SEQUENCE LISTING

This application includes a nucleotide and amino acid sequence listing in computer readable form (CRF) as an ASC I I text (.txt) file according to “Standard for the Presentation of Nucleotide and Amino Acid Sequence Listings in International Patent Applications Under the Patent Cooperation Treaty (PCT)” ST.25. The sequence listing is identified below and is hereby incorporated by reference into the specification of this application in its entirety and for all purposes.

FIELD OF THE INVENTION

The present invention relates to a recombinant cell for producing heterologous proteins and for screening of expression of the heterologous proteins. The invention further relates to a method for making the recombinant cell having deletion of multiple genes, a method for producing the heterologous proteins in the recombinant cell, and to the use of the recombinant cell for screening of expression of the heterologous proteins and in the production of the heterologous proteins.

BACKGROUND OF THE INVENTION

Advances in genetic engineering techniques have allowed the improvement of microbial cells as producers of heterologous proteins. Protein production is typically achieved by the manipulation of gene expression in a microorganism such that it expresses large amounts of a recombinant protein. For example, Bacillus species cells produce and secrete a large amount of heterologous proteins and metabolites. Bacillus strains are widely employed as cell factories to produce useful enzymes such as proteases, amylases, lipases as well as biochemicals such as riboflavin, nucleotides, antibiotics, and insecticides. However, an important factor limiting the use of Bacillus cells for the screening and production of the heterologous proteins has been the high level of cell lysis that occurs in liquid cultures. Cell lysis contributes to high background observed in supernatants and results in very impure protein samples thereby making the effective screening of proteins a difficult task. Cell lysis effects both protein expression screening as well as production and/ or fermentation of the proteins.

Effective screening of expression of the heterologous proteins and their production has always been a challenge. Attempts have been made to study the effect of various native genes on lysis of the Bacillus cells.

Stephenson et at. discloses that inactivation of genes encoding for extracellular proteases increases cell lysis in B. Subt/l/s following transition to stationary phase (Stephenson et at. Cellular Lysis in Bacillus Subt/l/s; the Affect of Multiple Extracellular Protease Deficiencies, Letters in Applied Microbiology, 1999, 29, 141- 145).

J P2011155950 A provides a microorganism having suppressed lytic tendency obtained by suppressing expression of at least one gene among cell wall teichoic acid synthase- associated gene of Bacillus Subti/is.

Wang etaL provides that deletion of genes encoding PG hydrolases, prophage genes and that encode cannibalism factors in B. Subt/l/s improves biomass yield of the recombinant proteins and reduces cell lysis when compared with the wild type B. Subt/l/s (Wang et ai Deleting Multiple Lytic Genes Enhances Biomass Yield and Production of Recombinant Proteins by B. Subti/is, Microbial Cell Factories, 2014, 13: 129).

Nevertheless, there is a need for optimized recombinant cells with lower cell lysis and improved production of the heterologous protein, which aids in more accurate screening of the heterologous protein expression and which can produce heterologous proteins at large scale fermentations.

Thus, it was an object of the presenty claimed invention to provide a recombinant cell for producing heterologous proteins, which exhibits lower cell lysis and aids in more accurate screening of the heterologous protein expression.

SUMMARY OF THE INVENTION

Surprisingly, it has been found that combined deletion of multiple genes coding for extracellular proteases, sporulation factors, and autolysins in Bacillus species cell provides a recombinant cell with reduced cell lysis. The recombinant cell exhibited more accurate screening of the heterologus proteins and less background was observed in the supernatant. Additionally, further deletion of specific native proteins of Bacillus species reduces the native background activity of specific proteins and aids in further improving the screening of expression of the heterologous proteins.

The present invention is illustrated in more detail by the following embodiments and combinations of embodiments which result from the corresponding dependency references and links:

1. A recom bina nt cel l for producing a heterologous protein, having a deletion of one or more genes coding for an extracel lu lar protease, a deletion of one or more genes coding for a sporulation factor, a nd a deletion of one or more genes coding for an autolysin, wherein the recombinant cel l differs from a natural ly occurring cel l in that the deletion of the one or more genes coding for the extracel l ular protease, the deletion of one or more genes coding for the sporulation factor, and the deletion of the one or more genes coding for an autolysin have been introd uced in the natural ly occu rring cel l by recombinant D NA techniq ues.

2. The recom binant cel l according to em bodiment 1, wherein the one or more genes coding for the extracel l ular protease is selected from the group consisting of an aprE, nprE, vpr, epr, m pr, nprB, bpr, wprA, ywa D, htr A, htrB, and htrC gene.

3. The recom binant cel l according to em bodiment 1 or 2, wherein the one or more genes coding for the extracel lu lar protease com prises a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence represented by SEQ I D No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by S EQ I D No: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; c) a nucleic acid sequence coding for a polypeptide com prising an amino acid sequence represented by SEQ I D No: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; and, d) a nucleic acid sequence coding for a polypeptide com prising an amino acid sequence having at least 70% identity to the amino acid sequence represented by S EQ I D No: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

4. The recombinant cell according to any of embodiments 1 to 3, wherein the one or more genes coding for the sporulation factor is selected from the group consisting of a sigE, sigF, sigG, sigH , sigK, prkA, ybaN, yhbH, ykvV, ylbJ, ypj B, ytrl, ytrH, and yqfC gene.

5. The recom binant cel l according to a ny of em bodiments 1 to 4, wherein the one or more genes coding for the sporulation factor com prises a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence represented by SEQ I D No: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38; b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ I D No: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38; c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52; and, d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52.

6. The recombinant cell according to any of embodiments 1 to 5, wherein the one or more genes coding for the autolysin is selected from the group consisting of a skfA, sdpC, xpf, lytC, lytD, lytF, lytG, cwlA, cwlC, cwlD, cwlH, cwlJ, cwlK, cwlO, cwlS, xepA, xlyA, and xlyB gene.

7. The recombinant cell according to any of embodiments 1 to 6, wherein the one or more genes coding for the autolysin comprises a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence represented by SEQ ID No: 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70; b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70; c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88; and, d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88.

8. The recombinant cell according to any of embodiments 1 to 7, having further deletion of at least one gene selected from the genes coding for proteins having amylase activity, lipase activity, xylanase activity, and mannanase activity.

9. The recombinant cell according to embodiment 8, wherein the gene coding for a protein having amylase activity is an amyE gene.

10. The recombinant cell according to embodiment 9, wherein the gene coding for a protein having amylase activity comprises a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence represented by SEQ ID No: 89; b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 89; c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 90; and, d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 90. 11. The recombinant cell according to any of embodiments 8 to 10, wherein the gene coding for a protein having lipase activity is selected from the group consisting of an estA, estB, lipC, yprmR, yodD, and ytpA gene.

12. The recombinant cell according to embodiment 11, wherein the gene coding for a protein having lipase activity comprises a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence represented by SEQ ID No: 91, 92, 93, 94, 95, or 96; b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 91, 92, 93, 94, 95, or 96; c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 97, 98, 99, 100, 101, or 102; and, d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 97, 98, 99, 100, 101, or 102.

13. The recombinant cell according to any of embodiments 8 to 12, wherein the gene coding for a protein having xylanase activity is selected from the group consisting of a xynA, xynB, xynC, and xynD gene.

14. The recombinant cell according to embodiment 13, wherein the gene coding for a protein having xylanase activity comprises a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence represented by SEQ ID No: 103, 104, 105, or 106; b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 103, 104, 105, or 106; c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 107, 108, 109, or 110; and, d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 107, 108, 109, or 110.

15. The recombinant cell according to any of embodiments 8 to 14, wherein the gene coding for a protein having mannanase activity is a gmuG gene.

16. The recombinant cell according to embodiment 15, wherein the gene coding for a protein having mannanase activity comprises a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence represented by SEQ ID No: 111; b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 111; c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 112; and, d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 112. 17. The recombinant cell according to any of embodiments 1 to 16, wherein the recombinant cell is a bacterial cell.

18. The recombinant cell according to embodiment 17, wherein the bacteria is selected from the group consisting of Bacillus Hcheniformis, Bacillus lentus, Bacillus subt/l/s, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus stearothermophilus, Bacillus dausii, Bacillus alkalophilus, Bacillus coagulans, Bacillus circulans, Bacillus pumilus, Bacillus thuringiensis, Bacillus psuedofirmus, Bacillus marmarensis, Bacillus

cellulolyticus, Bacillus hemicellulolyticus, Geobacillus stearothermophilus, Bacillus darkia, Bacillus akibai, Bacillus gibsonii, Bacillus lautus, Bacillus megaterium, and Bacillus halodurans.

19. The recombinant cell according to any of embodiments 1 to 18, wherein the heterologous protein is selected from the group consisting of enzymes, hormones, growth factors and cytokines.

20. The recombinant cell according to embodiment 19, wherein the heterologous protein is an enzyme.

21. The recombinant cell according to embodiment 20, wherein the enzyme is selected from the group consisting of phytases, proteases, beta-glucanases, xylanases, mannanases, lipases, cellulases, glucoarmylases, amylases, alpha- amylases, beta-amylases, hydrolases, isomerases, ligases, lyases, oxidoreductases, transferases, aminopeptidases, carbohydrases, carboxypeptidases, catalases, cellobiohydrolases, cellulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, endoglucanases, esterases, alpha- galactosidases, beta-galactosidases, alpha-glucosidases, beta-glucosidases, invertases, laccases, lactases, lipases, mannosidases, mutanases, oxidases, pectinolytic enzymes, peroxidases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminases, xylanases, and beta-xylosidases.

22. The recombinant cell according to any of embodiments 1 to 21, wherein the recombinant cell comprises a nucleic acid sequence coding for a heterologous protein according to any of embodiments 19 to 21.

23. The recombinant cell according to any of embodiments 1 to 22, wherein the nucleic acid sequence coding for a heterologous protein according to any of embodiments 19 to 21 is comprised in a vector transformed into the recombinant cell or wherein the nucleic acid sequence coding for a heterologous protein according to any of embodiments 19 to 21 is integrated in the genome of the recombinant cell. 24. The recombinant cell according to embodiment 23, wherein the vector further comprises a selection marker, a polyadenylation signal, a multiple cloning site, an origin of replication, a promoter, and a termination signal.

