILLUS STRAINS DOES NOT ALTER REPORTER PROTEIN PRODUCTION

[0586] In the instant example, Applicant assessed production of the reporter proteins (Example 4) in the strains co-expressing a nuclease. More specifically, B. subtilis strains expressing a subtilisin protease (i.e., SEQ ID NO: 110, SEQ ID NO: 112 or SEQ ID NO: 114), or a P-glucanase (SEQ ID NO: 116), and co-expressing a nuclease (SEQ ID NO: 31 or SEQ ID NO: 133) were compared to the parental strain not expressing a nuclease. In particular, the B. subtilis strains were grown in 24-well microtiter plates (MTPs) in cultivation medium (enriched semi-defined media based on MOPs buffer, with urea as major nitrogen source, maltodextrin as the main carbon source, supplemented with 3% soy tone for robust cell growth) for two (2) days at 37°C, 250 rpm, with 80% humidity in shaking incubator. After 48 hours, culture supernatants were used to measure (assay or SDS-PAGE) reporter activity to determine productivity levels.

[0587] Protease Activity Assays

[0588] Protease activity was determined by measuring the hydrolysis of the synthetic suc-AAPF-pNA peptide substrate. For the AAPF assay, the reagent solutions used were 100 mM Tris pH 8.6, 10 mM CaCl2, 0.005% Tween®-80 (Tris / Ca buffer) and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution; Sigma: S-7388). To prepare a working solution, 1 mL suc-AAPF-pNA stock solution was added to 100 mL Tris / Ca buffer and mixed. An enzyme sample was added to a microtiter plate (MTP) containing 1 mg / mL suc-AAPF-pNA working solution and assayed for activity at 405 nm over three-five (3-5) minutes using a SpectraMax plate reader in kinetic mode at room temperature. The protease activity was expressed as relative absorbance at 405 nm.

[0589] Supernatants of B. subtilis strains CB455 (control, no nuclease co-expression) and AP514 (nuclease co-expression) were compared for production of the B. amyloliquefaciens subtilisin variant (SEQ ID NO: 110). As presented below in TABLE 10, similar to slightly higher subtilisin production was observed in strain AP514 co-expressing the Bacillus sp. nuclease (SEQ ID NO: 31) and CB472 co-expressing T. reesei nuclease (SEQ ID NO: 133), compared to the parent (control) strain CB455.TABLE 10PROTEASE ACTIVITY ASSAYMature Nuclease Relative ProteaseStrain NameSEQ ID NO ActivitiesCB455 (control) N / A 1.00AP514 31 1.02CB472 133 1.00NB42146-WO-PCT[2]

[0590] Additionally, supernatants of B. subtilis strains CZ508 (control, no nuclease co-expression) and CZ507 (nuclease co-expression) were compared for production of the B. gibsonii subtilisin valiant (SEQ ID NO: 112). As set forth below in TABLE 11, similar to higher subtilisin production in strain CZ507 (coexpressing a nuclease (SEQ ID NO: 31) was observed compared to the parent (control) strain CZ508.TABLE 11PROTEASE ACTIVITY ASSAYMature Nuclease Relative ProteaseStrain NameSEQ ID NO ActivitiesCZ508 (control) N / A 1.00CZ507 31 1.14

[0591] Likewise, supernatants of B. subtilis strains CZ509 (control, no nuclease co-expression) and CZ510 (nuclease co-expression) were compared for production of the T. cellulosilytica subtilisin protease (SEQ ID NO: 114). As shown below in TABLE 12, similar subtilisin production in strain CZ509 (control) and strain CZ510 that co-expresses the nuclease (SEQ ID NO: 31) was observed.TABLE 12PROTEASE ACTIVITY ASSAYMature Nuclease Relative ProteaseStrain NameSEQ ID NO ActivitiesCZ509 (control) N / A 1.00CZ510 31 0.93

[0592] Beta-glucanase Activity Test

[0593] The P-glucanase activity test measures the release of reducing sugars by the action of P-glucanase on a Barley P-Glucan (medium viscosity) substrate. For instance, 3,5-dinitrosalicyclic acid (DNS) reacts with reducing sugars to form 3-amino-5-nitrosalicyclic acid which absorbs light at 540 nm.

[0594] The rate of reducing sugar release, as measured by the reaction with 3, 5, dinitrosalicylic acid (DNS), is proportional to the enzyme activity. The p-glucanase activity is measured as relative absorbance at 540 nm. A calibration curve was developed using P-glucanase enzyme standard. The activity in this procedure is proportional to the amount of enzyme expressed and measured relative to an enzyme standard with assigned P-glucanase units defined as BBUs (Beta-Glucanase Units). More particularly, native expression of P-glucanase enzyme in the parent B. subtilis strain was compared to the strains overexpressing P-glucanase (i.e., strains CZ483, CZ493 and CZ502) as presented below in TABLE 13, wherein the nuclease co-expression in strains CZ493 and CZ502 do not negatively affect expression of the p-glucanase.NB42146-WO-PCT[2]TABLE 13p-GLUCANASE ACTIVITY ASSAYStrain Relative ActivityName BBUsParent 0CZ483 1.00CZ493 1.19CZ502 1.18

[0595] Nuclease Activity Test

[0596] The nuclease activity test was carried out as described herein. Five-hundred (500) ng of lambda DNA (gDNA) from ThermoScientific (Catalogue No. SD0011) was added to 10 pl of broth supernatant and held at 37°C for one (1) hour. Integrity of the added gDNA was determined by running the samples on a ZAG DNA Analyzer System (Agilent) per manufacturer’s instructions. The remaining DNA concentration in each sample and the relative DNA size are shown below in TABLE 14.TABLE 14RELATIVE DNA CONCENTRATION AND AVERAGE DNA SIZEStrain Target Enzyme Mature Relative DNA Relative DNA Name SEQ ID NO Nuclease Concentration SizeSEQ ID NO CZ483 116 N / A 1.00 1.00CZ493 116 133 1.00 0.57CZ502 116 31 0.43 0.57CB455 110 N / A 1.00 1.00CB472 110 133 0.75 0.87AP514 110 31 0.37 0.70CZ508 112 N / A 1 1CZ507 112 31 0.45 0.34CZ509 114 N / A 1 1CZ510 114 31 0.59 0.31

[0597] Thus, as presented in TABLE 14 above, reduction of DNA concentration and / or DNA size was observed in the strains co-cxpressing a nuclease (i.e., CZ493, CZ502, CB472, AP514, CZ507, CZ510) compared to their respective parent (control) strains (i.e., CZ483, CB455, CZ508, CZ509; not expressing the nuclease).NB42146-WO-PCT[2]EXAMPLE 6CONSTRUCTION OF TRICHODERMA STRAINS CO-EXPRESSING AN ENZYME OF INTEREST AND NUCLEASE

[0598] In the instant example, a parent Trichoderma reesei strain expressing a trehalase (PCT Publication No. WO2015 / 065978) was modified herein to include the co-expression of a nuclease. More particularly, the nuclease expression cassette comprises an upstream T. reesei gpdl gene promoter sequence (SEQ ID NO: 137) operably linked to downstream DNA sequence (SEQ ID NO: 138) encoding a pepl signal peptide (pepl_sp) operably linked to a downstream DNA sequence (SEQ ID NO: 136) encoding a heterologous nuclease (SEQ ID NO: 135) operably linked to a downstream DNA sequence comprising a CBHI terminator (SEQ ID NO: 139) and was integrated in T. reesei at the sdil locus. This linear DNA fragment was used to transform T. reesei cells as described in PCT Publication No. WO202323644. More particularly, these DNA fragments were assembled using standard molecular biology techniques and were used as templates to develop linear DNA expression cassettes for integration in the T. reesei genome. Four (4) resulting transformed clones were isolated. Thus, the T. reesei strains constructed, expressing the trehalase enzyme in absence or presence of the co-expressed nuclease (SEQ ID NO: 135) are listed below TABLE 15.TABLE 15TRICHODERMA STRAINS EXPRESSING A TREHALASE IN THE ABSENCE AND PRESENCE OF A CO-EXPRESSED NUCLEASEStrain Name Target Enzyme Mature NucleaseSEQ ID NO SEQ ID NO FS65-1 140 135FS65-1 140 135FS65-2 140 135FS65-2 140 135FS65-3 140 135FSOO 140 N / AFS65-3 140 135FS65-4 140 135FSOO (control) 140 N / AFS65-4 140 135NB42146-WO-PCT[2]EXAMPLE 7NUCLEASE CO-EXPRESSION IN TRICHODERMA STRAINS DOES NOT ALTER PRODUCTION OF THE REPORTER PROTEIN

[0599] In the instant example, Applicant assessed production of the reporter protein in the strain coexpressing a nuclease, wherein the T. reesei strains (FS65) expressing the trehalase reporter protein and a nuclease (SEQ ID NO: 135) were compared to the parental strain (FS00) not expressing the nuclease. Several transformants were grown in duplicate microtiter plate fermentations. The supernatants of the T. reesei trehalase strains FSOO (control, no nuclease co-expression) and FS65 clones (with nuclease co-expression) were compared for production of trehalase reporter protein and nuclease activity.

[0600] Trehalase activity assay was performed in 50 mM acetate buffer, pH 5. The assay cocktail was prepared with 60 pL of 3% trehalose and 40 pL of samples in the assay buffer. The assay cocktail was incubated at 40°C for 15 minutes. At the end of incubation, glucose was measured using the ABTS assay. The ABTS assay measures glucose in a coupled reaction where glucose is oxidized to 5-D-gluconolactone and hydrogen peroxide in the presence of glucose oxidase (GOX). The production of hydrogen peroxide is coupled to a reaction catalyzed by horseradish peroxidase (HRP), where HRP oxidizes 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), producing a brilliant blue-green color product that can be measured kinetically at 420 nm in the plate reader.

