Abstract: The present disclosure is generally related to recombinant microbial cells expressing heterologous proteins of interest. Certain aspects of the disclosure are therefore related to, inter alia, recombinant Bacillus cells having enhanced protein production capabilities, novel protein signal sequences, recombinant polynucleotides encoding heterologous proteins of interest, and related compositions and/or methods thereof. Thus, as exemplified herein, the recombinant Bacillus cells of the instant disclosure are particularly suitable for use in the expression, production and secretion of heterologous proteins.

Abstract: Certain embodiments of the disclosure are related to recombinant Bacillus strains comprising enhanced protein productivity phenotypes, compositions and methods for constructing such recombinant Bacillus cells, and the like. More particularly, the recombinant Bacillus strains described herein are particularly useful for the enhanced production of proteins of interest when grown/cultivated/fermented under suitable conditions.

Abstract: The present disclosure is generally related to compositions and methods for obtaining Bacillus licheniformis cells/strains having increased protein production capabilities. Certain embodiments of the disclosure are related to genetically modified Bacillus licheniformis cells/strains derived from parental B. licheniformis cells/strains comprising a variant rghR2 gene.

Abstract: Certain embodiments of the disclosure are related to compositions and methods for producing proteins of interest in pigment deficient Bacillus cells. Certain other embodiments are related to compositions and methods for obtaining pigment deficient Bacillus cells. Certain other embodiments are related to compositions and methods for growing/cultivating/fermenting pigment deficient Bacillus cells. Certain other embodiments are therefore related to compositions and methods for producing, isolating, recovering and the like proteins of interest that are pigment deficient.

Abstract: Compositions and methods are provided for variant Cas systems and elements comprising such systems, including, but not limiting to, Cas endonuclease variants, guide polynucleotide/Cas endonuclease complexes comprising Cas endonuclease variants, as well as guide polynucleotides and guide RNA elements that can interact with Cas endonuclease variants. Compositions and methods are provided for genome modification of a target sequence in the genome of a cell. The methods and compositions employ a guide polynucleotide/Cas endonuclease system comprising a Cas9 endonuclease variant to provide an effective system for modifying or altering target sequences within the genome of a cell or organism.

Abstract: The present disclosure is generally related to compositions and methods for obtaining Bacillus licheniformis cells/strains having increased protein production capabilities. Certain embodiments of the disclosure are related to genetically modified Bacillus licheniformis cells/strains derived from parental B. licheniformis cells/strains comprising a variant rghR2 gene.

Abstract: The present disclosure is generally related to compositions and methods for constructing and/or obtaining B. licheniformis cells (e.g., protein production hosts) comprising enhanced protein production capabilities. Thus, certain embodiments are related to genetically modified Bacillus licheniformis strains derived from parental B. licheniformis strains producing increased amounts of one or more proteins of interest.

Abstract: Compositions and methods are provided for variant Cas systems and elements comprising such systems, including, but not limiting to, Cas endonuclease variants, guide polynucleotide/Cas endonuclease complexes comprising Cas endonuclease variants, as well as guide polynucleotides and guide RNA elements that can interact with Cas endonuclease variants. Compositions and methods are provided for genome modification of a target sequence in the genome of a cell. The methods and compositions employ a guide polynucleotide/Cas endonuclease system comprising a Cas9 endonuclease variant to provide an effective system for modifying or altering target sequences within the genome of a cell or organism.

Abstract: The present disclosure is generally related to novel promoter sequences and methods thereof for enhanced protein production in Bacillus sp. (host) cells. As set forth herein, the novel promoter sequences of the disclosure, when operably linked to a gene or open reading frame encoding a protein of interest, are particularly well suited for use in large scale production of industrially relevant proteins.

Abstract: The present disclosure is generally related to compositions and methods for constructing and obtaining Bacillus licheniformis cells having increased protein production phenotypes. Thus, certain embodiments are related to modified B. licheniformis cells derived from parental B. licheniformis cells. Certain embodiments are related to modified B. licheniformis cells comprising a modified rghR locus. Certain embodiments are related to modified B. licheniformis cells having a modified rghR locus and comprising an increased protein productivity phenotype. In certain other embodiments, modified B. licheniformis cells having a modified rghR locus produce a reduced amount of red pigment. In certain other embodiments, modified B. licheniformis cells comprise an increased protein productivity phenotype and produce a reduced amount of red pigment.

Abstract: The instant disclosure is generally related to novel Bacillus sp. mutants capable of producing increased amounts of industrially relevant proteins of interest. Certain embodiments of the disclosure are related to modified Bacillus sp. cells comprising an introduced polynucleotide encoding a variant GlcT protein. Other embodiments are related to methods and compositions for producing endogenous and/or heterologous proteins of interest in the modified Bacillus sp. (daughter) cells, whereas certain other embodiments are directed to nucleic acid sequences, particularly polynucleotide open reading frame (ORF) sequences, vectors thereof and DNA expression constructs thereof, encoding variant GlcT proteins of the disclosure.