25. A method of making the recombinant cell, comprising the steps of:

a) deleting in a cell one or more genes coding for an extracellular protease, one or more genes coding for a sporulation factor, and one or more genes coding for an autolysin according to any of embodiments 1 to 7; and

b) transforming the recombinant cell with a nucleic acid sequence coding for a heterologous protein according to any of embodiments 19 to 21.

26. The method according to embodiment 25, further comprising deleting at least one gene selected from the genes coding for a protein having amylase activity, lipase activity, xylanase activity, and mannanase activity according to any of embodiments 8 to 16.

27. A method of producing a heterologous protein in a recombinant cell, comprising the steps of:

a) obtaining a recombinant cell by deleting one or more genes coding for an extracellular protease, deleting one or more genes coding for a sporulation factor, and deleting one or more genes coding for an autolysin according to any of embodiments 1 to 7;

b) transforming the recombinant cell with a nucleic acid sequence coding for a heterologous protein according to any of embodiments 19 to 21, preferably an enzyme, preferably selected from the group consisting of lipase, amylase, mannanase, and xylanase;

c) growing the recombinant cell under conditions suitable for producing the heterologous protein; and

d) recovering the heterologous protein, wherein the deletion of the one or more genes coding for the extracellular protease, the deletion of the one or more genes coding for the sporulation factor, and the deletion of the one or more genes coding for the autolysin according to any of embodiments 1 to 7 results in reduction in the lysis of the recombinant cell.

28. The method according to embodiment 27, wherein the recombinant cell in step a) contains further deletion of at least one gene selected from the genes coding for a protein having amylase activity, lipase activity, xylanase activity, and mannanase activity according to any of embodiments 8 to 16. 29. Use of the recom binant cel l according to any of em bodiments 1 to 24 or a recom bina nt cell obtained according to the method of em bodiment 25 or 26, for screening the expression of a heterologous protein according to any of em bodiments 19 to 21.

30. Use of the recombinant cell according to any of embodiments 1 to 24 or a recombinant cell obtained according to the method of embodiment 25 or 26, for production of a heterologous protein according to any of embodiments 19 to 21.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain aspects of the following detailed description are best understood when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:

Figure 1) Schematic outline for the creation of genetic construct for gene deletion. First, individual nucleotides are amplified by PCR, and then spliced together by splicing by overhang extension PCR (SOE-PCR) technique. The resulting product is transformed into recombinant cells where it recombines in the genome. Successful transformants are selected for with kanamycin resistance (kan R) phenotype. Afterwards, kan R is recycled by excising the cassette with expression of Cre recombinase.

Figure 2) DNA agarose electrophoresis gel verifying deletion of lytC gene and insertion of the kanamycin resistance (kanR) marker.

Figure 3) Schematic diagram for the preparation of B. subtihs QZ120 strain.

Figure 4) Gel image comparison for screening of enzyme expression of QZ111 ( B subtihs PY79 A nprE A aprE A epr A rmpr A nprB A vpr A bpr A sigF) and the recombinant strain QZ120 {B. subtil is PY79

A nprE A aprE A epr A rmpr A nprB A vpr A bpr A sigF A IytC A sdpC A xpf A skfA). Lane 0- no enzyme (negative control), Lane 1- amylase, Lane 2- amylase, Lane 3- amylase, Lane 4- cellulose, Lane 5- mannanase, Lane 6- xylanase, Lane 7- xylanase, Lane 8- xylanase, Lane 9- xylanase, Lane 10- lipase. Arrows indicate band of enzyme of interest being expressed. More bands are visible, and are expressed with a higher purity, using the recombinant B. subtihs strain QZ120.

Figure 5) Workflow for quantifying cell lysis. Figure 6) B. subtihs ICDH activity in the supernatant at different dilutions. Long linear range observed with lOx and 20x dilutions of the sample.

Figure 7) B. subtihs ICDH activity measured in cell extract and supernatant to calculate lysed fraction of cells.

Figure 8) Background amylase activity in recombinant B. subtihs strain QZ120 with and without native amyE gene.

Figure 9) Background lipase activity in recombinant B. subtihs strain QZ120 with and without native genes estA, estB.

Figure 10) Background xylanase activity in recombinant B. subtihs strain QZ120 with and without native genes xynA, xynB, xynC, xynD.

Figure 11) Background mannanase activity in recombinant B. subtihs strain QZ120 with and without native gene gmuG.

Figure 12 provides assesment of expression of enzymes amylase, mannanase, xylanase, and lipase in recombinant B. subtihs QZ120 strain in comparison with the wild type parent B. subtihs strain (PY79). Lane 0- host, Lane 1- amylase, Lane 2- amylase, Lane 3- amylase, Lane 4- mannanase, Lane 5- mannanase, Lane 6- xylanase, Lane 7- xylanase, Lane 8- xylanase, Lane 9- lipase. Arrows indicate expected protein size. Grey, thinner arrows show an absence of the desired protein, while black thick arrows show the desired protein

Figure 13 provides an analysis of the background protease activity of B. subtihs strain QZ120 when compared with the wild type parent B. subtihs strain (PY79).

Figures 14 a) and 14 b) provide comparative assesment of amylase supenatant activity and lipase activity, respectively of the wild type parent B. subtihs strain (PY79) and B. subtihs strain QZ120.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense. Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given. Unless stated otherwise or apparent from the nature of the definition, the definitions apply to all methods and uses described herein.

As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise. In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ± 20 %, preferably ± 15 %, more preferably ± 10 %, and even more preferably ± 5 %.

It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii" etc. relate to steps of a method or use or assay, there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.

It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.

Definitions

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this invention pertains.

The term "recombinant cell" as used herein means that the cell contains at least one nucleic acid sequence which is not naturally present in the cell or which is naturally present in the cell but linked to sequences to which it is not natural ly linked in the cell such as a promoter to which the nucleic acid sequence encoding a protein is not naturally linked. I n the context of the present invention, the recombinant cell also differs from the naturally occurring cell in that it contains deletion of multiple genes coding for native proteins which are present in the naturally occurring cell. The recombinant cell of the present invention also differs from a naturally occurring cell in a deletion of the one or more genes coding for the extracellular protease, a deletion of one or more genes coding for the sporulation factor, and a deletion of the one or more genes coding for an autolysin, which are present in the naturally occurring cell. Thus, the recombinant cell of the present invention also differs from a naturally occurring cell in that the deletion of the one or more genes coding for the extracellular protease, the deletion of one or more genes coding for the sporulation factor, and the deletion of the one or more genes coding for an autolysin have been introduced in the naturally occurring cell by recombinant DNA techniques. In other words, the Bacillus cell of the present invention comprises a non native deletion of one or more genes coding for an extracellular protease, a non-native deletion of one or more genes coding for a sporulation factor, and a non-native deletion of one or more genes coding for an autolysin.

The term "heterologous” (or exogenous or foreign or recom bina nt or non- native) polypeptide or protein is defined herein as a polypeptide or protein that is not native to the host cel l, a polypeptide or protein native to the host cel l in which structural modifications, e.g., deletions, substitutions, and/or insertions, have been made by recom bina nt DNA tech niques to alter the native polypeptide, or a polypeptide or protein native to the host cel l whose expression is qua ntitatively a ltered or whose expression is directed from a genomic location different from the native host cel l as a result of manipulation of the DNA of the host cell by recombinant DNA techniques, e.g., a stronger promoter. Similarly, the term“heterologous” (or exogenous or foreign or recombinant or non-native) polynucleotide refers to a polynucleotide that is not native to the host cell, a polynucleotide native to the host cell in which structural modifications, e.g., deletions, substitutions, and/or insertions, have been made by recombinant DNA techniques to alter the native polynucleotide, or a polynucleotide native to the host cell whose expression is quantitatively altered as a result of manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques, e.g., a stronger promoter, or a polynucleotide native to the host cell, but integrated not within its natural genetic environment as a result of genetic manipulation by recombinant DNA techniques.

The term“native” (or wildtype or endogenous) cell or organism and“native” (or wildtype or endogenous) polynucleotide or polypeptide refers to the cell or organism as found in nature and to the polynucleotide or polypeptide in question as found in a cell in its natural form and genetic environment, respectively (i.e., without there being any human intervention).

The nucleic acid sequences used in the present invention further encompass codon- optimized sequences. A nucleic acid is codon-optimized by systematically altering codons in recombinant DNA to be expressed in a host cell other than the cell from which the nucleic acid was isolated so that the codons match the pattern of codon usage in the organism used for expression and thereby to enhance yields of an expressed protein. The codon-optimized sequence nevertheless encodes a protein with the same amino acid sequence as the native protein.

A particular nucleotide sequence, for example a sequence underlying a particular heterologous gene or a promoter sequence etc. can either be amplified by polymerase chain reaction from the genomic sequences of a particular organism from which they are derived from, or it can be chemically synthesized by method known the art.

Sequence identity usually is provided as“% sequence identity” or“% identity”. To determine the percent-identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e., a pairwise global

alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program“N EEDLE” (The European Molecular Biology Open Software Suite (EM BOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EBLOSU M62). The preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined.

After aligning two sequences, in a second step, an identity value is determined from the alignment produced. For purposes of this description, percent identity is calculated by %-identity = (identical residues / length of the alignment region which is showing the respective sequence of this invention over its complete length) *100. Thus, sequence identity in relation to comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give“%-identity

To determine the percent-identity between two nucleic acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e., a pairwise global

alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program“N EEDLE” (The European Molecular Biology Open Software Suite (EM BOSS)) with the programs default parameters for nucleic acid alignments (gapopen=10.0, gapextend=0.5 and matrix=EDNAFU LL).

For calculating the percent identity of two nucleic acid sequences sequences the same applies as for the calculation of percent identity of two amino acid sequences with some specifications.

For nucleic acid sequences encoding for a protein or a peptide, the pairwise alignment shall be made over the complete length of the coding region of the sequence of this invention. Introns present in the other sequence may be removed for the pairwise alignment to allow comparison with the sequence of this invention. Percent identity is then calculated by:

%-identity = (identical residues / length of the alignment region which is showing the coding region of the sequence of this invention over its complete length) *100.