[0601] As presented below in TABLE 16, similar to trehalase production was observed in strain FS65 (coexpressing a nuclease; SEQ ID NO: 140) compared to the parent (control) strain FSOO.TABLE 16TREHALASE ACTIVITY ASSAYStrain Name Target Enzyme Mature Nuclease Relative TrehalaseSEQ ID NO SEQ ID NO ActivityFS65-1 140 135 3.0FS65-1 140 135 3.0FS65-2 140 135 2.9FS65-2 140 135 3.0FS65-3 140 135 3.1FSOO 140 N / A 3.0FS65-3 140 135 3.1FS65-4 140 135 2.8FSOO (control) 140 N / A 3.1FS65-4 140 135 3.0NB42146-WO-PCT[2]

[0602] Nuclease Activity TestThe nuclease activity test was carried out as described herein: Nuclease activity measurement was conducted using the Promega® QuantiFluor® ONE dsDNA System detection kit. The assay was performed at room temperature for 5 minutes using a 15ppm lambda DNA substrate in 25mM MES, 10mM MgCl2 and 1mM CaCl2, pH 5.4 assay buffer. Nuclease activity is quantified by measuring the residual DNA amount using QuantiFluor dye fluorescence at 504nmEx & 531nmEm. Purified nuclease enzyme standard was used to calculate the nuclease concentration in tested samples.TABLE 17NUCLEASE ACTIVITY ASSAYMatureStrain Name Target Enzyme Nuclease Relative NucleaseSEQ ID NO SEQ ID NO ActivityFS65-1 140 135 50.7FS65-1 140 135 47.7FS65-2 140 135 43.3FS65-2 140 135 44.9FS65-3 140 135 20.9FS00 140 N / A 0.2FS65-3 140 135 18.8FS65-4 140 135 9.2FS00 140 N / A 0.0FS65-4 140 135 7.9EXAMPLE 8EXPRESSION, FERMENTATION AND PURIFICATION OF NUCLEASES

[0603] In the present example, various natural isolates from Applicant’s microbial strain collection were selected as a potential source for nuclease enzymes useful in industrial applications, wherein DNA sequences encoding the mature protein sequence of nucleases (FIG. 2) were codon-optimized for the expression in B. subtilis. In particular, the polynucleotides were synthesized by Sangon (Sango Biotech Co., Ltd, Shanghai, China) or Azenta (AzentaLife Sciences, Shanghai, China) and inserted into the p2JM expression vector, a derivative vector from p2JM103BBI plasmid described in Vogtentanz et al. (2007). Subsequently, the expression plasmids containing nucleases were transformed to B. subtilis cells, and the transformed cells were spread on Luria Agar plates supplemented with five (5) ppm Chloramphenicol.

[0604] The colony with correct insertion, confirmed by PCR and sequencing, was selected and subjected to fermentation in an ultra-yield shake flask with Grant’s II medium (a MOPS-based defined medium). Supernatants from these fermentation cultures were used to confirm the protein expression by SDS-PAGENB42146-WO-PCT[2]analysis. The broth from the shake flasks was concentrated and buffer-exchanged into appropriate buffer system for downstream purification processes. For instance, the purification procedure will depend on the specific nuclease, and as such, diverse chromatography methods were used, aiming to improve the purity of target nuclease to approximately 70%. The final purified enzyme solution was added to 40% glycerol and stored at -20°C for future usage.EXAMPLE 9SEQUENCES OF THE NUCLEASES

[0605] The mature protein sequence of nucleases presented below in TABLE 18 were used for codon-optimized expression in Bacillus subtilis. In particular, Applicant constructed expression plasmids encoding the mature nuclease sequences shown in FIG. 2 and TABLE 18, wherein the name, sequence identification number (SEQ ID), molecular weight (Da), total number of amino acid residues and domain family for each nuclease are shown.NB42146-WO-PCT[2]TABLE 18MATURE NUCLEASE SEQUENCE DETAILSName SEQ ID Molecular Residues Domain Family Weight (Da)PtaNucl 142 20096.25 182 DUF1524 MabNucl 143 20272.42 182 DUF1524 TspNuc5 144 20136.13 182 DUF1524 TlaNucl 145 30223.39 266 Endonuclease_NS MetNuclO 146 20280.32 182 DUF1524 MetNucll 147 20082.26 182 DUF1524 MetNucl8 148 19994.13 182 DUF1524 MetNuc26 149 20047.25 182 DUF1524 MetNuc27 150 20272.47 182 DUF1524 MetNuc35 151 20231.42 182 DUF1524 MetNuc39 152 20266.47 182 DUF1524 MetNuc55 153 19742.77 182 DUF1524 MetNuc66 154 19851.14 182 DUF1524 MetNuc67 155 19815.07 182 DUF1524 BspNucll 156 20308.37 182 DUF1524 PspNuc6 157 20027.18 182 DUF1524 BspNucl4 158 20231.32 182 DUF1524 PmuNuc4 159 20158.31 182 DUF1524 MidNuc2 160 20000.02 182 DUF1524 BspNucl6 161 20098.37 182 DUF1524 PfrNucl 162 20079.07 182 DUF1524 MidNuc3 163 20004.99 182 DUF1524 BspNucl7 164 20511.79 182 DUF1524 BspNucl8 165 20512.78 182 DUF1524 BvaNucl 166 20550.86 182 DUF1524 BspNuc35 167 20507.79 182 DUF1524 BspNucl9 168 20006.97 182 DUF1524 BspNuc21 169 20450.74 182 DUF1524 BvaNuc2 170 20562.91 182 DUF1524BspNuc23 171 20534.81 182 DUF1524NB42146-WO-PCT[2]TABLE 18 (Continued)MATURE NUCLEASE SEQUENCE DETAILSName SEQ ID Molecular Residues Domain Family Weight (Da)BspNuc26 172 19874.99 182 DUF1524 BceNuc2 173 20583.89 182 DUF1524 BspNuc27 174 20519.75 182 DUF1524 CfiNucl 175 19894.98 182 DUF1524 BspNuc28 176 19913.88 182 DUF1524 TasNucl 177 20364.93 188 DUF1524 CpaNuc4 178 11984.32 110 DNase_NucA_NucB TauNucl 179 20403.54 186 DUF1524 SspNuc5 180 19397.49 180 DUF1524 EsiNucl 181 19781.86 182 DUF1524 EspNucl 182 19848.91 182 DUF1524 UstNucl 183 19841.1 182 DUF1524 PfiNucl 184 20268.47 182 DUF1524 MetNuc42 185 19898.97 182 DUF1524 MetNuc44 186 19897.84 182 DUF1524 MetNuc53 187 19728.75 182 DUF1524 MetNuc54 188 19756.8 182 DUF1524 MetNuc62 189 19910.22 182 DUF1524 MetNuc63 190 19937.29 182 DUF1524 MetNuc64 191 19808.89 182 DUF1524 MetNuc68 192 19885.16 182 DUF1524 MetNuc69 193 19748.01 182 DUF1524 NspNuc3 194 20192.33 182 DUF1524 JspNucl 195 20149.17 182 DUF1524 RaqNuc2 196 20097.05 182 DUF1524 BspNuc20 197 20080.06 182 DUF1524 BspNuc22 198 19825.73 182 DUF1524 BspNuc25 199 20493.73 182 DUF1524 RarNuc5 200 19936.92 182 DUF1524 BspNuc29 201 19854.81 182 DUF1524 BspNuc32 202 19911.82 182 DUF1524 PcuNucl 203 20873.35 195 DUF1524 CthNucl 204 20681.8 188 DUF1524 TpuNucl 205 20774.16 186 DUF1524 AspNuc7 206 28616.31 257 Endonuclease_NS RdeNucl 207 29088.6 270 Endonuclease_N S CecNucl 208 27498.66 252 Endonuclease_NSTspNucl4 209 28840.2 260 Endonuclease_N SNB42146-WO-PCT[2]TABLE 18 (Continued)MATURE NUCLEASE SEQUENCE DETAILSName SEQ ID Molecular Residues Domain Family Weight (Da)SenNuc2 210 16990.29 154 PLDc_2 PsyNuc2 211 16651.78 153 PLDc_2 MplNucl 212 30953.74 278 Endonuclease_N S GbuNuc1 213 28025.25 258 Endonuclease_NS BciNuc3 214 27697.09 247 Endonuclease_NS PniNucl 215 28503.07 260 Endonuclease_NS AglNuc2 216 28087.42 257 Endonuclease_N S GpcNucl 217 30456.15 276 Endonuclease_NS ZmeNucl 218 29645.98 263 Endonuclease_NS TspNucl 219 20069.97 182 DUF1524PmuNuc3 220 20064.24 182 DUF1524NB42146-WO-PCT[2]TABLE 19NUCLEIC ACID (DNA) SEQUENCES ENCODING MATURE NUCLEASES Name DNA PRT SEQSEQ ID IDPtaNucl 221 142MabNucl 222 143TspNuc5 223 144TlaNucl 224 145MetNuclO 225 146MetNucll 226 147MetNucl8 227 148MetNuc26 228 149MetNuc27 229 150MetNuc35 230 151MetNuc39 231 152MetNuc55 232 153MetNuc66 233 154MetNuc67 234 155BspNucll 235 156PspNuc6 236 157BspNucl4 237 158PmuNuc4 238 159MidNuc2 239 160BspNucl6 240 161PfrNucl 241 162MidNuc3 242 163BspNucl7 243 164BspNucl8 244 165BvaNucl 245 166BspNuc35 246 167BspNucl9 247 168BspNuc21 248 169BvaNuc2 249 170BspNuc23 250 171BspNuc26 251 172BceNuc2 252 173BspNuc27 253 174CfiNucl 254 175BspNuc28 255 176TasNucl 256 177CpaNuc4 257 178NB42146-WO-PCT[2]TABLE 19 (Continued)NUCLEIC ACID (DNA) SEQUENCES ENCODING MATURE NUCLEASES Name DNA PRT SEQSEQ ID IDTauNucl 258 179SspNuc5 259 180EsiNucl 260 181EspNucl 261 182UstNucl 262 183PfiNucl 263 184MetNuc42 264 185MetNuc44 265 186MetNuc53 266 187MetNuc54 267 188MetNuc62 268 189MetNuc63 269 190MetNuc64 270 191MetNuc68 271 192MetNuc69 272 193NspNuc3 273 194JspNucl 274 195RaqNuc2 275 196BspNuc20 276 197BspNuc22 277 198BspNuc25 278 199RarNuc5 279 200BspNuc29 280 201BspNuc32 281 202PcuNucl 282 203CthNucl 283 204TpuNucl 284 205AspNuc7 285 206RdeNucl 286 207CecNucl 287 208TspNucl4 288 209SenNuc2 289 210PsyNuc2 290 211MplNucl 291 212NB42146-WO-PCT[2]TABLE 19 (Continued)NUCLEIC ACID (DNA) SEQUENCES ENCODING MATURE NUCLEASES Name DNA PRTSEQ ID SEQ IDGbuNucl 292 213BciNuc3 293 214PniNucl 294 215AglNuc2 295 216GpeNuc 1 296 217ZmeNucl 297 218TspNucl 298 219PmuNuc3 299 220EXAMPLE 10EXPRESSION, FERMENTATION AND PURIFICATION OF NUCLEASES