Abstract: Methods and compositions are provided for integrating donor DNA sequences into the genome of a Bacillus sp. cell without the integration of a selectable marker into said genome. The methods employ a linear recombinant DNA construct comprising a donor DNA flanked by long homology arms (each of at least 1000 nucleotides in length) in combination with a recombinant DNA construct encoding a Cas9 endonuclease and a guide RNA, for the introduction of a guide RNA/Cas endonuclease into a Bacillus sp. cell, and as such providing a highly effective system for integrating donor DNA sequences into the genome of said Bacillus sp. cell, without the need to integrate a selectable marker in the genome of said Bacillus sp. cell.

Abstract: Methods and compositions are provided for integrating genes of interest into the genome of a Bacillus sp. cell without the integration of a selectable marker into said genome. The methods employ a dual circular recombinant DNA system for introduction of a guide RNA/Cas endonuclease system (also referred to as an RNA guided endonuclease, RGEN) as well as a donor DNA into a Bacillus sp. cell, and providing a highly effective system for inserting genes of interest into the genome of said Bacillus sp. cell.

Abstract: The present disclosure is generally related to novel promoter sequences and methods thereof for enhanced protein production in Bacillus sp. (host) cells. As set forth herein, the novel promoter sequences of the disclosure, when operably linked to a gene or open reading frame encoding a protein of interest, are particularly well suited for use in large scale production of industrially relevant proteins.

Abstract: The present disclosure is generally modified Bacillus strains and host cells thereof capable of producing increased amounts of industrially relevant proteins of interest. Other embodiments of the disclosure are related to isolated polynucleotides comprising modified Bacillus subtilis aprE 5?-untranslated region (5?-UTR) nucleic acid sequences, vectors thereof, DNA (expression) constructs thereof, modified Bacillus (daughter) cells thereof, and methods of making and using the same.

Abstract: The instant disclosure is generally related to novel Bacillus sp. mutants capable of producing increased amounts of industrially relevant proteins of interest. Certain embodiments of the disclosure are related to modified Bacillus sp. cells comprising an introduced polynucleotide encoding a variant GlcT protein. Other embodiments are related to methods and compositions for producing endogenous and/or heterologous proteins of interest in the modified Bacillus sp. (daughter) cells, whereas certain other embodiments are directed to nucleic acid sequences, particularly polynucleotide open reading frame (ORF) sequences, vectors thereof and DNA expression constructs thereof, encoding variant GlcT proteins of the disclosure.

Abstract: Compositions and methods are provided for variant Cas systems and elements comprising such systems, including, but not limiting to, Cas endonuclease variants, guide polynucleotide/Cas endonuclease complexes comprising Cas endonuclease variants, as well as guide polynucleotides and guide RNA elements that can interact with Cas endonuclease variants. Compositions and methods are provided for genome modification of a target sequence in the genome of a cell. The methods and compositions employ a guide polynucleotide/Cas endonuclease system comprising a Cas9 endonuclease variant to provide an effective system for modifying or altering target sequences within the genome of a cell or organism.

Abstract: Compositions and methods are provided for editing nucleotides and/or altering target sites in the genome of a cell. The methods and compositions employ a recombinant DNA construct comprising a Pol-II promoter operably linked to a polynucleotide encoding a single guide RNA, wherein said guide RNA is capable of forming a guide RNA/Cas endonuclease complex, wherein said complex can bind to and cleave a target site sequence in the genome of a cell such as a eukaryote cell. The present disclosure further describes methods and compositions employing said recombinant DNA construct to modify the genome of non-conventional yeast.

Abstract: A composition is disclosed that comprises at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein the RGEN protein component and CPP are covalently or non-covalently linked to each other in an RGEN protein-CPP complex. The RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell. The RGEN protein component of an RGEN protein-CPP complex in certain embodiments can be associated with a suitable RNA component to provide an RGEN capable of specific DNA targeting. Further disclosed are compositions comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one CPP, as well as methods of delivering RGEN proteins into microbial cells, as well as methods of targeting DNA with RGENs.

Abstract: A composition is disclosed that comprises at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein the RGEN protein component and CPP are covalently or non-covalently linked to each other in an RGEN protein-CPP complex. The RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell. The RGEN protein component of an RGEN protein-CPP complex in certain embodiments can be associated with a suitable RNA component to provide an RGEN capable of specific DNA targeting. Further disclosed are compositions comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one CPP, as well as methods of delivering RGEN proteins into microbial cells, as well as methods of targeting DNA with RGENs.