As used herein the term "gene" means a segment of DNA involved in producing a polypeptide chain that may or may not include regions preceding and following the coding regions (e.g. 5' untranslated (5 UTR) or leader sequences and 3' untranslated (3 UTR) or trailer sequences, as well as intervening sequence (introns) between individual coding segments (exons)). The term“coding for" as used herein has its usual meaning and may include, but are not limited to, for example, the property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein if transcription and translation of m RNA corresponding to that gene produces the protein in a cell or other biological system.

As used herein, "deletion" of a gene refers to the deletion of the entire coding sequence, deletion of part of the coding sequence, or deletion of the coding sequence including flanking regions. The end result is that the deleted gene is effectively non-functional. I n simple terms, a "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, have been removed (i.e., are absent). Thus, a deletion strain has fewer nucleotides or amino acids than the respective wild-type organism.

“Extracellular proteases” are enzymes that break peptide bonds between amino acids of protein and which are secreted by the cell into the extracellulare space. Extracellular proteases are important for the hydrolysis of proteins in the extracellular space and enable the cells to absorb and utilize hydrolytic products.

Sporulation allows bacterias to survive adverse conditions and is essential to the lifecycle of some obligate anaerobes. “Sporulation factors” activate compartment- specific transcriptional programs that drive sporulation through its morphological stages.

The term“autolysins” refer to enzymes that hydrolyse the components of a biological cells or tissues in which they are produced. Autolysins exist in all bacteria containing peptidoglycan. Autolysins hydrolyze the /5 - (1,4) bond between N-acetylmuramic acid and N-acetylglucosamine molecules. Gram-positive bacteria regulate autolysins with teichoic acid molecules attached to the tetrapeptide of the peptidoglycan matrix.

The term“amylase activity” refers to the ability of the protein to catalyse the hydrolysis of a -1 4 glycosidic linkages of starch and other related polysaccharides to produce maltose and other oligosaccharides. The amylase activity of a protein can be determined by incubating the protein with a suitable amylase substrate, such as Ethylidene-p- Nitrophenyl-G7 (E-pN P-G7), or 4-Nitrophenyl a -D-maltoside.

The term "lipase activity" means that the protein can cleave ester bonds in lipids. The lipase activity of a protein can be determined by incubating the protein with a suitable lipase substrate, such as 4-PNP-octanoate, 1-olein, galactolipids, phosphatidylcholine or triacy Iglycerols.

The term "xylanase activity" means that the protein can degrade the linear polysaccharide xylan into xylose. The xylanase activity of a protein can be determined by incubating the protein with a suitable xylanase substrate, such as azo-xylan, or insoluble xylan.

The term“mannanase activity” refers to the ability of the protein to catalyse the random hydrolyses of 1,4-beta-D-mannosidic linkages in mannans, galactomannans, glucomannans, and galactoglucomannans. The mannanase activity of a protein can be determined by incubating the protein with a suitable mannanase substrate, such as azo- carob galactomannan substrate.

As used herein the term "screening" refers to a systematic study of expression of the heterologous protein.

As used herein the term "expression" refers to a process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

As used herein, the terms "genetic construct" refers 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 one embodiment, a genetic construct of the invention comprises a selection marker and deletion of genes as defined herein.

As used herein in the context of introducing a nucleic acid sequence into a cell, the term "introduced" refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, conjugation, and transduction.

As used herein, the term "transformed" refers to a cell that has a non-native (heterologous) polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained for at least two generations.

As used herein, the term "vector" refers to a polynucleotide designed to introduce nucleic acids into one or more host cells. Vectors may autonomously replicate in different host cells. The term is intended to encompass, but is not limited to cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, and the like. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.

As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. In preferred embodiments, the promoter is appropriate to the host cell in which the target gene is being expressed. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also termed "control sequences") is necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

As used herein, the term "operably linked" refers to an arrangement of elements that allows them to be functionally related. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

As used herein, the term "selection marker" refers to a protein capable of expression in a host that allows for ease of selection of those hosts containing an introduced nucleic acid or vector. Examples of selectable markers include but are not limited to antimicrobials (e.g., hygromycin, kanamycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.

As used herein, a "flank/ flanking" with reference to a sequence refers to having a sequence that is either upstream or downstream of the sequence in question (e.g., for genes A-B-C, gene B is flanked by the A and C gene sequences).

"Homolgous recombination" refers to an exchange of DNA fragments between two DNA molecules at the site of identical or nearly identical nucleotide sequence.

Description The presently claimed invention provides a recombinant cell for producing heterologous proteins, having combined non-native deletion of one or more genes coding for an extracellular protease, deletion of one or more genes coding for a sporulation factor, and deletion of one or more genes coding for an autolysin, and preferably having further non native deletion of at least one gene coding for proteins having amylase activity, lipase activity, xylanase activity, and mannanase activity. Preferably, the recombinant cell comprises a nucleic acid sequence coding for a heterologous protein.

I n one em bodiment, the one or more genes coding for the extracell u lar protease to be deleted is selected from the group consisting of a aprE, n prE, vpr, epr, m pr, nprB, bpr, wprA, ywa D, htr A, htrB, and htrC gene. Preferably, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least

11, or al l genes coding for the extracel lu lar protease selected from the grou p consisting of a aprE, nprE, vpr, epr, m pr, n prB, bpr, wprA, ywa D, htr A, htrB, and htrC gene are deleted. Preferably, at least 3, at least 4, at least 5, at least 6, or al l genes coding for the extracel l ular protease selected from the group consisting of a aprE, n prE, vpr, epr, m pr, n prB, and bpr gene are deleted. Preferably, the aprE, nprE, vpr, epr, m pr, nprB, a nd bpr genes are deleted.

Preferably, the aprE, nprE, vpr, epr, mpr, nprB, bpr, wprA, ywaD, htrA, htrB, and htrC gene is respresented by a sequence having at least 70% identity to a sequence shown in SEQ I D No. 1 to 12, respectively.

In an embodiment of the present invention, the genes coding for the extracel l ular proteases com prises a nucleic acid sequence selected from the grou p consisting of a) nucleic acid sequence represented by S EQ I D No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or

12, b) a nucleic acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the nucleic acid sequence represented by SEQ I D No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, c) a nucleic acid seq uence coding for a polypeptide com prising an amino acid sequence represented by SEQ I D No: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, a nd d) a n ucleic acid sequence coding for a polypeptide com prising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least

76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least

98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the amino acid sequence represented by SEQ I D No: SEQ I D No: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

In one embodiment, the one or more genes coding for the sporulation factor to be deleted is selected from the group consisting of a sigE, sigF, sigG, sigH, sigK, prkA, yba N, yhbH, ykvV, ylbJ, ypjB, ytrl, ytrH, and yqfC gene. Preferably, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, ast least 13, or all genes coding for the sporulation factor selected from the group consisting of a sigE, sigF, sigG, sigH, sigK, prkA, ybaN, yhbH, ykvV, ylbJ, ypj B, ytrl, ytrH, and yqfC gene are deleted. Preferably, the sigF genes is deleted.

Preferably, the sigE, sigF, sigG, sigH, sigK, prkA, ybaN, yhbH, ykvV, ylbJ, ypjB, ytrl, ytrH, and yqfC gene is respresented by a sequence having at least 70% identity to a sequence shown in SEQ ID No. 25 to 38, respectively.

In an embodiment of the present invention, the genes coding for the sporulation factors comprises a nucleic acid sequence selected from the group consisting of a) nucleic acid sequence represented by SEQ I D No: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38, b) a nucleic acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the nucleic acid sequence represented by SEQ I D No: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38, c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ I D No: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, and d) a nucleic acid sequence coding for a polypeptide com prising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the amino acid seq uence represented by S EQ I D No: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52.

I n one em bodiment, the one or more genes coding for the autolysin to be deleted is selected from the group consisting of a skfA, sdpC, xpf, lytC, lytD, lytF, lytG, cwlA, cwlC, cwl D, cwl H , cwlJ , cwl K, cwlO, cwlS, xepA, xlyA, and xlyB gene. Preferably, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or a l l genes coding for the autolysin selected from the group consisting of a skfA, sdpC, xpf, lytC, lytD, lytF, lytG, cwlA, cwlC, cwl D, cwl H , cwlJ , cwl K, cwlO, cwlS, xepA, xlyA, and xlyB gene are deleted. Preferably, at least 2, at least 3, or al l genes coding for the autolysin selected from the group consisting of a skfA, sd pC, xpf, a nd lytC gene are deleted. Prefera bly, the skfA, sd pC, xpf, and lytC genes are deleted.

Preferably, the skfA, sdpC, xpf, lytC, lytD, lytF, lytG, cwlA, cwlC, cwl D, cwl H, cwlJ, cwlK, cwlO, cwlS, xepA, xlyA, and xlyB gene is respresented by a sequence having at least 70% identity to a sequence shown in SEQ I D No. 53 to 70, respectively.

In an embodiment of the present invention, wherein the genes coding for the autolysins com prises a nucleic acid seq uence selected from the group consisting of a) nucleic acid sequence represented by SEQ I D No: 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 or 70, b) a n ucleic acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the nucleic acid sequence represented by S EQ I D No: 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 or 70, c) a nucleic acid seq uence coding for a polypeptide com prising an amino acid sequence represented by S EQ I D No: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 or 88, and d) a nucleic acid sequence coding for a polypeptide com prising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the amino acid sequence represented by SEQ I D No: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 or 88.

I n one em bodiment, the gene coding for the a mylase to be deleted is an amyE gene.

In an embodiment of the present invention, wherein the genes coding for proteins having amylase activity com prises a nucleic acid seq uence selected from the grou p consisting of a) nucleic acid sequence represented by S EQ I D No: 89, b) a nucleic acid sequence having at least 70% identity, preferably having at least 75%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the nucleic acid sequence represented by S EQ I D No: 89, c) a nucleic acid sequence coding for a polypeptide com prising an am ino acid sequence represented by S EQ I D No: 90, a nd d) a n ucleic acid sequence coding for a polypeptide com prising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the amino acid sequence represented by SEQ I D No: 90.

In one embodiment, the one or more genes coding for the lipase to be deleted is selected from the group consisting of an estA, estB, lipC, ypm R, yodD, and ytpA gene. Preferably, at least 1, at least 2, at least 3, at least 4, at least 5, or all genes coding for the lipase selected from the group consisting of an estA, estB, lipC, ypm R, yodD, and ytpA gene are deleted. Preferably, at least 1, or all genes coding for the lipase selected from the group consisting of an estA and estB gene are deleted. Preferably, the estA and estB genes are deleted.