[0606] In other embodiments, the mature protein sequence of nucleases (SEQ ID NO: 135, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 204, SEQ ID NO: 205. SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218) were used for codon optimized for the expression in Trichoderma reesei. The polynucleotides were synthesized by Sangon (Sango Biotech Co., Ltd, Shanghai, China) or Azenta (Azenta life sciences, Shanghai, China) and inserted into pGX256 expression vector, a derivative vector from pTTT (see, US Patent Publication No. 20110020899).

[0607] The pGX256 derived expression plasmids containing nucleases were transformed into a suitable Trichoderma reesei strain using protoplast transformation (Te’o et al., 2002). The transformants were selected and fermented by the methods described in PCT Publication No. WO2016 / 138315. Supernatants from these cultures were used to confirm the Protein expression by SDS-PAGE analysis.

[0608] The broth from fermentations of T. reesei transformants was concentrated and buffer-exchanged into an appropriate buffer system for downstream purification processing. The purification procedure depends on specific nucleases, and diverse chromatography methods were used, aiming to improve the purity of target nuclease to approximately 70%. The final purified enzyme solution was added to 40% glycerol and stored at -20°C for future usage.EXAMPLE 11

[0609] Nuclease Activity Determination Before and After Preincubation with Protease for Purified Enzyme Samples

[0610] The protease stability of mature nuclease sequences was identified by measuring the residual activities after incubating the purified nuclease samples with protease. Specifically, five (5) ppm purifiedNB42146-WO-PCT[2]nuclease samples were preincubated with five (5) ppm B. amyloliquefaciens subtilisin protease in 50 mM HEPES (pH 8.0) buffer in 96 well plates (Axygen, half skirt). The samples were then preincubated at 50°C in a PCR thermocycler. To exclude the possibility that the loss of the nuclease activity may also be caused by the incubation temperature, plates with the same prepared nucleases, but without protease, were preincubated at 4°C and 50°C as controlled experimental groups. The preincubated enzymes were sampled for the residual activity assay after incubation for sixty (60) minutes. The detailed activity assay was conducted by incubating 0.05 ppm nucleases with 100 ng / pL lambda DNA (Thermo Scientific, SD0011) in the presence of 10 mM Mg2+and 1 mM Ca2+in 50 mM Tris-HCl buffer (pH 7.0) (Axygen, full skirt). The reactions were carried out in iEMS incubator at 50°C for 10 minutes with the shake speed of 1150 rpm. After reactions, four (4) pL of the mixtures were transferred into 96 pL 2x SYBR Green I nucleic acid gel stain Mili-Q water solution (Invitrogen, S7563), mixing well for fluorescence detection (Corning, #3505). The excitation and emission wavelength were 490 nm and 530 nm respectively, using spectrophotometer. The activity of nuclease was indicated by the change in fluorescence before and after reaction, which was calculated by the following equation:F0— FRCorrected Delta Fluorescence Slqnal = - “ 10e8

[0611] Where Fo represents the initial fluorescence of substrate DNA, FR represents the detected fluorescence after reaction. 10e8 is a self-defined parameter to normalize the fluorescence data.

[0612] The residual activities of nuclease were also calculated using the following equation:Corrected Delta Fluorescence Signal (after stress) Residual Activity = - - - - - - - — - - — x 100%Corrected Delta Fluorescence Signal (control)

[0613] The results are shown below in TABLE 20. For instance, as presented in TABLE 20, among the seventy-six (76) nuclease candidates, forty-nine (49) demonstrated enhanced protease stability, with over 60% residual activities remaining after the protease stress conditions.NB42146-WO-PCT[2]TABLE 20NUCLEASE ACTIVITIES AFTER INCUBATION WITH PROTEASE AT 50°C FOR 60MINUTES SEQ Corrected Corrected Corrected Residual Residual ID NO delta delta delta activity w / activity w / fluorescence fluorescence fluorescence preincubatio preincubatio signal w / o signal w / signal w / n at 50°C n at 50°C preincubation preincubati preincubatio for 60 min with protease on at 50°C n at 50°C for 60 min for 60 min with proteasefor 60 min27 0.32 0.32 0.30 100% 96%31 0.53 0.55 0.53 104% 97%33 0.54 0.53 0.51 98% 97%41 0.54 0.51 0.48 96% 94%43 0.51 0.52 0.50 101% 96%45 0.55 0.53 0.51 96% 97%47 0.53 0.53 0.52 100% 98%49 0.46 0.47 0.43 103% 91%133 0.63 0.56 0.00 89% 0%142 0.55 0.53 0.52 97% 96%143 0.27 0.21 0.24 77% 86%144 0.48 0.46 0.46 97% 96%145 0.45 0.27 0.21 61% 46%146 0.54 0.49 0.53 90% 97%147 0.54 0.54 0.53 100% 99%148 0.53 0.52 0.48 98% 91%149 0.50 0.46 0.41 91% 81%150 0.54 0.54 0.53 100% 96%151 0.54 0.54 0.53 100% 98%152 0.52 0.48 0.40 92% 76%153 0.55 0.52 0.46 95% 85%154 0.58 0.55 0.54 95% 93%155 0.57 0.54 0.25 95% 43%156 0.52 0.51 0.44 97% 84%157 0.54 0.28 0.13 52% 24%NB42146-WO-PCT[2]TABLE 20 (Continued)NUCLEASE ACTIVITIES AFTER INCUBATION WITH PROTEASE AT 50°C FOR 60MINUTES SEQ Corrected Corrected Corrected Residual Residual ID delta delta delta activity w / activity w / NO fluorescence fluorescence fluorescence preincubatio preincubatio signal w / o signal w / signal w / n at 50°C n at 50°C preincubation preincubatio preincubatio for 60 min with protease n at 50°C for n at 50°C for 60 min 60 min with proteasefor 60 min158 0.52 0.49 0.47 95% 91%159 0.51 0.51 0.48 100% 94%160 0.53 0.50 0.49 94% 93%161 0.53 0.51 0.52 95% 97%162 0.50 0.50 0.50 101% 101%163 0.52 0.49 0.50 95% 97%164 0.56 0.54 0.50 97% 89%165 0.57 0.56 0.51 98% 90%166 0.46 0.31 0.34 68% 73%167 0.56 0.51 0.52 91% 93%168 0.51 0.45 0.42 87% 82%169 0.57 0.56 0.53 98% 93%170 0.57 0.57 0.49 99% 85%171 0.58 0.55 0.52 96% 90%172 0.53 0.51 0.42 95% 79%173 0.55 0.52 0.50 95% 91%174 0.58 0.53 0.52 90% 89%175 0.56 0.51 0.47 92% 84%176 0.54 0.10 0.09 18% 17%177 0.63 0.61 0.02 98% 0%178 0.52 0.52 0.23 101% 45%179 0.52 0.53 0.00 101% 0%180 0.58 0.52 0.00 89% 0%181 0.55 0.14 0.02 26% 0%NB42146-WO-PCT[2]TABLE 20 (Continued)NUCLEASE ACTIVITIES AFTER INCUBATION WITH PROTEASE AT 50°C FOR 60MINUTES SEQ ID Corrected Corrected Corrected Residual Residual NO delta delta delta activity w / activity w / fluorescence fluorescence fluorescence preincubation preincubation signal signal w / signal w / at 50° C for at 50° C with w / o preincubatio preincubatio 60 min protease for preincubation n at 50°C for n at 50°C 60 min 60 min withproteasefor 60 min182 0.56 0.54 0.11 97% 20%183 0.53 0.56 0.55 105% 103%184 0.51 0.48 0.46 95% 91%185 0.52 0.50 0.02 95% 0%186 0.52 0.50 0.00 95% 0%187 0.54 0.52 0.16 97% 30%188 0.52 0.51 0.45 99% 87%189 0.57 0.43 0.35 75% 62%190 0.57 0.50 0.44 88% 77%191 0.55 0.50 0.04 90% 7%192 0.57 0.55 0.53 96% 93%193 0.57 0.54 0.52 95% 91%194 0.47 0.49 0.47 104% 99%203 0.58 0.01 0.00 0% 0%204 0.56 0.12 0.02 22% 0%205 0.49 0.51 0.00 105% 0%206 0.70 0.00 0.00 0% 0%135 0.71 0.01 0.00 0% 0%207 0.55 0.00 0.02 0% 0%208 0.65 0.06 0.00 9% 0%209 0.25 0.01 0.01 0% 0%210 0.06 0.05 0.05 75% 75%211 0.50 0.02 0.00 0% 0%213 0.63 0.21 0.00 34% 0%218 0.63 0.01 0.01 0% 0%219 0.48 0.49 0.00 102% 0%220 0.50 0.51 0.49 100% 98%NB42146-WO-PCT[2]EXAMPLE 12IDENTIFYING NUCLEASES WITH ENHANCED PROTEASE STABILITY IN DIGESTING DOUBLE STRANDED DNA

[0614] Nuclease Activity Determination Before and After Preincubation with Protease for Crude Enzyme Samples

[0615] Forty-three (43) nuclease crude samples were harvested for protease stability (stress) measurements. The nuclease crude samples were firstly diluted by lOOx of the initial concentration with 100 ppm of B. amyloliquefaciens subtilisin in 50 mM HEPES (pH 8.0) buffer in 96 well plates (Axygen, half skirt). The plates were then preincubated at 50°C in iEMS incubator for one (1) hour. Plates with the same prepared samples, but no protease were preincubated at 4°C for one (1) hour as control.

