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Title:
RECOMBINANT MANNANASE EXPRESSION
Document Type and Number:
WIPO Patent Application WO/2023/247514
Kind Code:
A1
Abstract:
The present invention relates to nucleic acid constructs comprising a first polynucleotide encoding a signal peptide from a bacterial galactanase and a second polynucleotide encoding a polypeptide having mannanase activity; expression vectors and host cells comprising said nucleic acid constructs; and methods for producing polypeptides having mannanase activity.

Inventors:
KOEBMANN BRIAN (DK)
RASMUSSEN MICHAEL (DK)
MICHAELSEN SOEREN (DK)
KIM CHE LIN (DK)
Application Number:
PCT/EP2023/066600
Publication Date:
December 28, 2023
Filing Date:
June 20, 2023
Export Citation:
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Assignee:
NOVOZYMES AS (DK)
International Classes:
C12N9/24; C12N9/28; C12N15/63; C12N15/75; C12N15/90; C12R1/01
Domestic Patent References:
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Claims:
Claims

1. A nucleic acid construct comprising: a) a first polynucleotide encoding a signal peptide from a bacterial galactanase; and b) a second polynucleotide encoding a polypeptide having mannanase activity; wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion.

2. The nucleic acid construct according to claim 1 , wherein the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, and the second polynucleotide are operably linked.

3. The nucleic acid construct according to claim 2, wherein the promoter is a P3 promoter or a P3-based promoter.

4. The nucleic acid construct according to any of the preceding claims, wherein the signal peptide is a naturally occurring signal peptide, or a functional fragment or functional variant of a naturally occurring signal peptide.

5. The nucleic acid construct according to any of the preceding claims, wherein the signal peptide is obtained from an endo-beta-1 ,4-galactanase, preferably expressed by a Bacillus species, more preferably expressed by a Bacillus licheniformis cell.

6. The nucleic acid construct according to any of the preceding claims, wherein the signal peptide is obtained from a bacterial endo-beta-1 ,4-galactanase comprising or consisting of an amino acid sequence having a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:21 , preferably the bacterial endo-beta-1 , 4-galactanase comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:21 .

7. The nucleic acid construct according to any of the preceding claims, wherein the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:2; preferably the signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:2.

8. The nucleic acid construct according to any of the preceding claims, wherein the polypeptide having mannanase activity is a microbial polypeptide; preferably a bacterial polypeptide.

9. The nucleic acid construct according to claim 8, wherein the polypeptide having mannanase activity is obtained from Bacillus circulans (group 6), or Paenibacillus illinoisensis.

10. The nucleic acid construct according to any of claims 8-9, wherein the polypeptide having mannanase activity comprises or consists of an amino acid sequence having a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:6, preferably the polypeptide having mannanase activity comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:6. 11. An expression vector comprising a nucleic acid construct according to any of claims 1-10.

12. A bacterial host cell comprising in its genome: a) a nucleic acid construct according to any of claims 1-10; and/or b) an expression vector according to claim 11.

13. The bacterial host cell of claim 12, wherein the bacterial host cell is a Gram-positive host cell. 14. A method of producing a polypeptide having mannanase activity, the method comprising: a) cultivating a bacterial host cell according to any of claims 12-13 under conditions conducive for production of the polypeptide having mannanase activity; and optionally b) recovering the polypeptide having mannanase activity.

Description:
RECOMBINANT MANNANASE EXPRESSION

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

Background of the Invention

Field of the Invention

The present invention relates to nucleic acid constructs comprising a first polynucleotide encoding a signal peptide from a bacterial galactanase and a second polynucleotide encoding a polypeptide having mannanase activity; expression vectors and host cells comprising said nucleic acid constructs; and methods for producing polypeptides of interest.

Description of the Related Art

Product development in industrial biotechnology includes a continuous challenge to increase enzyme yields at large scale to reduce costs. Two major approaches have been used for this purpose in the last decades. The first one is based on classical mutagenesis and screening. Here, the specific genetic modification is not predefined, and the main requirement is a screening assay that is sensitive to detect increments in yield. High-throughput screening enables large numbers of mutants to be screened in search for the desired phenotype, /.e., higher enzyme yields. The second approach includes numerous strategies ranging from the use of stronger promoters and multi-copy strains to ensure high expression of the gene of interest to the use of codon-optimized gene sequences to aid translation. However, high-level production of a given protein may in turn trigger several bottlenecks in the cellular machinery for secretion of the enzyme of interest into the medium, emphasizing the need for further optimization strategies.