In an embodiment of the present invention, wherein the genes coding for proteins having lipase activity comprises a nucleic acid sequence selected from the group consisting of a) nucleic acid sequence represented by SEQ I D No: 91, 92, 93, 94, 95 or 96, b) a nucleic acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the nucleic acid sequence represented by SEQ I D No: 91, 92, 93, 94, 95 or 96, c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ I D No: 97, 98, 99, 100, 101 or 102, and d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the amino acid sequence represented by SEQ I D No: 97, 98, 99, 100, 101 or 102. I n one em bodiment, the one or more genes coding for the xylanase to be deleted is selected from the group consisting of a xynA, xyn B, xynC, and xyn D gene. Preferably, at least 1, at least 2, at least 3, or al l genes coding for the xylanase selected from the group consisting of a xynA, xyn B, xynC, and xyn D gene a re deleted. Preferably, at least 1 , or al l genes coding for the xylanase selected from the grou p consisting of a xyn B und xyn D gene are deleted. Prefera bly, the xyn B und xyn D genes are deleted. More preferably, at least 1, or all genes coding for the xylanase selected from the group consisting of a xynA und xynC, preferably xynA, gene are deleted. More preferably, the xynA u nd xynC genes are deleted.

In an embodiment of the present invention, wherein the genes coding for proteins having xylanase activity com prises a nucleic acid sequence selected from the grou p consisting of a) nucleic acid sequence represented by SEQ I D No: 103, 104, 105 or 106, b) a nucleic acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the nucleic acid sequence represented by S EQ I D No: 103, 104, 105 or 106, c) a n ucleic acid sequence coding for a polypeptide com prising an amino acid sequence represented by S EQ I D No: 107, 108, 109 or 110, and d) a nucleic acid sequence coding for a polypeptide com prising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, o r at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, fu rthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the amino acid sequence represented by S EQ I D No: 107, 108, 109 or 110.

I n one em bodiment, the gene coding for the mannanase to be deleted is a n gm uG gene. In an embodiment of the present invention, wherein the genes coding for proteins having mannanase activity com prises a nucleic acid sequence selected from the grou p consisting of a) a nucleic acid sequence represented by S EQ I D No: 111 , b) a nucleic acid seq uence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, o r at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, fu rthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the nucleic acid sequence represented by SEQ I D No: 111, c) a nucleic acid seq uence coding for a polypeptide com prising a n amino acid sequence represented by S EQ I D No: 112, and d) a nucleic acid sequence coding for a polypeptide comprising an a mino acid seq uence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, furthermore preferably having at least 90%, and most preferably having at least 95% sequence identity to the amino acid sequence represented by S EQ I D No: 111.

In an embodiment of the present invention the recombinant cell is a bacterial cell, preferably a Bacillus cell, more preferably a Bacillus subitilis.

As used herein, the term " Bacillus species" refers to any species of the genus Bacillus, including but not limited to Bacillus subtihs (B. subti/is), B. Hcheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. dausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. gibsonii, B. agaradhaerens, B. akibai, B. darkiiand 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 been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named " Geobacillus stearothermophilus." The production of resistant endospores under stressful environmental conditions is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, AmphibaciHus, AneurinibacMus, AnoxybaciHus, BrevibaciHus, Filobacillus, Gracilibacillus, Halobacillus, PaenibaciHus, SalibacHlus, Thermobacillus, UreibaciHus, and VirgibaciHus.

Preferably, the bacteria is selected from the group consisting of Bacillus Hcheniformis , Bacillus lentus, Bacillus subt/l/s, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus stearothermophilus, Bacillus dausii, Bacillus alkalophilus, Bacillus coagulans, Bacillus circulans, Bacillus pumilus, Bacillus thuringiensis, Bacillus psuedofirmus, Bacillus marmarensis, Bacillus cellulolyticus, Bacillus hemicellulolyticus, Geobacillus stearothermophilus, Bacillus darkia, Bacillus akibai, Bacillus gibsonii, Bacillus lautus, Bacillus megaterium, and Bacillus halodurans, more preferably the bacteria is selected from the group consisting of Bacillus Hcheniformis, Bacillus coagulans, Bacillus circulans, Bacillus lentus, Bacillus thuringiensis, Bacillus subt/l/s, Bacillus dausii, Bacillus alkalophilus, Bacillus halodurans, even more more preferably the bacteria is selected from the group consisting of Bacillus Hcheniformis, Bacillus subt/l/s, Bacillus dausii, Bacillus alkalophilus, Bacillus halodurans, and most preferably, the bacteria is Bacillus subtiHs.

In another embodiment, the present invention provides Bacillus species cells for producing heterologous proteins, having combined deletion of multiple genes coding for extracellular proteases, sporulation factors, and autolysins.

In another embodiment, the present invention provides further deletion of at least one gene coding for native proteins having amylase activity, lipase activity, xylanase activity, and mannanase activity.

In an embodiment of the present invention, genes are deleted from the Bacillus species cell by homologous recombination and splicing by overhang extension (SOE) PCR. In a preferred embodiment, three nucleotide fragments namely, 5’ homology arm similar to a segment on the chromosome upstream of the gene to be deleted, an antibiotic selection marker flanked by lox P sites, and 3’ homology arm similar to a segment on the chromosome downstream of the gene to be deleted are amplified by PCR, and then spliced together by SOE-PCR technique. The resulting product is transformed into recombinant cells where it recombines in the genome. Successful transformants are selected for with kanamycin resistance (kanR) phenotype. Afterwards, kanR is recycled by excising the cassette with expression of Cre recombinase.

Table 1 provides preferred embodiments i.e. combination of genes (a to h) coding for (A) extracellular proteases, (B) sporulation factors, and (C) autolysins, which are deleted to obtain the recombinant Ba ciHus species cell, for example recombinant B. subtih ^' s, of the presently claimed invention, which exhibits reduced cell lysis.

In another embodiment, the recombinant Bacillus species cell of the presently claimed invention having deletion of genes according to embodiments a to h is subjected to further deletion of genes coding for native proteins, which lowered the background activity of the native proteins and resulted in higher sensitivity in screening of the heterologous proteins.

In yet another embodiment, the recombinant Bacillus species cell of the presently claimed invention contains further deletion of at least one gene selected from the genes coding for proteins having amylase activity, lipase activity, xylanase activity, and mannanase activity.

In another embodiment of the present invention, the gene coding for proteins having amylase activity is amyE, which is preferably represented by SEQ I D No: 89.

In another embodiment of the present invention, the genes coding for proteins having lipase activity are selected from the group consisting of estA, estB, lipC, ypm R, yodD, and ytpA, which are preferably represented by SEQ I D No: 91, 92, 94, 93, 95, and 96, respectively, more preferably estA and estB represented by S EQ I D No: 91 a nd 92, respectively.

I n yet another em bodiment of the present invention, the genes coding for proteins having xylanase activity a re selected from the group consisting of xynA, xyn B, xynC, and xyn D, which are preferably represented by SEQ I D No: 103, 104, 105 and 106, respectively, more prefera bly xynA and xynC represented by SEQ I D No: 103 and 105, respectively.

I n a further em bodiment of the present invention, the gene coding for proteins having mannanase activity is gm uG, which is preferably represented by S EQ I D No: 111.

I n one em bodiment of the present invention, the recom bina nt cel l com prises a nucleic acid sequence coding for a heterologous protein.

In an embodiment of the present invention, the heterologous protein is selected from the group consisting of enzymes, hormones, growth factors and cytokines.

I n another em bodiment of the present invention, the heterologous protein is an enzyme.

Preferably, the enzyme is selected from the group consisting of phytases, proteases, beta-glucanases, xylanases, mannanases, lipases, cel l ulases, gl ucoa mylases, amylases, al pha-amylases, beta-amylases, hydrolases, isomerases, ligases, lyases, oxidoreductases, transferases, aminopeptidases, carbohydrases, carboxypeptidases, cata lases, cel lobiohydrolases, cel l ulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, endogl uca nases, esterases, al pha- galactosidases, beta-galactosidases, al pha-glucosidases, beta-glucosidases, invertases, laccases, lactases, lipases, mannosidases, m utanases, oxidases, pectinolytic enzymes, peroxidases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transgl utaminases, xyla nases, and beta-xylosidases, more prefera bly the enzymes are selected from the group consisting of lipases, amylases, xylanases, mannanases, proteolytic enzymes, carbohydrases, isomerases, transferases, kinases, phosphatases, and most prefera bly selected from the group consisiting of lipases, amylases, xylanases, and man nanases.

I n a n em bodiment, the present invention provides a recom binant B. subtihs ce\ \ for producing an enzyme selected from the group consisting of lipase, amylase, mannanase, and xylanase, having deletion of one or more genes coding for extracellular proteases selected from the group consisting of aprE, nprE, vpr, epr, mpr, nprB, bpr, wprA, ywaD, htrA, htrB, and htrC; deletion of one or more genes coding for sporulation factors selected from the group consisting of sig E, sigF, sigG, sigH, sigK, prkA, ybaN, yhbH, ykvV, ylbJ, y p j B , ytrl, ytrH, and yqfC; and deletion of one or more genes coding for autolysins selected from the group consisting of skfA, sdpC, xpf, lytC, lytD, lytF, lytG, cwlA, cwlC, cwlD, cwlH, cwlJ, cwlK, cwlO, cwlS, xepA, xlyA, and xlyB, preferably having deletion of genes aprE, nprE, vpr, epr, mpr, nprB, bpr, sigF, skfA, sdpC, xpf, and lytC.

In an embodiment, the present invention provides a recombinant B. subtihs ce\\ for producing an enzyme selected from the group consisting of lipase, amylase, mannanase, and xylanase, having a deletion of one or more genes coding for an extracellular protease comprising a nucleic acid sequence selected from the group consisting of a) nucleic acid sequence represented by SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, and d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; a deletion of one or more genes coding for a sporulation factor comprising a nucleic acid sequence selected from the group consisting of a) nucleic acid sequence represented by SEQ ID No: 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38, b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38, c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52, and d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52; and a deletion of one or more genes coding for an autolysin comprising a nucleic acid sequence selected from the group consisting of a) nucleic acid sequence represented by SEQ ID No: 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 , b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70, c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 or 88, and d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 or 88.