[0616] After preincubations, the nuclease crude samples were further diluted to lOOOx for residual activity measurement. Specifically, the nuclease crude samples were incubated with 100 ng / pL lambda DNA (Thermo Scientific, SD0011) in the presence of 10 mM Mg2+and 1 mM Ca2+in 50 mM Tris-HCl buffer (pH 7.0) (Axygen, full skirt). The reactions were carried out in iEMS incubator at 50°C for ten (10) minutes with the shake speed of 1150 rpm. After reactions, four (4) pL of the mixtures were transferred into 96 pL 2x SYBR Green I nucleic acid gel stain Mili-Q water solution (Invitrogen, S7563), mixing well for fluorescence detection (Corning, #3505). The excitation and emission wavelength were 490 nm and 530 nm respectively using spectrophotometer. The activity of nuclease was indicated by the change in fluorescence before and after reactions, which was calculated using the following equation:F0— FRCorrected Delta Fluorescence Signal = (F₀ — F_R) / 10e8

[0617] Where F0represents the initial fluorescence of substrate DNA, FRrepresents the detected fluorescence after reaction. 10e8 is a self-defined parameter to normalize the fluorescent data.

[0618] The residual activities of nuclease were also calculated using the following equation:Residual Activity = Corrected Delta Fluorescence Signal (after stress) / Corrected Delta Fluorescence Signal (control) × 100%Corrected Delta Fluorescence Signal (control)

[0619] The detected corrected delta fluorescence signal and the calculated residual activity are shown in TABLE 21. In particular, as presented in TABLE 21, among the forty-three (43) nuclease crude samples, twenty-four (24) of them retained over 80% residual activities after stressing with the protease.NB42146-WO-PCT[2]TABLE 21NUCLEASE CRUDE SAMPLE ACTIVITIES BEFORE AND AFTER PREINCUBATION WITH PROTEASE AT 50° C FOR 60 MINUTESSEQ Corrected Corrected delta Residual activity w / ID NO delta fluorescence signal preincubation at fluorescence w / preincubation at 50° C with proteasesignal w / o 50° C with protease for 60 min preincubation for 60 min31 0.5 0.49 98%156 0.41 0.45 108%157 0.47 0.48 102%158 0.36 0.37 101%159 0.45 0.39 87%160 0.35 0.35 100%161 0.48 0.46 95%162 0.38 0.38 100%163 0.42 0.41 98%164 0.48 0.48 101%165 0.52 0.50 96%166 0.57 0.52 92%167 0.54 0.52 96%168 0.45 0.40 89%169 0.53 0.52 98%170 0.54 0.53 97%171 0.54 0.52 96%172 0.44 0.38 87%173 0.51 0.45 88%174 0.53 0.50 94%175 0.49 0.46 93%176 0.50 0.43 88%194 0.34 0.36 106%195 0.48 0.00 0%196 0.46 0.18 39%NB42146-WO-PCT[2]TABLE 21 Continued)NUCLEASE CRUDE SAMPLE ACTIVITIES BEFORE AND AFTER PREINCUBATION WITH PROTEASE AT 50° C FOR 60 MINUTESSEQ Corrected Corrected delta ResidualID NO delta fluorescence signal activity w / fluorescence w / preincubation at preincubationsignal w / o 50° C with protease at 50° C with preincubation for 60 min protease for 60min197 0.42 0.23 55%198 0.48 0.20 43%199 0.54 0.24 44%200 0.42 0.00 0%201 0.47 0.14 29%202 0.46 0.00 0%206 0.02 0.00 0%135 -0.02 0.00 0%207 0.58 0.00 0%208 0.22 0.00 0%212 0.69 0.00 0%213 0.75 0.00 0%214 0.56 0.00 0%215 0.69 0.00 0%216 0.75 0.00 0%217 0.74 0.00 0%218 0.66 0.00 0%220 0.50 0.41 82%NB42146-WO-PCT[2]EXAMPLE 13IDENTIFYING NUCLEASES WITH ENHANCED THERMOSTABILITY

[0620] Nuclease Activity Determination Before and After Preincubation at 60°C for Purified Enzyme Samples

[0621] The thermostability of nucleases was identified by measuring the residual activities after preincubation at 60°C for sixty (60) minutes. Specifically, five (5) ppm purified nuclease samples were prepared in 50 mM Tris-HCl (pH 7.0) buffer in 96 well plates (Axygen, half skirt). The plates were then preincubated at 60°C in a PCR thermocycler. The control plates with the same prepared samples were preincubated at 4°C for the same time. The enzymes were sampled for residual activity assay after incubation for 60 minutes. For the activity assay, 0.05 ppm nucleases were incubated with 100 ng / pL lambda DNA (Thermo Scientific, SD0011) in the presence of 10 mM Mg2+and 1 mM Ca2+in 50 mM Tris-HCl buffer (Axygen, full skirt). The reactions were carried out in iEMS incubator at 50°C for 10 minutes with the shake speed of 1150 rpm. After reactions, 4 pL of the mixtures were transferred into 96 pL 2x SYBR Green I nucleic acid gel stain Mili-Q water solution (Invitrogen, S7563), mixing well for fluorescence detection (Corning, #3505). The excitation and emission wavelength were 490 nm and 530 nm respectively using spectrophotometer. The activity of nuclease was indicated by the change in fluorescence before and after reactions, which was calculated as follows:Fo-FRCorrected Delta Fluorescence Siyqnal = - 10e8

[0622] Where F0represents the initial fluorescence of substrate DNA, FRrepresents the detected fluorescence after reaction. 10e8 is a self-defined parameter to normalize the fluorescent data.

[0623] The residual activities of nuclease were also calculated using the following equation:Corrected Delta Fluorescence Siqnal (after stress) Residual Activity = - - - - - ■ - — - - — x 100%Corrected Delta Fluorescence Signal (control)

[0624] The results are shown below in TABLE 22. Higher corrected delta fluorescence signal indicates a nuclease having better activity towards the substrate DNA. For instance, after stressing under 60°C for 60 minutes, most of the nucleases lost their activities at varying degrees. However, as presented in TABLE 22, 40% of the nuclease candidates could maintain over 60% of the activity after significant heat stress.NB42146-WO-PCT[2]TABLE 22NUCLEASE ACTIVITIES BEFORE AND AFTER PREINCUBATION AT 60°C FOR 60MINUTESSEQ Corrected Corrected delta Residual ID NO delta fluorescence activity after fluorescence signal w / preincubationsignal w / o preincubation at at 60° C for 60 preincubation 60°C for 60 min min27 0.24 0.26 110%31 0.55 0.54 98%33 0.51 0.52 102%41 0.49 0.04 8%43 0.52 0.52 99%45 0.48 0.31 65%47 0.54 0.53 99%49 0.44 0.42 95%133 0.58 0.33 57%142 0.54 0.51 94%143 0.25 0.22 91%144 0.46 0.48 105%145 0.27 0.24 89%146 0.53 0.28 53%147 0.54 0.54 100%148 0.55 0.34 62%149 0.45 0.09 19%150 0.55 0.49 90%151 0.55 0.48 87%152 0.44 0.09 21%153 0.48 0.20 41%154 0.58 0.25 43%155 0.58 0.22 38%156 0.50 0.23 45%157 0.41 0.45 110%158 0.51 0.12 23%NB42146-WO-PCT[2]TABLE 22 (Continued)NUCLEASE ACTIVITIES BEFORE AND AFTER PREINCUBATION AT 60° C FOR 60MINUTES SEQ Corrected Corrected delta Residual activityID NO delta fluorescence afterfluorescence signal w / preincubation at signal w / o preincubation at 60°C for 60 min preincubation 60° C for 60 min159 0.45 0.51 113%160 0.47 0.23 48%161 0.49 0.50 102%162 0.44 0.16 37%163 0.47 0.03 0%164 0.57 0.28 49%165 0.57 0.53 92%166 0.40 0.05 13%167 0.51 0.13 26%168 0.43 0.04 0%169 0.57 0.55 96%170 0.58 0.25 43%171 0.57 0.50 87%172 0.50 0.29 58%173 0.55 0.20 36%174 0.58 0.15 26%175 0.52 0.46 89%176 0.53 0.01 0%177 0.65 0.57 88%178 0.51 0.49 97%179 0.48 0.42 88%180 0.59 0.44 74%181 0.55 0.12 22%182 0.57 0.26 45%183 0.58 0.48 83%184 0.40 0.47 118%NB42146-WO-PCT[2]TABLE 22 (Continued)NUCLEASE ACTIVITIES BEFORE AND AFTER PREINCUBATION AT 60° C FOR 60MINUTES SEQ ID Corrected Corrected delta Residual activityNO delta fluorescence afterfluorescence signal w / preincubation at signal w / o preincubation at 60°C for 60 min preincubation 60° C for 60 min185 0.48 0.06 12%186 0.45 0.02 0%187 0.51 0.05 10%188 0.49 0.32 64%189 0.54 0.09 17%190 0.56 0.25 45%191 0.55 0.23 42%192 0.56 0.26 46%193 0.58 0.23 40%194 0.42 0.47 110%203 0.57 0.02 0%204 0.54 0.02 0%205 0.51 0.08 16%206 0.72 0.00 0%135 0.71 0.00 0%207 0.61 0.00 0%208 0.66 0.01 0%209 0.20 0.02 0%210 0.07 0.01 0%211 0.14 0.00 0%213 0.65 0.00 0%218 0.64 0.00 0%219 0.50 0.36 72%220 0.45 0.49 108%NB42146-WO-PCT[2]EXAMPLE 14NUCLEASE ACTIVITY DETERMINATION BEFORE AND AFTER PREINCUBATION AT60°C FOR CRUDE ENZYME SAMPLES

[0625] Forty-three (43) nuclease crude samples were harvested for thermostability measurement. The nuclease crude samples were firstly diluted by lOOx of the initial concentration in 50 mM Tris-HCl (pH 7.0) buffer in 96 well plates (Axygen, half skirt). The plates were then preincubated at 60°C in a PCR thermocycler for twenty-four (24) hours. The control plates with the same prepared samples were preincubated at 4°C for twenty-four (24) hours. After preincubations, the nuclease crude samples were further diluted to lOOOx for residual activity detection. In the activity assay, nuclease crude samples were incubated with 100 ng / pL lambda DNA (Thermo Scientific, SD0011) as double-stranded DNA substrate in the presence of 10 mM Mg2+and 1 mM Ca2+in 50 mM Tris-HCl buffer (Axygen, full skirt). The reactions were carried out in iEMS incubator at 50°C for 10 minutes with the shake speed of 1150 rpm. After reactions, 4 pL of the mixtures were transferred into 96 pL 2x SYBR Green I nucleic acid gel stain Mili-Q water solution (Invitrogen, S7563), mixing well for fluorescence detection (Corning, #3505). The excitation and emission wavelength were 490 nm and 530 nm respectively using spectrophotometer. The activity of nuclease was indicated by the change in fluorescence before and after reaction, which was calculated as follows:F0— FRCorrected Delta Fluorescence Siyqnal = - 10e8

[0626] Where F0represents the initial fluorescence of substrate DNA, FRrepresents the detected fluorescence after reaction. 10e8 is a self-defined parameter to normalize the fluorescent data.

[0627] The residual activities of nuclease were also calculated using the following equation:Corrected Delta Fluorescence Signal (after stress) Residual Activity = - - - - - - - — - - — x 100Corrected Delta Fluorescence Signal (control)

[0628] The results are listed below in TABLE 23. All the crude samples were harvested in the same fermentation batch and were considered with the similar expression levels. The fermentation broth was then directly diluted for further activity assay use. The results (TABLE 23) demonstrate >60% residual activity after stressing at 60°C for 24 hours in twenty (20) out of the forty-three (43) crude samples.NB42146-WO-PCT[2]TABLE 23NUCLEASE CRUDE SAMPLES ACTIVITY BEFORE AND AFTER PREINCUBATION AT60° C FOR 24 HOURSSEQ Corrected delta Corrected delta Residual activity ID NO fluorescence fluorescence signal aftersignal w / o w / preincubation at preincubation at preincubation 60° C for 24 hours 60° C for 24 hours31 0.51 0.41 81%156 0.49 0.07 14%157 0.52 0.15 29%158 0.47 0.31 66%159 0.48 0.38 78%160 0.45 0.33 74%161 0.50 0.03 6%162 0.47 0.35 73%163 0.48 0.11 23%164 0.54 0.09 16%165 0.55 0.36 65%166 0.58 0.09 16%167 0.56 0.12 22%168 0.48 0.35 73%169 0.58 0.07 12%170 0.59 0.01 0%171 0.57 0.03 6%172 0.48 0.34 71%173 0.55 0.04 8%174 0.56 0.36 65%175 0.52 0.40 77%176 0.52 0.43 82%194 0.47 0.31 66%195 0.48 0.38 80%196 0.48 0.40 82%197 0.46 0.37 80%198 0.49 0.41 85%NB42146-WO-PCT[2]TABLE 23 (Continued)NUCLEASE CRUDE SAMPLES ACTIVITY BEFORE AND AFTER PREINCUBATION AT60° C FOR 24 HOURSSEQ Corrected Corrected ResidualID delta delta activity after NO fluorescence fluorescence preincubationsignal w / o signal w / at 60°C for 24preincubation preincubation hoursat 60°C for 24hours199 0.56 0.41 73%200 0.48 0.35 72%201 0.47 0.37 77%202 0.48 0.36 75%206 0.75 0.00 0%135 0.75 0.01 0%207 0.68 0.01 0%208 0.20 0.00 0%212 0.71 0.01 0%213 0.75 0.01 0%214 0.58 0.00 0%215 0.72 0.00 0%216 0.75 0.00 0%>217 0.75 0.02 0%218 0.69 0.01 0%220 0.48 0.12 25%EXAMPLE 15IDENTIFYING NUCLEASES WITH ENHANCED PHOSPHATE TOLERANCE IN DIGESTING DOUBLE STRANDED DNA

[0629] Nuclease Activity Determination under 20 mM and 100 mM Phosphate Stress for Purified Samples

[0630] The phosphate tolerance of nucleases was identified by measuring the enzyme acti vibes in different concentrations of sodium phosphate buffer, wherein the phosphate stress comes from the sodium phosphate reaction buffer. For the activity assay, 0.05 ppm nucleases were incubated with 100 ng / pL lambda DNA (Thermo Scientific, SD0011) as double-stranded DNA substrate in the presence of 10 mM Mg2+and 1 mM Ca2+in 20 mM and 100 mM sodium phosphate buffer (pH 7.0) (Axygen, full skirt). The same reactions conducted in 20 mM and 100 mM Tris-HCl buffer (pH 7.0) were used as control groups. The reactions were carried out in iEMS incubator at 50°C for 10 minutes with the shaker speed of 1150 rpm. AfterNB42146-WO-PCT[2]reactions, 4 gL of the mixtures were transferred into 96 giL 2x SYBR Green I nucleic acid gel stain Mili-Q water solution (Invitrogen, S7563), mixing well for fluorescence detection (Corning, #3505). The excitation and emission wavelength were 490 nm and 530 nm respectively using a spectrophotometer. The activity of nuclease was indicated by the change in fluorescence before and after reactions, which was calculated as follows:F0- FRCorrected Delta Fluorescence Siyqnal = - 10e8

[0631] Where F0represents the initial fluorescence of substrate DNA, FRrepresents the detected fluorescence after reaction. 10e8 is a self-defined parameter to normalize the fluorescent data.

[0632] The residual activities of nuclease were also calculated using the following equation:Corrected Delta Fluorescence Signal (under stress) Residual Activity = - - - - - - - — - - — x 100%Corrected Delta Fluorescence Signal (control)

[0633] The detected results are shown in TABLE 24. For instance, as presented in TABLE 24, as compared with the control groups, the activity of nuclease was inhibited greatly in the presence of phosphates, wherein twenty-one (21) of fifty-three (53) candidates retained over 40% activity in the presence of 20 mM phosphates. As the concentration of phosphates increased to 100 mM, seven (7) of the nucleases continued to retain over 50% of their activity.NB42146-WO-PCT[2]TABLE 24NUCLEASE ACTIVITIES UNDER 20 mM AND lOOmM PHOSPHATE STRESS SEQ Corrected Corrected Corrected Corrected Residual Residual ID delta delta delta delta activity in activity in NO fluorescence fluorescence fluorescence fluorescence 20 mM lOOmM signal in 20 signal in 20 signal inlOO signal in phosphate phosphate mM Tris mM mM Tris 100 mM buffer buffer buffer phosphate buffer phosphatebuffer buffer27 0.48 0.00 0.42 0.00 0% 0% 31 0.55 0.38 0.48 0.01 68% 2% 33 0.53 0.20 0.46 0.00 39% 0% 41 0.53 0.12 0.46 0.00 22% 0% 43 0.55 0.20 0.46 0.01 36% 2% 45 0.50 0.18 0.48 0.00 35% 0% 47 0.54 0.42 0.47 0.02 78% 5% 49 0.49 0.01 0.40 0.00 1% 0% 133 0.53 0.48 0.50 0.21 92% 42% 142 0.66 0.15 0.63 0.00 23% 0% 143 0.55 0.00 0.26 0.00 0% 0% 144 0.53 0.01 0.54 0.00 0% 0% 145 0.64 0.33 0.27 0.02 52% 0% 146 0.67 0.08 0.64 0.00 12% 0% 147 0.65 0.14 0.60 0.00 22% 0% 148 0.63 0.10 0.58 0.00 16% 0% 149 0.57 0.03 0.41 0.01 6% 0% 150 0.61 0.09 0.57 0.00 15% 0% 151 0.54 0.09 0.57 0.00 17% 0% 152 0.61 0.04 0.60 0.00 6% 0% 153 0.62 0.13 0.60 0.00 22% 0% 154 0.71 0.24 0.64 0.02 34% 0% 155 0.67 0.36 0.66 0.02 53% 0% 177 0.76 0.70 0.72 0.58 92% 81% 178 0.63 0.62 0.55 0.57 97% 103% 179 0.65 0.07 0.59 0.00 11% 0% 180 0.72 0.61 0.64 0.42 85% 65%81 0.64 0.07 0.59 0.00 11% 0%NB42146-WO-PCT[2]TABLE 24 (Continued)NUCLEASE ACTIVITIES UNDER 20 mM AND lOOmM PHOSPHATE STRESS SEQ Corrected Corrected Corrected Corrected Residual Residual ID NO delta delta delta delta activity in activity in fluorescence fluorescence fluorescence fluorescence 20 mM 100 mM signal in 20 signal in 20 signal inlOO signal in phosphate phosphate mM Tris mM mM Tris 100 mM buffer buffer buffer phosphate buffer phosphatebuffer buffer182 0.66 0.09 0.58 0.00 13% 0% 183 0.71 0.44 0.65 0.02 63% 0% 184 0.66 0.01 0.51 0.00 0% 0% 185 0.64 0.04 0.57 0.00 7% 0% 186 0.63 0.02 0.58 0.00 0% 0% 187 0.64 0.16 0.59 0.00 25% 0% 188 0.59 0.05 0.56 0.00 9% 0% 189 0.69 0.29 0.65 0.00 41% 0% 190 0.70 0.30 0.65 0.00 43% 0% 191 0.66 0.11 0.63 0.00 16% 0% 192 0.69 0.28 0.64 0.01 41% 0% 193 0.67 0.41 0.65 0.00 61% 0% 203 0.71 0.62 0.59 0.10 88% 16% 204 0.70 0.53 0.58 0.00 76% 0% 205 0.63 0.36 0.55 0.00 57% 0% 206 0.81 0.78 0.80 0.80 97% 100% 135 0.82 0.82 0.81 0.79 100% 97% 207 0.78 0.74 0.69 0.10 95% 15% 208 0.79 0.78 0.73 0.65 100% 89% 209 0.45 0.41 0.17 0.03 91% 0% 210 0.50 0.50 0.50 0.08 99% 16% 211 0.39 0.20 0.23 0.10 50% 43% 213 0.77 0.80 0.77 0.36 103% 47% 218 0.68 0.58 0.68 0.40 86% 59%19 0.64 0.28 0.27 0.00 45% 0%