In another embodiment of the present invention the recombinant B. subtihs cell further contains deletion of at least one gene selected from amyE, estA, estB, lipC, ypmR, yodD, and ytpA, xynA, xynB, xynC, xynD, and gmuG, preferably deletion of at least one gene selected from amyE, estA, estB, xynA, xynC, and gmuG.

In another embodiment of the present invention the recombinant B. subtihs cell further contains deletion of at least one gene coding for a protein having amylase activity and the gene coding for a protein having amylase activity comprises a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence represented by SEQ ID No: 89, b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 89, c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 90, and d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 90; a deletion of at least one gene coding for a protein having lipase activity and the gene coding for a protein having lipase activity comprises a nucleic acid sequence selected from the group consisting of a) nucleic acid sequence represented by SEQ ID No: 91, 92, 93, 94, 95 or 96, b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 91, 92, 93, 94, 95 or 96, c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 97, 98, 99, 100, 101 or 102, and d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 97, 98, 99, 100, 101 or 102; a deletion of at least one gene coding for a protein having xylanase activity and the gene coding of a protein having xylanase activity comprises a nucleic acid sequence selected from the group consisting of a) nucleic acid sequence represented by SEQ ID No: 103, 104, 105 or 106, b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by SEQ ID No: 103, 104, 105 or 106, c) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence represented by SEQ ID No: 107, 108, 109 or 110, and d) a nucleic acid sequence coding for a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence represented by SEQ ID No: 107, 108, 109 or 110; and a deletion of at least one gene coding for a protein having mannanase activity and the gene coding for a protein having mannanase activity comprises a nucleic acid sequence selected from the grou p consisting of a) a nucleic acid sequence represented by SEQ I D No: 111, b) a nucleic acid sequence having at least 70% identity to the nucleic acid sequence represented by S EQ I D No: 111, c) a nucleic acid sequence coding for a polypeptide com prising an amino acid sequence represented by SEQ I D No: 112, and d) a nucleic acid sequence coding for a polypeptide com prising an amino acid sequence having at least 70% identity to the amino acid seq uence represented by S EQ I D No: 112.

In another embodiment, the gene coding for the heterologous enzyme may be introduced into the genome of the recombinant B. subtilis cell or may also be introduced into the recombinant B. subtilis cell as a vector. Furthermore, the number of such gene to be introduced herein may be one or a plural number thereof. When a plurality of genes is introduced, a plurality of genes may be introduced via arrangement thereof in a line on one DNA fragment, or may also be introduced as different DNA fragments. In one embodiment, the plurality of genes may be introduced by multiple chromosomal integrations. I n another embodiment, the plurality of genes may be introduced by using a multi-copy plasmid. A technique for introduction of genes is not particularly limited. Conventionally known transformation methods, transduction methods, and the like can be used.

I n another em bodiment of the present invention, the recom bina nt B. subtilis cel l com prises a nucleic acid sequence coding for an enzyme selected from the grou p consisting of phytases, proteases, beta-glucanases, xyla nases, mannanases, lipases, cel l ulases, gl ucoa mylases, amylases, a l pha-amylases, beta-amylases, hydrolases, isomerases, ligases, lyases, oxidoreductases, transferases, aminopeptidases, carbohydrases, ca rboxypeptidases, catalases, cel lobiohydrolases, cel l ulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, endogl uca nases, esterases, al pha-galactosidases, beta-galactosidases, al pha- glucosidases, beta-gl ucosidases, invertases, laccases, lactases, lipases, mannosidases, m utanases, oxidases, pectinolytic enzymes, peroxidases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transgl utaminases, xyla nases, and beta-xylosidases.

I n another em bodiment of the present invention, the recom bina nt B. subtilis cel l com prises a nucleic acid sequence coding for a heterologous enzyme selected from the grou p consisting of lipases, amylases, xylanases, and mannanases.

I n an em bodiment, the present invention provides a method of producing a recom bina nt cel l for prod ucing heterologous proteins, the method com prises the fol lowing steps: a) deleting the genes coding for extracel l ular proteases, sporulation factors, and autolysins and b) transforming the recombinant cell with a nucleic acid sequence coding for a heterologous protein. In a preferred embodiment, step a) is performed prior step b). However, step a) and b) can also be performed simultaneously or step b) can be performed before step a).

In another embodiment, the method of producing the recombinant cell of the present invention further comprising deleting at least one endogenous genes selected from the genes coding for proteins having amylase activity, lipase activity, xylanase activity, and mannanase activity.

In another embodiment of the present invention, the recombinant B. subtihs cell comprises a nucleic acid sequence coding for a heterologous enzyme selected from the group consisting of lipases, amylases, xylanases, and mannanases and the recombinant B. subtihs cell comprises a deletion of one or more endogenous gene selected from the group of genes coding for a lipase, amylase, xylanase, and mannanase.

In another embodiment, the present invention provides a method of producing a recombinant Bacillus species cell for producing enzymes, wherein the step of deleting the genes coding for extracellular proteases, sporulation factors, and autolysins comprises preparing a genetic construct for deletion of genes, wherein the genetic construct comprises an 5’ upstream homologous fragment, a selection marker gene that could recombine for excision of the selection marker through expression of a ere recombinase, a 3’ downstream homologous fragment in sequence; and transforming the genetic construct into the Bacillus species cell.

Preferably, the selection marker gene is a spectinomycin resistance gene spcR, a kanamycin resistance gene kanR, or an ampicillin resistance gene amp R, more preferably, the resistance gene is kanR.

In an embodiment, the present invention provides a method of producing a B. subtihs recombinant cell for producing enzymes, the method comprises the following steps: a) deleting the genes coding from extracellular proteases selected from the group consisting of aprE, nprE, vpr, epr, mpr, nprB, bpr, wprA, ywaD, htrA, htr B, and htrC; genes coding for sporulation factors selected from the group consisting of sigE, sigF, sigG, sigH, sigK, prkA, ybaN, yhbH, ykvV, ylbJ, y p j B , ytrl, ytrH, and yqfC; and genes coding for autolysins selected from the group consisting of skfA, sdpC, xpf, lytC, lytD, lytF, lytG, cwlA, cwlC, cwlD, cwlH, cwlJ, cwlK, cwlO, cwlS, xepA, xlyA, and xlyB, preferably deleting genes aprE, nprE, vpr, epr, mpr, nprB, bpr, sigF, skfA, sdpC, xpf, and lytC; and b) transforming the recombinant cell with a nucleic acid sequence coding for an enzyme selected from the group consisting of lipase, amylase, mannanase, and xylanase.

In another embodiment of the present invention a method of producing a B. subtihs recombinant cell further comprises deletion of at least one gene selected from amyE, estA, estB, lipC, ypmR, yodD, and ytpA, xynA, xynB, xynC, and xynD, and gmuG, preferably deletion of at least one gene selected from amyE, estA, estB, xynA, xynC, and gmuG.

In an embodiment, the present invention provides a method of producing heterologous enzymes in a recombinant B. subtihs cell, comprising the steps of: a) obtaining a recombinant B. subtihs cell having one or more deletions of a gene coding for an extracellular protease selected from the group consisting of a aprE, nprE, vpr, epr, mpr, nprB, bpr, wprA, ywaD, htr A, htr B, and htrC gene; one or more deletions of a gene coding for a sporulation factor selected from the group consisting of a sigE, sigF, sigG, sigH, sigK, prkA, ybaN, yhbH, ykvV, ylbJ, y p j B , ytrl, ytrH, and yqfC gene; and one or more deletions of a gene coding for an autolysin selected from the group consisting of a skfA, sdpC, xpf, lytC, lytD, lytF, lytG, cwlA, cwlC, cwlD, cwlH, cwlJ, cwlK, cwlO, cwlS, xepA, xlyA, and xlyB gene, preferably having a deletion of the aprE, nprE, vpr, epr, mpr, nprB, bpr, sigF, skfA, sdpC, xpf, and lytC gene, and having a further deletion of at least one gene selected from the group consisting of an amyE, estA, estB, lipC, ypmR, yodD, and ytpA, xynA, xynB, xynC, and xynD, and gmuG gene, more preferably deletion of at least one gene selected from the group consisting of an amyE, estA, estB, xynA, xynC, and gmuG gene; b) transforming the recombinant B. subtihs ce\\ with a nucleic acid sequence coding for one or more enzyme selected from the group consisting of lipase, amylase, mannanase, and xylanase; c) growing the recombinant B. subtihs ce\\ under conditions suitable for producing said one or more enzymes selected from the group consisiting of lipases, amylases, xylanases, and mannanases; and d) recovering the enzymes.

Recombinant B. subtihs cells transformed with polynucleotide sequences coding for enzymes may be cultured under conditions suitable for the expression and recovery of the encoded protein from the cell culture.

The protein produced by a recombinant B. subtihs cell comprising a deletion of multiple genes as discussed above will be secreted into the culture media. I n an em bodiment the screening of enzyme expression is done by SDS page gel or ana lytical enzyme activity assay.

I n a nother em bodi ment, the present invention provides use of the recom binant cell for screening the expression of a heterologous protein, preferably an enzyme selected from the group consisting of phytases, proteases, beta-glucanases, xyla nases, mannanases, lipases, cel l ulases, gl ucoa mylases, amylases, al pha- amylases, beta-amylases, hyd rolases, isomerases, ligases, lyases, oxidored uctases, transferases, aminopeptidases, carbohydrases, carboxypeptidases, cata lases, cel lobiohydrolases, cel l ulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, endogl uca nases, esterases, al pha- galactosidases, beta-galactosidases, al pha-glucosidases, beta-glucosidases, invertases, laccases, lactases, lipases, mannosidases, m utanases, oxidases, pectinolytic enzymes, peroxidases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transgl utaminases, xyla nases, and beta-xylosidases.