[0634] Nuclease Reaction Dose Response under 100 mM, 150 mM and 200 mM Phosphates Stress

[0635] Nine (9) nuclease molecules with enhanced phosphate tolerance were selected for further characterization under higher phosphate stress. To better distinguish the performance of these molecules, a series of nuclease reaction dosages from 0-0.5 ppm were set. The phosphate concentrations were set to 100 mM, 150 mM, and 200 mM. Specifically, the nuclease was incubated with 100 ng / pL lambda DNA (Thermo Scientific, SD0011) in the presence of 10 mM Mg2+and 1 mM Ca2+in 100 mM, 150 mM and 200NB42146-WO-PCT[2]mM sodium phosphate buffers (Axygen, full skirt). The reactions were carried out in iEMS incubator at 50°C for 10 minutes with the shake speed of 1150 rpm. After reactions, 4 pL of the mixtures were transferred into 96 pL 2x SYBR Green I nucleic acid gel stain Mili-Q water solution (Invitrogen, S7563), mixing well for fluorescence detection (Corning, #3505). The excitation and emission wavelength were 490 nm and 530 nm respectively using spectrophotometer. The activity of nuclease was indicated by the change in fluorescence before and after reactions, which was calculated as follows:F0— FRCorrected Delta Fluorescence Siyqnal = - 10e8

[0636] Where F0represents the initial fluorescence of substrate DNA, FRrepresents the detected fluorescence after reaction. 10e8 is a self-defined parameter to normalize the fluorescent data.

[0637] The lambda DNA standards were also included, and the corrected DNA standard fluorescence signal was calculated using the following equation:p

[0638] Corrected DNA Standard Fluorescence Signal =

[0639] Where Forepresents the detected fluorescence signal of DNA standards, 10e8 is a self-defined parameter to normalize the fluorescent data.

[0640] The results of nuclease reaction dose responses are presented below in TABLES 25-27.TABLE 25NUCLEASE REACTION DOSE RESPONSES UNDER 100 mM PHOSPHATE STRESS Correc Correc Correc Correc Correc Correc Correc Correc Correc Correc ted ted ted ted ted ted ted ted ted ted Reac Delta Delta Delta Delta Delta Delta Delta Delta Delta DNA DNA S tion Fluores Fluores Fluores Fluores Fluores Fluores Fluores Fluores Fluores Std, td Dose cence cence cence cence cence cence cence cence cence ng / jiL Fluores Signal Signal Signal Signal Signal Signal Signal Signal Signal cence PPm SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ Signal NO: NO:17 NO: NO: NO: NO: NO: NO: NO:177 8 180 203 206 135 208 210 211 4 0.76 0.5 0.56 0.48 0.54 0.47 0.77 0.77 0.68 0.58 0.73 2 0.62 0.25 0.56 0.47 0.5 0.37 0.76 0.76 0.65 0.39 0.590.121 0.27 0.53 0.49 0.35 0.23 0.76 0.76 0.63 0.11 0.0350.060.5 0.11 0.48 0.41 0.22 0.12 0.76 0.76 0.6 0.03 0.07250.030.25 0.05 0.39 0.28 0.13 0.06 0.73 0.74 0.57 0.02 0.031250.010.125 0.01 0.3 0.1 0.07 0.04 0.62 0.64 0.48 0.02 0.0256250.062 0.000 0.24 0.04 0.05 0.02 0.39 0.49 0.34 0.02 0.03 5 78130 0 0 0 0 0 0 0 0 0 0 0NB42146-WO-PCT[2]TABLE 26NUCLEASE REACTION DOSE RESPONSES UNDER 150 mM PHOSPHATE STRESSCorrec Correc Correc Correc Correc Correc Correc Correc Correc Corrcc ted ted ted ted ted ted ted ted ted ted Reac Delta Delta Delta Delta Delta Delta Delta Delta Delta DNA DNA S tion Fluores Fluores Fluores Fluores Fluores Fluores Fluores Fluores Fluores Std, td Dose cence cence cence cence cence cence cence cence cence ng / jiL Fluores Signal Signal Signal Signal Signal Signal Signal Signal Signal cence ppm SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ Signal NO: NO: NO: NO: NO: NO: NO: NO: NO:177 178 180 203 206 135 208 210 211 4 0.76 0.5 0.56 0.49 0.4 0.13 0.77 0.77 0.6 0.04 0.14 2 0.67 0.25 0.49 0.34 0.23 0.07 0.76 0.77 0.57 0.02 0.030.121 0.34 0.32 0.19 0.14 0.03 0.74 0.76 0.5 0.02 0.0250.060.5 0.14 0.21 0.11 0.07 0.02 0.75 0.74 0.46 0.01 0.02250.030.25 0.06 0.13 0.05 0.05 0.02 0.64 0.57 0.28 0 0.011250.010.125 0.02 0.07 0.04 0.03 0.01 0.14 0.25 0.14 0 056250.062 0.000.01 0.05 0.02 0.01 0.01 0.13 0.09 0.07 0 0 5 78130 0 0 0.01 0.01 0 0.01 0 0 0 0 0TABLE 27NUCLEASE REACTION DOSE RESPONSES UNDER 200 mM PHOSPHATE STRESS DNA Correc Reac Correc Correc Correc Correc Correc Correc Correc Correc Correc Std, ted tion ted ted ted ted ted ted ted ted ted ng / pL DNA S Dose Delta Delta Delta Delta Delta Delta Delta Delta Delta td Fluores Fluores Fluores Fluores Fluores Fluores Fluores Fluores Fluores Fluores PPm cence cence cence cence cence cence cence cence cence cence Signal Signal Signal Signal Signal Signal Signal Signal Signal Signal SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ NO: NO: NO: NO: NO: NO: NO: NO: NO: 177 178 180 203 206 135 208 210 211 4 0.77 0.5 0.32 0.19 0.25 0.08 0.77 0.77 0.54 0.01 0.01 2 0.65 0.25 0.27 0.12 0.12 0.04 0.77 0.76 0.43 0 0 1 0.28 0.12 0.13 0.04 0.05 0.03 0.73 0.68 0.33 0 050.5 0.11 0.06 0.06 0.02 0.03 0.01 0.53 0.47 0.17 0 0250.25 0.05 0.03 0.04 0.01 0.01 0.01 0.19 0.13 0.08 0 01250.125 0.02 0.01 0.02 0.01 0 0 0.07 0.05 0.05 0 056250.062 0.01 0.00 0.02 0.01 0.01 0 0.04 0.03 0.04 0 0 5 78130 0 0 0 0.01 0 0 0 0 0 0 0NB42146-WO-PCT[2]EXAMPLE 16DETERMINING THE ACTIVITIES OF NUCLEASE CRUDE SAMPLES UNDER 20 mM AND 100 mM PHOSPHATE STRESS

[0641] Forty-three (43) nuclease crude samples were harvested for phosphate tolerance measurements. For nuclease crude activity assay, the nuclease crude samples were diluted by lOOOx then incubated with 100 ng / pL lambda DNA (Thermo Scientific, SD0011) as double-stranded DNA substrate in the presence of 10 mM Mg2+ and 1 mM Ca2+ in 20 mM and 100 mM sodium phosphate buffers (Axygen, full skirt). 20 mM and 100 mM Tris-HCl buffer were used as control buffer for paralleled reaction groups. The reactions were carried out in iEMS incubator at 50°C for 10 min with the shake speed of 1150 rpm. After reactions, 4 pL of the mixtures were transferred into 96 pL 2x SYBR Green I nucleic acid gel stain Mili-Q water solution (Invitrogen, S7563), mixing well for fluorescence detection (Corning, #3505). The excitation and emission wavelength were 490 nm and 530 nm respectively using spectrophotometer. The activity of nuclease was indicated by the change in fluorescence before and after reactions, which was calculated as follows:F0— FRCorrected Delta Fluorescence Siyqnal = - 10e8

[0642] Where F0represents the initial fluorescence of substrate DNA, FRrepresents the detected fluorescence after reaction. 10e8 is a self-defined parameter to normalize the fluorescent data.