I n a nother em bodi ment, the present invention provides use of the recom binant cel l for production of a heterologous protein, preferably an enzyme selected from the group consisting of phytases, proteases, beta-glucanases, xyla nases, mannanases, lipases, cel l ulases, gl ucoa mylases, amylases, al pha-amylases, beta-amylases, hydrolases, isomerases, ligases, lyases, oxidoreductases, transferases, aminopeptidases, carbohydrases, carboxypeptidases, catalases, cel lobiohydrolases, cel lu lases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, endogl uca nases, esterases, al pha-galactosidases, beta- galactosidases, al pha-glucosidases, beta-glucosidases, invertases, laccases, lactases, lipases, mannosidases, m utanases, oxidases, pectinolytic enzymes, peroxidases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transgl utaminases, xylanases, and beta-xylosidases.

By means of the above technical solutions, as compared with the prior art the recombinant Bacillus species cell of the present invention provides more accurate screening of enzymes due to reduced cell lysis and less background activity of the native enzymes.

The following examples are provided for illustrative purposes. It is thus understood that the examples are not to be construed as limiting. The skilled person will clearly be able to envisage further modifications of the principles laid out herein.

EXAMPLES Example 1: Construction of genetic construct for deletions of genes of B. subtihs

Genetic constructs for deletion of genes from B. subtihs genome were prepared through homologous recombination of a recombinant nucleic acid containing a selection marker. The following genes of B. subtihs PY79 strain were targeted for deletion: genes coding for extracellular proteases (nprE, aprE, epr, mpr, nprB, vpr, bpr), genes coding for sporulation factors (sigF), and the genes coding for autolysins (lytC, xpf, sdpC, skfA).

The genetic construct was prepared by fusion of three nucleotides into a single molecule i.e. i) a 5’ homology arm highly similar to a segment on the chromosome upstream of the gene to be deleted, ii) an antibiotic selection marker flanked by loxP sites that could recombine for excision of the selection marker through expression of a Cre recombinase and iii) a 3’ homology arm highly similar to a segment of the chromosome downstream of the gene to be deleted. The methods described in Koo BM, et ai, Construction and Analysis of Two Genome Scale Libraries for B. subtihs, Cell Systems 2017, 4, 291- 305, and Datsenko KA, hereinafter referred to as Koo BM, et ai, and Wanner BL, PNAS, One Step Inactivation of Chromosomal Genes in £ Coii K-12 using PCR products, 2000, 97 (12), 6640- 6645, are used for construction of the genetic constructs.

To create this construct, each of the three nucleotides is first individually amplified by polymerase chain reaction (PCR) to obtain DNA products. Table 2 provides the primers used for creation of the genetic constructs. The binding sites of primers used for amplification are visualized on the final construct. The homology arm regions are amplified directly from isolated genomic DNA of the B. subtihs strain to be modified i.e. B. subtihs PY79. For primer design for the two homology arms, both the 5’ homology arm forward primer and the 3’ homology arm reverse primer are designed with only a binding region for this exact homology arm. However, the reverse primer on the 5’ arm and the forward primer on the 3’ arm should contain a binding region for that specific homology arm on their 3’ end and approximately 20 base pairs of homology for the antibiotic selection marker cassette as well. In practice, these primers were designed with selection marker-homology segment that had a nucleic acid melting temperature of approximately 60 °C. Homology arms were designed to be approximately 1 kilobase pair long, and to flank the targeted gene for deletion without disrupting regulatory and expression components of neighboring genes on the chromosome. Such design considerations have been discussed extensively by Koo BM, et ai. For simplicity, the antibiotic selection marker was amplified with primers that did not contain a homology region for individual homology arms so that a single pair of primers could amplify the genetic construct for multiple genes. The template DNA for amplifying the selection marker could be a custom synthesized molecule or any appropriate template resistance gene flanked by lox sites, such as the kanamycin-resistance gene (KanR) flanked by loxP71 and loxP66 sites found on the plasmid DNA pDR240a (created by Koo BM, eta/.). A schematic outline of the construction of the genetic construct is provided in figure 1.

All individual DNA products were amplified with Phusion ^® High- Fidelity DNA polymerase, available from New England Biolabs Inc. USA. 50 pi Phusion reactions were prepared with 32.5 mI double distilled water, 10 mI Phusion 5x H F Buffer, 4 mI 10 m M dNTP mix, 2.5 mI lOm M primer pair mix, 0.5 mI to 1.0 mI template DNA, and 0.5 mI Phusion DNA Polymerase. The reaction mixtures were prepared on ice and run in a thermal cycler according to the following program: 98 °C for 1 minute, 30 cycles of 98 °C for 10 seconds, 58 °C for 10 seconds, and 72 °C for 1 minute, 72 °C for 5 minutes, and held at 16 °C until removed from the machine. 2 mI of the PCR reaction were checked by running on agarose gel. Each gel was verified for having a single product band at the expected size. If multiple bands appeared on the gel, the correct size band was purified from the gel and cleaned with a Zymo Research Zymoclean Gel DNA Recovery Kit. All other PCR reactions were cleaned with a DNA Clean and Concentrator Kit, available from Zymo Research, USA. The final construct as mentioned in figure 1 was made by a splicing by overlap extension (SOE) PCR of the individual DNA components made according to the description above. In this reaction, a normal Phusion PCR was performed as used on the individual DNA products with 2 modifications: the DNA template now consisted of 0.5 mI of a 10-fold dilution of each individual DNA part purified above and the 72 °C extension time was increased from 1 minute to 2 minutes. These SOE- PCR reaction products were directly transformed into competent B. subtihs as described in example 2.

Table 2: Primers used for the creation of genetic constructs for deletion of genes of B. subtihs

Example 2: Method for creating a B. subtihs strain with multiple gene deletions

Genes were deleted from the B. subtihs PY79 strain by adaption of the widely-used homologous DNA recombination technique. With this technique, the selection marker was continuously recycled by excision with the Cre recombinase protein. For genomic deletion, a recombinant DNA construct for the gene to be deleted was constructed as described in example 1. The 50 pi SOE- PCR product of example 1 was directly transformed into naturally competent B. subtihs PY79. The competent cells were made by growing a single colony from an LB agar plate overnight in 3 ml of liquid media (comprising 10.7 g/l dibasic potassium phosphate, 5.2 g/l monobasic potassium phosphate, 20 g/l D-glucose, 0.88 g/l trisodium citrate dihydrate, 0.022 g/l ferric ammonium citrate, 1 g/l Bacto casamino acids, 2.2 g/l potassium glutamate monohydrate, 20 m M magnesium sulfate, 300 nM manganese chloride, and 20 mg/I L- tryptophan) at 37 °C and 250 rpm agitation. Thereafter, next day a fresh 250 ml flask was filled with 30 ml of medium containing 10.7 g/l dibasic potassium phosphate, 5.2 g/l monobasic potassium phosphate, 20 g/l D-glucose, 0.88 g/l tri sodium citrate dihydrate, 0.022 g/l ferric ammonium citrate, 2.4 g/l aspartic acid, 10 m M magnesium sulfate, 150 nM manganese chloride, 40 mg/I L-tryptophan, and 0.05 %w/v yeast extract. This medium was inoculated with the overnight culture to reach an optical density at 600 nm wavelength equal to 0.1 absorbance units. The culture was then incubated at 37 °C with shaking at 250 rpm until an optical density at 600 nm wavelength between 1.6 and 2.0 absorbance units was reached. Then, the DNA SOE-PCR product as described above was added to 500 mI of the cell culture in a 14 ml snap cap tube, available from BD Biosciences ^® , USA. This mixture was incubated at 37 °C with shaking for 2 to 3 hours before selection by plating on LB agar plates supplemented with 7.5 mg/I kanamycin sulfate. These plates were incubated at 37 °C overnight, with colonies forming within 24 hours.

Following successful selection, a few colonies in each genomic deletion were streaked onto a fresh LB agar plate supplemented with 7.5 mg/I kanamycin sulfate and grown at 37 °C overnight. Single colonies formed on this plate were re-streaked onto another plate, also incubated at 37 °C. As colonies formed on this plate, selected representatives were analyzed by colony PCR (amplification of genomic DNA direct from the genome of living colonies) using ExTaq DNA Polymerase Kit, available from Takara Bio. Inc., USA. The Kit was used according to the manufacturer’s protocol, and the 5’ homology arm forward primer and 3’ homology arm reverse primer were used for amplification according to example 1 and figure 1.

In most cases, the deletion of the gene and replacement with the kanamycin resistance selection marker through homologous recombination of the deletion construct created a shift in the size of DNA, as could be identified in the DNA agarose electrophoresis gel of figure 2 verifying deletion of lytC gene. Lanes 1, 2, and 3 are colonies produced by transformation of the gene deletion SOE- PCR product which were re-streaked twice as described above. The 4 ^th lane“wt” shows amplification of the wild type gene. Here, the deletion creates a reduction in size as seen in lanes 2 and 3, compared to the wild type. Either colony would work to move forward with further experiments. Lane 1 shows a mixture that is not suitable to move forward with further experiments. Similarly, verification of deletion of other genes targeted for deletion is done.

Following identification of correct colonies, the strain could be again transformed with a plasmid containing the widely-used Cre recombinase gene to remove the kanamycin marker. An example plasmid is pDR244 which is described in Koo BM, et aL This plasmid is transformed in the same manner as the SOE- PCR product, however selection and re streaking of colonies is performed with 100 mg/I spectinomycin instead of kanamycin sulfate, and all growth post-DNA addition is carried out at 30 °C. Once clean colonies a re achieved, they are re-streaked on an LB agar only plate and grown overnight at 42 °C to cure the plasmid, then re-streaked once again on LB agar only to obtain individual colonies to verify the complete deletion. In this case, the complete deletion and excision of the kanamycin marker is verified by streaking colonies onto fresh LB agar only, LB agar with 7.5 mg/I kanamycin sulfate, and LB agar with 100 mg/I spectinomycin for phenotypic verification of loss of antibiotic resistance, and colony PCR to verify deletion of the gene of interest - similar to that of figure 2, but in all cases the deleted construct will be shorter since the kanamycin resistance marker and original gene target have been excised. The kanamycin resistance phenotype will be recycled for subsequent deletions. The above technique was used on B. subtiHs PY79 strain (wild type) to create a sporulation-deficient strain lacking extracellular proteases, by sequentially deleting the following genes: nprE, aprE, epr, mpr, nprB, vpr, bpr, and s/g ^' F Deletion of these genes created a strain designated as QZ111. QZ111 is a high lysing strain and is used as a reference. The recombinant B. subtih ^' s strain designated as QZ120 was generated by further sequential deletion of the following autolysin genes: lytC, xpf, sdpC, skfA. Thus, strain QZ120 contained deletion of the following genes: nprE, aprE, epr, mpr, nprB, vpr, bpr, and sigF and lytC, xpf, sdpC, skfA. The strain QZ120 was evaluated for the production of enzymes and cell lysis in examples 3 and 4 below. Figure 3 provides a schematic diagram for the preparation of B. Subtih ^' s strain QZ120 from the wild type strain QZ111, which involves sequential deletion of multiple genes.

Example 3: Methods for the production of enzymes

Strains created using Examples 1 and 2 were further engineered to produce heterologous enzymes of interest. To do this, these strains were genetically transformed with recombinant DNA which contains genes and regulatory elements to express the enzymes of interest. This recombinant DNA could be directly synthesized by a DNA provider (e.g. Twist Biosciences Corporation, San Francisco, California) or cloned into a suitable expression vector using routine molecular cloning techniques (e.g. polymerase chain reaction [PCR] , restriction-ligation plasmid construction, Golden Gate plasmid construction). The expression vectors can be either self-replicating plasmids, such as the widely used pU BllO vector (NCBI GenBank accession No. M 19465.1), or direct genomic integration constructs. Genomic integration constructs can be generated like those in Example 1 and Figure 1, but with additional DNA sequences coding for the expression of the gene of interest also in between the 5’ and 3’ homology arms (alongside the selection marker -“kan R” in Figure 1). The genetic regulatory components to drive expression of a gene include a promoter, ribosome binding site (RBS), and secretion signal peptide. An example promoter and RBS used for production of broad range of heterologous enzymes is those of the Bacillus subtih ^' s Veg protein. Additionally, the example secretion signal peptide sequences widely-used in literature include those of the Bacillus subtih ^' s EstA, NprE, AprE, and Ydj M proteins. Once created by synthesis or molecular cloning techniques, the recombinant DNA constructs can be transformed into the engineered Bacillus strain by the same transformation method as detailed in Example 2, with minor modifications to add DNA of the synthesized or molecularly-cloned vector instead of the final PCR reaction and adjusting antibiotic selection on LB-agar plates as appropriate (for pU BllO vectors, LB with 20 pg/ml kanamycin sulfate plates serve for selection). With overnight incubation of selection plates at 37°C, successfully transformed Bacillus colonies will form by the within 24 hours. From here, individual colonies can be directly inoculated into liquid medium for expression. This expression can be achieved with sub examples 3a and 3b below.

Example 3a: Method for the production of enzymes at a microliter to milliliter scale

Microliter to a few milliliter scale production for enzyme screening is performed in 96- deep well plates, available from GE Healthcare, USA, sealed with a breathable, sterile adhesive plate films. To start expression, a seed culture was first grown in the deep well plates. To grow the seed cultures, plate layouts and quantities were determined ahead of time. According to the plate layout, 600 pi of a rich medium (for example, 0.5 %w/v yeast extract, 1 % w/v sodium chloride, 1 % w/v tryptone, 2 % w/v D-glucose with appropriate antibiotic selection supplementation was added to all wells. Each well is inoculated with a single colony from a fresh (less than 2 weeks old) LB agar plate of the strain to be tested. When all wells are inoculated, the plate is sealed and incubated at 37 °C overnight. The plate is mixed by shaking in a Titer Plate Shaker, available form Thermo Scientific, USA, at a speed of “7.” To start the expression culture, the seed is removed from the incubator the following morning and used to inoculate a fresh, sterile plate filled with expression medium. The expression medium can be any appropriate mixture, but for example, a rich medium consisting of 1 % w/v yeast extract, 1 % w/v sodium chloride, 1 % w/v tryptone, and 5 % w/v glucose with appropriate antibiotic selection markers, can be used. Each well of the new plate is filled with 600 mI of the expression medium and inoculated with 15 mI of the seed culture. When all wells are inoculated, the plate is sealed and continually mixed in an incubator. Different conditions can be used for expression of different enzymes. For exam ple, expression can be done for 48 hrs at 37 °C with mixing from a Titer Plate Shaker, available from Thermo Scientific, USA, set to a speed of “7.” Following the expression method, enzyme screening studies are performed by SDS- PAGE gel or analytical enzyme activity assays. An exam ple with SDS-PAGE gel analysis is given below in Example 4. For production of more than 600 mI, the seed well can be used to inoculate multiple expression medium wells.

Example 3b: Method for the production of enzymes milliliters to liter scale

An example of enzyme production at a larger scale is given in the following method. A 3 ml seed culture is grown in a 14-ml snap-cap culture tube, available from BD Biosciences, USA, in 0.5 % w/v yeast extract, 1 % w/v sodium chloride, 1 % w/v tryptone, 2 % w/v D-glucose with appropriate antibiotic selection supplementation at 37 °C, at 250 rpm shaking overnight. The next day, the seed is expanded with growth in 25 m l of the same medium in a 250 ml baffled shake flask at 37 °C, at 250 rpm. This culture is inoculated with 0.5 ml of the first seed, and then grown for 8 hours. When ready, the second culture is used to inoculate the semi-batch feeding expression culture. The semi batch feeding expression culture is grown in a 4 I, in a 3-necked glass vessel. The largest, upright neck is fitted with a custom made 6-port head. The head includes an agitation shaft with 2 Rushton impellers, available from Fusion Fluid Equipment, USA (agitating at “high” speed with a 3.5A electric motor, available from Lin Engineering, USA), a sparger to allow addition of filter-sterilized ambient air at 5 standard cubic feet per hour (scfh), a cooling loop connected to the cold-water line (i.e. to cool temperature to 30 °C), a temperature probe, and a sampling port. The other two necks were covered with breathable, sterile films. Each could be used as a sampling port as needed. Additionally, the vessel is wrapped with an electric heating blanking to raise temperature as needed. With this setup, 2 I of rich expression medium as described in the example above is added to the vessel, and the entire second seed culture is added when ready. The time of this culture can be extended up to 7 days by feeding an additional carbon source (e.g. 50 % w/v D-glucose) and neutralizing the pH (i.e. addition of appropriate acid or base), both done through the feeding port.

Example 4: Assessment of cell lysis

Example 4a: Qualitative assessment of cell lysis reduction by SDS- PAGE gels analysis

Qualitative assessment of cell lysis was performed using SDS-Polyacrylamide gel electrophoresis (SDS- PAGE) analysis. Recombinant B. subtiHs strains were obtained as provided in examples 1 and 2, and figure 3, and enzyme samples were generated as provided in example 3, and then analyzed by SDS-PAGE. Enzyme-containing supernatant was extracted from samples by cooling cell cultures on ice for about 20 minutes, and pelleting cells by centrifugation at 5,000 x g for 15 minutes at 4°C. Supernatant was mixed in a 1:1 volumetric ratio with Novex ^® Tris-Glycine SDS sample buffer (2x), available from Thermo Fischer, USA, supplemented with 5% beta-mercaptoethanol, available from Sigma-Aldrich, USA, and heated at 98°C for 10 minutes in a thermal cycler. 10 pi of the above mixture was loaded into a Criterion Pre-Cast Gel (available from Bio- Rad Laboratories, USA, Catalog #3450044) in a Criterion Cell gel bath (available from Bio- Rad Laboratories, USA). Electrophoresis was run on the gel at 150 V for 90 minutes. After, the gel was rinsed with distilled water and removed from the casing, the naked gel was further cleaned with distilled water, stained with Novex SimplyBlue SafeStain, available from Life Technologies, USA), and de-stained with distilled water. The gel was imaged with a EZ Imager Gel Doc run Imager software, available from Bio-Rad Laboratories, USA.

Qualitative cell lysis was observed by differences in the background i.e. undesired proteins found throughout a given gel lane. A gel comparing the expression of various enzymes in the strain QZ111 ( B subtHis

PY79 A nprE A aprE A eprA mpr A nprB A vpr A bpr A sigF) and recombinant strain QZ120

given in figure 4. The recombinant B. subtHis strain QZ120 exhibited lower cell lysis when compared with the B. subtHis strain QZ111. The higher level of cell lysis in the B. subtHis strain QZ111 is evident from the darker background of all lanes of QZ111 strain. This trend was further quantified and verified with the example 4b as provided below.

Example 4b: Quantitative assessment of cell lysis reduction by B. subtHis Isocitrate Dehydrogenase method

The extent of cell lysis was quantified by adapting the method of Coxon RD, etai Protein Export During Growth of B. subtiiis\ the Effect of Extracellular Protease Deficiency, Letters Appl. Microbiol., 1991, 12, 91- 94, referred hereinafter as“Coxon". In this assay, the fractional activity measurement of isocitrate dehydrogenase (ICDH) in the culture supernatant is compared with the total activity of the supernatant and cell pellet for quantification of cell lysis. This method is based on findings that the B. subtHis ICDH is a cytoplasmic enzyme and is resistant to hydrolysis from its own native proteases (as detailed in Coxon and references cited therein). Therefore, it is concluded that the ICDH activity found in the supernatant comes from leaking cytoplasm of lysed cells, and the ICDH activity of any given lysed cell should remain constant through a reasonable enzyme expression timeline or experiment. The assay monitors the conversion of NADP to NADPH as a change in absorbance at 340 nm, using isocitrate as substrate.

To perform this assay, cells were first grown according to a given enzyme expression protocol, such as those given in example 3. The workflow for measurement is given in figure 5. 1ml of the culture broth was extracted and chilled on ice for 15 to 30 minutes. Meanwhile, a centrifuge was precooled to 4°C. When the incubation on ice was over, the samples were moved to the centrifuge and spun down at 8,000 x g for 4 minutes at 4°C. Supernatant was then extracted and stored in a fresh microcentrifuge tube on ice until later assaying. The cell pellet was resuspended in an equal volume of ice-cold 0.1M potassium phosphate at pH 8.0. The spin-and-resuspension cycle was repeated twice to wash the cells. The resulting cell suspension was then lysed open by incubation with 200 mg/I lysozyme at 37 °C for 15 minutes, re-cooling for 15 minutes on ice, and sonication with a Q125 sonicator, available from Qsonica, USA, set for 15 seconds on, 10 seconds off for 5 cycles. A 423-A tip was submerged in the sample during sonication, and the pellet was kept on ice. Insoluble debris left from sonication was removed by centrifugation at 16,000 x g for 10 minutes at 4°C. Remaining soluble fraction was removed by pipette and placed in a fresh, p re-chi I led microcentrifuge tube.