[0643] The residual activities of nuclease were also calculated using the following equation:Corrected Delta Fluorescence Signal (under stress') Residual Activity = - - - - - - - — - - — x 100%Corrected Delta Fluorescence Signal (control)

[0644] The detected corrected delta fluorescence signal and the calculated residual activity are shown in TABLE 28. For instance, among the forty-two (42) nuclease crude samples, thirteen (13) and 10 of them maintained over 50% of their activities in the presence of 20 mM and 100 mM phosphates, respectively.NB42146-WO-PCT[2]TABLE 28NUCLEASE CRUDE SAMPLES UNDER 20 mM AND lOOmM PHOSPHATE STRESS SEQ Corrected Corrected Corrected Corrected Residual Residual ID NO delta delta delta delta activity activity fluorescen fluorescen fluorescen fluorescen in 20 in 100 ce signal in ce signal in ce signal ce signal in mM mM 20 mM 20 mM inlOO mM 100 mM phospha phospha Tris buffer phosphate Tris buffer phosphate te buffer te buffer buffer buffer12 0.51 0.05 0.45 0.00 9% 0% 156 0.50 0.03 0.41 0.01 7% 0% 157 0.53 0.06 0.46 0.01 11% 0% 158 0.47 0.02 0.25 0.01 0% 0% 159 0.48 0.03 0.36 0.00 5% 0% 160 0.42 0.01 0.27 0.00 0% 0% 161 0.50 0.07 0.35 0.01 14% 0% 162 0.46 0.01 0.23 0.01 0% 0% 163 0.47 0.01 0.33 0.00 0% 0% 164 0.53 0.17 0.46 0.01 32% 0% 165 0.55 0.19 0.48 0.01 34% 0% 166 0.56 0.25 0.53 0.00 44% 0% 167 0.56 0.25 0.52 0.01 44% 0% 168 0.47 0.01 0.35 0.00 0% 0% 169 0.56 0.31 0.54 0.01 54% 0% 170 0.56 0.30 0.59 0.02 54% 0% 171 0.56 0.22 0.52 0.01 39% 0% 172 0.47 0.04 0.34 0.00 7% 0% 173 0.53 0.20 0.49 0.01 37% 0% 174 0.54 0.19 0.47 0.01 35% 0% 175 0.52 0.03 0.42 0.00 7% 0% 176 0.47 0.02 0.45 0.00 4% 0% 194 0.43 0.02 0.21 0.01 0% 0% 195 0.49 0.02 0.42 0.00 0% 0% 196 0.48 0.00 0.36 0.00 0% 0% 197 0.49 0.00 0.32 0.00 0% 0% 198 0.49 0.00 0.34 0.00 0% 0% 199 0.56 0.27 0.51 0.00 47% 0% 200 0.46 0.00 0.22 0.00 0% 0%201 0.49 0.00 0.35 0.01 0% 0%NB42146-WO-PCT[2]TABLE 28 (Continued)NUCLEASE CRUDE SAMPLES UNDER 20 mM AND lOOmM PHOSPHATE STRESS SEQ Corrected Corrected Corrected Corrected Residual Residual ID NO delta delta delta delta activity activity fluorescen fluorescen fluorescen fluorescen in 20 in 100 ce signal in ce signal in ce signal ce signal in mM mM 20 mM 20 mM inlOO mM 100 mM phospha phospha Tris buffer phosphate Tris buffer phosphate te buffer te buffer buffer buffer202 0.47 0.00 0.34 0.00 0% 0% 206 0.73 0.72 0.73 0.7 99% 96% 135 0.73 0.72 0.73 0.72 99% 99% 207 0.69 0.64 0.61 0.09 93% 15% 208 0.25 0.25 0.24 0.16 100% 67% 212 0.7 0.66 0.67 0.57 94% 85% 213 0.73 0.72 0.72 0.64 99% 89% 214 0.56 0.56 0.51 0.5 100% 98% 215 0.71 0.69 0.66 0.59 97% 89% 216 0.73 0.72 0.73 0.68 99% 93% 217 0.73 0.71 0.71 0.58 97% 82% 218 0.69 0.65 0.64 0.57 94% 89%219 0.48 0.03 0.51 0.00 5% 0%NB42146-WO-PCT[2]EXAMPLE 17NUCLEASE IMMOBILIZATION SCREEN

[0645] Immobilized nuclease is one desirable format for reducing the presence of contaminating DNA (e.g., genomic DNA. recombinant DNA) in industrial microbial cell fermentation broths and / or its presence in any down-stream protein recovery processes. As described herein Applicant determined that certain nuclease enzymes exhibited poor immobilization efficiency. Accordingly, a subset of nucleases characterized by an elevated number of lysine residues was selected for further evaluation. In the instant example, purified nuclease protein was immobilized in microtiter plates (MTPs) (Pall Corporation 8019) using a known crosslinking method described in U. S. Patent No. 4,355,105. In particular, bentonite (Cholino, Patagonia, Argentina; P / N F30) was hydrated in water overnight with stirring. Polyethyleneimine (PEI) (Sigma Aldrich P / N 181978), glutaraldehyde (GA) (Sigma Aldrich), and CELITE 505™ diatomaceous earth (Imerys) were added to the bentonite and mixed for 30 minutes to create a master mix. Twenty (20) pl of this mix was dispersed into MTPs containing nuclease protein and mixed up and down by a pipet.

[0646] Secondary additions (10 pl each) of PEI and GA were added and mixed on an external MTP mixer for five (5) minutes. The MTP was spun down and the liquid filtrate was collected in a MTP catch plate and the immobilized particles stayed on the filter in the MTP filter plate. The immobilized particles were washed by pipetting water into the well and spinning them down in a centrifuge and collecting the wash liquid. This wash procedure was performed two times.

[0647] A nuclease assay was conducted on the filtrate and the two wash fractions. The underlying rationale is that any nuclease not detected in the filtrate or washes is assumed to be immobilized within the particle matrix. Therefore, lower nuclease concentrations measured in the filtrate and washes indicate a higher proportion of nuclease successfully immobilized in the particles (see, TABLE 29).

[0648] Nuclease activity was measured using the Promega QuantiFluor® dsDNA detection kit. The assay was performed at room temperature for five (5) minutes using a fifteen (15) ppm lambda DNA substrate in 25 mM MES, 10 mM MgCE, and 1 mM CaCE, pH 5.4 assay buffer. Nuclease activity was quantified by measuring the residual DNA amount using QuantiFluor dye fluorescence at 504 nm (excitation) and 531 nm (emission). A purified nuclease enzyme standard (TreNucl; SEQ ID NO: 133) was used to calculate the nuclease concentration in the tested samples. Enzyme immobilization was calculated using residual nuclease concentration in filtrate compared to that of the starting material, wherein the percent immobilized enzyme was calculated using the following equation:Percent (%) Immobilization = (Immobilized Enzyme) / (Inifial Starting Material)* 100

[0649] For example, as presented below in TABLE 29, a subset of nucleases, such as BspNuc28 (SEQ ID NO: 176), RdeNuc2 (SEQ ID NO: 302), RarNuc3 (SEQ ID NO: 300) and GbuNucl (SEQ ID NO: 213),NB42146-WO-PCT[2]displayed unexpectedly high immobilization yields (i.e., > 90%; TABLE 29), making them particularly suitable candidates for fixed-bed or recyclable nuclease applications in broth conditioning and downstream processing.TABLE 29NUCLEASE IMMOBILIZATION ASSAYStartingMaterial Filtrate post Wash Wash%Enzyme (purified Immobilization #1 #2Immobilization enzyme) PPM PPM PPMPPMBspNucl482 65.7 2.5 0.4SEQ ID NO: 158 19.88 BspNuc28412.4 21.7 19.4 0.7SEQ ID NO: 176 94.74 BspNuc2799.2 88.1 6.2 1.5SEQ ID NO: 174 11.19 BspNucl9146.7 121.9 21 1.1SEQ ID NO: 168 16.91 BspNucl6329.1 257.1 16.7 0.3SEQ ID NO: 161 21.88 PmuNuc4126.9 99.1 12.4 0.2SEQ ID NO: 159 21.91 PmuNuc3171.5 158.2 11.5 0.5SEQ ID NO: 220 7.76 PspNuc6120 45 8.5 0.1SEQ ID NO: 157 62.50 RdeNuc2592.6 10.6 0.8 0.1SEQ ID NO: 302 98.21 RarNuc3454.8 2.3 0.9 0SEQ ID NO: 300 99.49 BspNucH87.8 69.5 9 0.6SEQ ID NO: 164 20.84 MidNuc3145.9 116.2 7.4 0.9SEQ ID NO: 163 20.36 PfrNucl108 94.8 6.9 0.5SEQ ID NO: 162 12.22 MidNuc2133.3 68.6 6.1 0.2SEQ ID NO: 160 48.54 NspNuc3 82.2 66.7 4.4 0.1SEQ ID NO: 194 18.86 GbuNucl2455.2 66.9 8.8 0.4SEQ ID NO: 213 97.28NB42146-WO-PCT[2]TABLE 29 (Continued)NUCLEASE IMMOBILIZATION ASSAYStarting% Material Filtrate post Wash WashImmobilization Enzyme (purified Immobilization #1 #2enzyme / Start enzyme) PPM PPM PPMmaterialPPMMetNuc7010.4 2SEQ ID NO: 27 0.1 080.77 MetNuc69 600.4 465.8 20.8 8SEQ ID NO: 193 22.42 MetNuc67598.9 271.7 16.7 3.3SEQ ID NO: 155 54.63 MetNuc66 405.4 2.4SEQ ID NO: 154 274.5 19.132.29 MetNuc64406.7 273.1 14.5 0.5SEQ ID NO: 191 32.85 MetNuc63443.5 293.5 24.7 2.1SEQ ID NO: 190 33.82 MetNuc62279.5 200.8 28 2.6SEQ ID NO: 189 28.16 MetNuc55 258.4 177 20 0.8SEQ ID NO: 153 31.50 MetNuc54181.4 165.4 10.4 1.7SEQ ID NO: 188 8.82 MetNuc53276.5 196.3 22.2 0.6SEQ ID NO: 187 29.01 MetNuc44275.9 213 13.2 0.8SEQ ID NO: 186 22.80 MetNuc42199.7 161.8 12 0.3SEQ ID NO: 185 18.98