To measure ICDH activity from the two now isolated fractions, each sample was first diluted into a measurable linear range by addition of 0.1 M potassium phosphate at pH 8.0. Typical linear dilution range was between 5 and 100-fold (with the appropriate range determined after a first run of the activity assay as described below, example given in Figure 6 where the lx, 5x, lOx dilutions do not provide a linear reaction rate, while the 20x reaction does provide a linear reaction rate that can be used to accurately calculate ICDH activity in that fraction). The assay reaction mixture was composed of 420 pi of a diluted sample (isolated fractions described above), 500 mI pH 8.0 0.1 M potassium phosphate, 10 mI 0.1M sodium isocitrate at pH 7.0, 20 mI 0.25M magnesium chloride, and 50 mI 8m M b - Nicotinamide adenine dinucleotide phosphate disodium salt (NADP ⁺ ) each per 1 ml. All reactions were brought to room temperature before use. To run the assays, 87 mI of a mixture of the all components except, the sample itself, into a flat, clear, 96- well Costar plate. The reaction was initiated by mixing in 64 mI of the diluted sample into the other ingredients. The reaction was monitored by measuring kinetics of reduction of NADP ⁺ at 340 nm in a HI plate reader, available from Biotek Synergy, USA, running Gen5 microplate and imager software. And, as mentioned above and shown in Figure 6, samples were diluted into a linear reaction rate range accurate calculation of the ICDH activity.

Table 3 provides a comparison of % lysed cell fraction between the reference strain QZ 111 and selected strains of the recombinant B. subtihs used for enzyme production. To calculate the lysed cell fraction, the ICDH activity of the supernatant sample fraction was divided by the sum of the ICDH activity of the supernatant and of the sonicated cell pellet extract. The lysed cell fraction was then computed by dividing the ICDH activity of a given sample’s supernatant by the sum of the ICDH activity of the supernatant and the cell pellet. An example of how this calculation method is depicted in Figure 7. I n the figure, a 10-fold and 20-fold dilution were needed to bring ICDH activity of the supernatant and the cell pellet extraction, respectively, into a measurable linear range. From here, the reaction rate, given by the slope of the line (m) is found. The fraction of lysis is then given by the product of the supernantant ICDH activity slope and its dilution factor divided by the product of the cell pel le extract ICDH activity slope and its dilution factor. Table 3: Comparison of % lysed cell fractions in selected B. Subtih ^' s strains

QZ111*= B. subtih ^' s PY79 D nprEA aprEA eprA mprA nprBA vprA bprA sigF

Example 5: Deletion of additional native enzymes from recombinant B. subtih ^' s QZ120 strain

The native genes coding for proteins having amylase activity ( amyE) , lipase activity (estA, estB, UpC, ypmR, yodD, ytpA), xylanase activity {xynA, xynB, xynC, xynD) and mannanase activity (gmuG) were deleted according to the methods described in examples 1 and 2, using primers mentioned in table 4.

Table 4: Primers for gene deletion

For example, the native gene amyE, coding for proteins having amylase activity was deleted from the recombinant B. subtiHs QZ120 strain. The background amylase activity in the recombinant B. subtihs QZ120 strain with and without the native amyE gene was quantified using an in vitro activity assay with the Infinity Amylase Liquid Stable Reagent, available from Thermo Scientific™, USA. To run the assay, supernatant was extracted from strains as described in the above examples 3 and 4, and diluted 50-fold in 50 m M pH 8.0 3-(N-morpholino) propanesulfonic acid buffer (MOPS). The diluted supernatant was brought to room temperature by resting on a bench. 100 pi aliquots of these samples were added to a clear, 96-well Costar ^® assay plate, available from Sigma Aldrich, USA. The assay reaction was initiated by addition of 100 pi I nfinity reagent to each well. The amylase activity in each reaction was measured by monitoring the rate of change of absorbance at 405 nm wavelength, as described in the I nfinity Reagent manufacturer’s protocol. The absorbance was measured in a HI plate reader running Gen5 microplate and imager software, available from Biotek Synergy, USA. The measured loss of background amylase activity with additional amyE deletion is shown in figure 8. Thus, an additional deletion of the amyE gene from B. subtihs QZ120 strain reduces the background amylase activity and contributes to improved screening resolution of the amylase enzyme.

In an additional example, background lipase activity was lowered with deletion of the estA and estB gene from the recombinant B. subtihs QZ120 strain. Both native lipases were deleted with methods of exam ples 1 and 2 using primers in table 3. The decrease of background lipase activity was verified with an in vitro assay with 4-nitrophenol octanoate (pN P-08, available from Sigma Aldrich, USA). To run the assay pN P-08 stock solution was made at 4 M concentration in DMSO. The assay was run with a 1:1 volumetric mix of a supernatant buffer and a substrate buffer. The supernatant buffer was composed of 50 m M H EPES buffer at pH 7.0, 1 %w/v B- PER (Thermo Scientific), 0.01 %w/v Triton X-100, 4% D-sorbitol, and 10 to 0.001 %v/v supernatant prepared according to Example 3. The substrate buffer was composed of 14 ml of a solution of 50 m M H EPES at pH 7.0 and 5 mg/m l bovine serum albumin mixed with 1 ml of the pN P- 08 stock solution. The reagents were pre-heated to 30 °C. The reaction was initiated by mixing the two solutions together. The reaction rate was monitored kinetically using a plate reader as described above at 405 nm wavelength absorbance. Results confirming drop in background activity are provided in figure 9. Thus, an additional deletion of estA and estB genes from B. subtiiis QZ120 strain reduces the background lipase activity and contributes to improved screening resolution of the lipase enzyme.

In an yet another example, background xylanase activity was lowered successfully with deletion of xynA and xynC from the recombinant B. subtihs QZ120 strain. All native xylanases were deleted with methods of examples 1 and 2 using primers given in table 3. The removal of background xylanase activity was verified with an in vitro assay with Azo-Xylan substrate (AX, available from Megazymes, Ireland). To run the assay, A 1 %w/v solution of AX in pH 5.2 50 m M sodium acetate was mixed 1: 1 volumetric ratio was a dilution of enzyme substrate generated according to example 3. In practice, multiple dilutions were run to ensure enzyme activity measurements fell in an acceptable, linear range (typical 10-fold to 200-fold). Dilutions were made with a 50 m M sodium acetate solution at pH 5.2 The mixture was left to react at room temperature for 9 minutes and 30 seconds, before being quenched with addition of 1:2.5 reaction to 100% ethanol. The quenched reaction then rested for 5 minutes, before being centrifuged at 4,000 x g for 15 minutes to remove precipitate. The soluble fraction was removed by pipetting. Azo dye concentration, directly indicative of xylanase activity, was then measured with reading absorbance at 465 nm wavelength in a plate reader as described above. Drop in background activity with additional deletion of xynA and xynCmeas red with this method is shown in figure 10. Thus, an additional deletion of xynA and xynCgenes from B. subtiHs QZ120 strain reduces the background xylanase activity and contributes to improved screening resolution of the xylanase enzyme.

In an additional example, background mannanase activity was removed with deletion of the gm uG gene. gmuG from the recombinant B. subti/is QZ120 strain was deleted with methods of examples 1 and 2 using primers in table 3. The removal of background mannanase activity was verified with an in vitro assay with Azo-Carob Galactomannan substrate (AZCG, available from Megazymes, Ireland). To run the assay, a 2 %w/v solution of AZCG was mixed in 1:1 volumetric ratio and a dilution of enzyme substrate was generated according to Example 3. In practice, multiple dilutions were run to ensure enzyme activity measurements fell in an acceptable, linear range (typical 10-fold to 50- fold). Dilutions were made in a pH 7.0, 50 mM HEPES buffer. The mixture was left to react at room temperature for 9 minutes and 30 seconds, before being quenched with addition of 1:2.5 reaction to 100% ethanol. The quenched reaction was then rested for 5 minutes, before being centrifuged at 4,000 x g for 15 minutes to remove precipitate. The soluble fraction was removed by pipetting. Azo dye concentration, directly indicative of mannanase activity, was then measured with reading absorbance at 465 nm wavelength in a plate reader as described above. Drop in background activity with additional deletion of gmuG measured with this method is shown in figure 11. Thus, an additional deletion of gmuG gene from B. subti/is QZ120 strain reduces the background mannanase activity and contributes to improved screening resolution of the mannanase enzyme.

Example 6: Comparison of enzyme expression between wild type parent B. subti/is strain (PY79) and B. subtiiis QZllO strain.

To further study the effect of deletion of genes coding for extracellular proteases (nprE, aprE, epr, mpr, nprB, vpr, bpr), genes coding for sporulation factors (sigF), and the genes coding for autolysins (lytC, xpf, sdpC, skfA), the expression of enzymes amylase, mannanase, xylanase, and lipase in recombinant B. subti/is QZ120 strain was compared with the wild type parent B. subti/is strain (PY79). The assesment of enzyme expression was performed using SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Recombinant B. subti/is strain QZ120 was obtained as provided in examples 1 and 2, and figure 3, and enzyme samples were generated as provided in example 3, and then analyzed by SDS-PAGE. The results as provided in figure 12 indicate that all target enzymes are visible more clearly for B. subti/is strain QZ120, whereas only a few enzymes could be identified for the wild type parent B. subti/is strain (PY79). Further, experiments were conducted to analyze the background protease activity of B. subtiHs strain QZ120 when compared with the wild type parent B. subtilis strain (PY79). The results as shown in figure 13 demonstrate that the wild type parent B. subtilis strain (PY79) exhibits higher extracellular protease activity.

Furthermore, a comparative assesment of amylase supenatant activity and lipase activity of the wild type parent B. subtilis strain (PY79) and B. subtilis strain QZ120 was done. The results as shown in figures 14 a) and 14 b) demonstrate that B. subtilis strain QZ120 exhibited higher enzyme activity when expressing amylase and lipase enzymes, than the parent B. subtilis strain (PY79).