[0650] As described above in Example 9, the name, sequence identification number (SEQ ID), predicted molecular weight (Da), total number of amino acid residues and domain family of the BspNuc28 and GbuNucl nucleases immobilized in the present example are set forth in TABLE 18. Additionally, the name, sequence identification number (SEQ ID), predicted molecular weight (Da), total number of amino acid residues and domain family of the RdeNuc2 and RarNuc3 nucleases immobilized in the present example are shown below in TABLE 30.NB42146-WO-PCT[2]TABLE 30MATURE NUCLEASE SEQUENCE DETAILSName SEQ ID Molecular Residues Domain Family Weight (Da)RarNuc3 300 29716.22 271 Endonuclease_NSRdeNuc2 302 29677.27 271 Endonuclease_NSNB42146-WO-PCT[2]REFERENCES PCT Publication No. WO 1999 / 50389PCT Publication No. W02008 / 065200PCT Publication No. WO2011 / 015327PCT Publication No. WO2015 / 065978PCT Publication No. WO2018 / 210794PCT Publication No. W02019 / 040412PCT Publication No. WO2019 / 055261PCT Publication No. WO2021 / 254832PCT Publication No. W02022 / 272155PCT Publication No. WO2023 / 118565PCT Publication No. WO2023 / 23644U. S. Patent No. 3,796,634U. S. Patent No. 4,355,105U. S. Patent No. 4,713,333U. S. Patent No. 5,177,005U. S. Patent No. 5,437,993U. S. Patent No. 5,811,280U. S. Patent No. 5,916,789U. S. Patent No. 6,255,115U. S. Patent No. 7,297,510US Patent No. 5,173,418US Publication No. US20200140838Ausubel et al., “Current Protocols in Molecular Biology”, published by Greene Publishing Assoc, and Wiley-Interscience (1987).Kriegler, “Gene Transfer and Expression: A Laboratory Manual”, 1990.NB42146-WO-PCT[2]Mistry et al., “Pfam: The protein families database in 2021”, Nucleic Acids Research, 8;49(D1): D412-D419, 2021.Needleman and Wunsch, “A general method applicable to the search for similarities in the amino acid sequence of two proteins”, J. Mol. Biol. 48: 443-453, 1970.Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988.Penttila etal., “A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei”, Gene (Amst.) 61, 155- 164, 1987.Rice et al., “EMBOSS: The European Molecular Biology Open Software Suite”, Trends in Genetics 16: 276-277, 2000.Sambrook et al., “Molecular Cloning: A Laboratory Manual” Cold Spring Harbor Laboratory: Cold Spring Harbor, N. Y. (1989), (2001) and (2012).Smith and Waterman, Adv. Appl. Math. 2:482, 1981.Stuckey et al., “Crystal structure of a phospholipase D family member”, Nat Struct Biol., 6: 278-284, 1999. Te’o et al., J. Microbiol. Methods 51:393-99, 2002.Teufel et al., “SignalP 6.0 predicts all five types of signal peptides using protein language models”, Nature Biotechnology, Vol. 40, 1023-1023, 2022.Vogtentanz et al., “N Bacillus sublilis fusion protein system to produce soybean Bowman-Birk protease inhibitor”, Protein Expr Purif., 55(l):40-52, 2007.

Claims

NB42146-WO-PCT[2]CLAIMS1. A nuclease polypeptide comprising a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a mature amino acid sequence selected from the group consisting of SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 133, 135, 142-220, 300, and 302, wherein the polypeptide has nuclease activity.

2. The nuclease of claim 1, wherein the nuclease comprises a protein family or domain selected from a DUF1524 domain, an Endonuclease_NS domain, a PLDc_2 domain, and a DNase_NucA / NucB domain.

3. The nuclease of claim 1 or 2, wherein the nuclease has an enhanced performance property selected from thermotolerance, protease tolerance, phosphate tolerance, salt tolerance, immobilization compatibility, or combinations thereof.

4. The nuclease of any one of claims 1-3, wherein the nuclease is thermotolerant and retains at least about 55% residual nuclease activity after 60 minutes pre-incubation at 60°C in 50 mM Tris-HCl (pH 7).

5. The nuclease of any one of claims 1-3, wherein the nuclease is protease tolerant and retains at least about 10% residual nuclease activity after incubation of a purified nuclease with 5 ppm Bacillus amyloliquefaciens subtilisin protease in 50 mM HEPES (pH 8.0) at 50°C for 60 minutes.

6. The nuclease of any one of claims 1-3, wherein the nuclease is sodium phosphate tolerant and retains nuclease activity in the presence of at least about 20 mM sodium phosphate.

7. The thermotolerant nuclease of claim 4, comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature amino acid sequence of any one of SEQ ID NO: 27, 31, 33, 43, 41, 45, 47, 49, 55, 57, 61, 65, 67, 71, 73, 77, 79, 81, 83, 87, 89, 93, 95, 97, 99, 101, 103, 105, 142, 143, 144, 145, 146, 147, 148, 149. 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177- 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 205, 210, 219 and 220.

8. The protease tolerant nuclease of claim 5, comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NO: 27, 31, 33, 41, 43, 45, 47, 49, 142-176, 178, 182, 183, 184, 187, 188, 189, 190, 192, 193, 194, 196-199, 201, 210 and 220.NB42146-WO-PCT[2]9. The sodium phosphate tolerant nuclease of claim 6, comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the mature amino acid sequence of any one of SEQ ID NO: 31, 47, 133, 135, 145, 155, 177, 178, 180, 183, 189, 190, 192, 193, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 218 and 219.

10. A polynucleotide encoding the nuclease of any one of claims 1-9.

11. An expression construct comprising a promoter operably linked to a nucleic acid encoding a signal peptide operably linked to a nucleic acid encoding the nuclease of any one of claims 1-9.

12. A recombinant microbial host cell comprising the polynucleotide of claim 10 or the expression construct of claim 11.

13. The recombinant microbial host cell of claim 12, wherein the host cell is a Gram-positive bacterial cell, a filamentous fungal cell or a yeast cell.

14. The recombinant microbial host cell of claim 12 or 13, further comprising a nucleic acid encoding a protein of interest (POI), wherein the host cell co-expresses the POI and the nuclease.

15. A fermentation broth comprising the nuclease of any one of claims 1-9 and / or the recombinant microbial host cell of any one of claims 12-14.

16. A method for producing a protein of interest essentially free of DNA, comprising cultivating the recombinant microbial host cell of claim 14 under conditions suitable for expression of the POI and the nuclease and recovering the POI from the cultivation medium, wherein DNA in the cultivation medium and / or recovered POI is reduced by action of the nuclease.

17. A method for reducing DNA content in a fermentation broth, comprising contacting the broth with a nuclease of any one of claims 1-9 provided as an exogenous nuclease preparation and / or an immobilized nuclease composition, under conditions suitable for DNA degradation.

18. An immobilized nuclease composition comprising a nuclease immobilized on a solid support, wherein the immobilized nuclease retains nuclease activity and comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a mature amino acid sequence selected from the group consisting of SEQ ID NO: 176, 213, 300 and 302.

19. The immobilized nuclease composition of claim 18, wherein the solid support comprises a bead, resin, ion exchange resin, membrane, porous matrix, colloidal silica, activated charcoal, hydroxyapatite, alumina, bentonite, diatomaceous earth, or combinations thereof.NB42146-WO-PCT[2]20. A method for reducing DNA content in a fermentation broth or protein preparation, comprising contacting the fermentation broth or protein preparation with the immobilized nuclease composition of claim 18 under conditions suitable for DNA degradation.

21. The method of claim 20, wherein contacting is performed in a packed-bed reactor, fixed-bed reactor, column, vessel, tank reactor, or combinations thereof,22. The method of claim 20, wherein the immobilized nuclease composition is reused for multiple cycles of DNA degradation.

23. The method of claim 16, wherein the modified cell produces no less than 25% of the POI as compared to a control cell expressing the same POI, but not co-expressing the nuclease.

24. The method of claim 17, wherein the broth is contacted with an exogenous nuclease preparation, wherein the nuclease comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NO: 31, 53, 55, 57, 61, 63, 65, 67, 71, 73, 77, 79, 81, 83, 87, 89, 95, 97, 99, 101, 103 and 105, and the nuclease reduces the DNA concentration in the broth by at least 1.4x.

25. The method of claim 24, wherein the nuclease comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NO: 55, 67, 73, 79, 81, 83, 87, 89, 97, 101, 103, and 105 and the nuclease reduces the DNA concentration in the broth at least 1,4x following incubation at an elevated temperature for at least one hour.

26. The method of claim 17, wherein the broth is contacted with an exogenous nuclease preparation, wherein the nuclease comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 31, 53, 55, 61, 65, 67, 71, 73, 77, 79, 85, 87, 89, 93, 97, 103, 105, 107 and 133, and reduces the DNA size in the broth at least 1.4x.

27. The method of claim 26, wherein the nuclease comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NO: 55, 57, 61, 65, 67, 71, 77, 79, 81, 87, 93, 95, 99, 101 and 103, and reduces the DNA size in the broth by at least 1,4x following incubation at an elevated temperature for at least 1 hour.

28. A method for co-expressing a protein of interest (POI) and a nuclease in a Gram-positive bacterial cell comprising cultivating a Gram-positive cell expressing a POI and nuclease comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,NB42146-WO-PCT[2]51, 133, 135 and 142-220, under conditions for the expression of the POI and nuclease, wherein the mature nuclease is secreted into the fermentation broth.

29. The method of claim 28, wherein the cell produces no less than 25% of the POI as compared to a control cell expressing the same POI, but not co-expressing the nuclease.

30. The method of claim 28 or 29, wherein the POI is an amylase and the cell produces no less than 20% of the amylase as compared to a control cell expressing the same amylase, but not coexpressing the nuclease.

31. The method of claim 28 or 29, wherein the POI is a protease and the cell produces no less than 10% of the protease as compared to a control cell expressing the same protease, but not co-expressing the nuclease.

32. A method for co-expressing a protein of interest (POI) and a nuclease in a filamentous fungal cell comprising cultivating a filamentous fungal cell expressing a POI and nuclease comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a mature amino acid sequence of any one of SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 133, 135 and 142-220, under conditions for the expression of the POI and nuclease, wherein the mature nuclease is secreted into the fermentation broth.

33. The method of claim 32, wherein the cell produces no less than 25% of the POI as compared to a control cell expressing the same POI, but not co-expressing the nuclease.

34. The method of claim 32 or 33, wherein the POI is a trehalase and the cell produces no less than 5% of the trehalase as compared to a control cell expressing the same trehalase, but not co-expressing the nuclease.

35. The method of claim 33 or 34, wherein the POI is a protease and the cell produces no less than 10% of the protease as compared to a control cell expressing the same protease, but not co-expressing the nuclease.

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