Signal peptides (SPs) are short amino acid sequences present in the amino terminus of many newly synthesized polypeptides that target these into or across cellular membranes, thereby aiding maturation and secretion. The amino acid sequence of the SP influences secretion efficiency and thereby the yield of the polypeptide manufacturing process. Bioinformatic tools such as SignalP and SignalP5 can predict SPs from amino acid sequences, but most cannot distinguish between various types of SPs (Armenteros etal., Nat. Biotechnol. 37: 420-423, 2019). Moreover, a large degree of redundancy in the amino acid sequence of SPs makes it difficult to predict the efficiency of any given SP for production of enzymes at industrial scale. Hence, SP selection is an important step for manufacturing of recombinant proteins, but the optimal combination of signal peptide and mature protein is very context dependent and not easy to predict.

Mannanases are a particular class of enzymes, that catalyze the hydrolysis of (1 ->4)- beta-D-mannosidic linkages in mannans, galactomannans and glucomannans.

Improved recombinant mannanase expression has been reported previously by medium optimization (Zhou C, Xue Y, Ma Y. Microb Cell Fact. 2018 Aug 11 ;17(1):124. doi: 10.1186/s12934-018-0973-0), albeit with relatively low yields. Thus, in order to satisfy the growing demand for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides, it is necessary to provide recombinant expression systems with increased mannanase yields.

Summary of the Invention

The present invention is based on the surprising and inventive finding that expression of mannanase variants with a signal peptide obtained from a bacterial galactanase provides an improved yield of the mannanase variants compared to expression of the same mannanase variants with other signal peptides, i.e. a 2.1 -fold to 3.3-fold increase in mannanase expression. Notably, the increased mannanase yields were achieved using several, different mannanase variants.

In a first aspect, the present invention relates to nucleic acid constructs comprising: a) first polynucleotide encoding a signal peptide from a bacterial galactanase; and b) a second polynucleotide encoding a polypeptide having mannanase activity; wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion.

In a second aspect, the present invention relates to expression vectors comprising nucleic acid constructs of the first aspect.

In a third aspect, the present invention relates to bacterial host cells comprising nucleic acid constructs of the first aspect and/or expression vectors of the second aspect.

In a fourth aspect, the present invention relates to methods for producing polypeptides having mannanase activity.

Brief Description of the Drawings

Figure 1 illustrates the structure of the galactanaseSP-mannnanase_V1 fusion gene.

Figure 2 illustrates the structure of the galactanaseSP-mannnanase_V2 fusion gene. Figure 3 illustrates the structure of the aprHSP-mannnanase_V1 fusion gene.

Figure 4 illustrates the structure of the aprHSP-mannnanase_V2 fusion gene.

SEQUENCE OVERVIEW

Definitions

In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

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

Bacterial galactanase: The term “bacterial galactanase” means a polypeptide having galactanase activity (acting on beta-1 ,4 linkages in galactan and arabinogalactan branches; EC 3.2.1.89) which is naturally expressed by a bacterial cell. Many bacterial galactanases belong to the Glycosyl Hydrolase (GH) family GH53. A non-limiting example for a bacterial GH53 galactanase is the endo-beta-1 , 4-galactanase with SEQ ID NO:21 isolated from Bacillus licheniformis. Further bacterial galactanases are described in Ryttersgaard C. et al., J. Mol. Biol. (2004) 341 , 107-117. Typically, bacterial galactanases are secreted and thus first comprise a signal peptide which is cleaved off after/during secretion, leaving a matured galactanase. In the wildtype B. licheniformis, the galactanase with SEQ ID NO:21 is encoded together with the signal peptide shown in SEQ ID NO:2. As shown in the examples, such signal peptides from a bacterial galactanase has been shown to increase recombinant protein yield when fused to different polypeptides having mannanase activity. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA. Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native (/.e., from the same gene) or heterologous (/.e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide. A non-limiting example for a promoter is the P3 promoter with SEQ ID NO: 17.

Expression: The term “expression” means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

Extension: The term “extension” means an addition of one or more amino acids to the amino and/or carboxyl terminus of a polypeptide, wherein the “extended” polypeptide has mannanase activity. Persons skilled in the art will know that a polypeptide having a given amino acid sequence and enzymatic activity may be produced with one or a few additional amino acids at the N- and/or C-terminus, and that such a polypeptide can have essentially the same enzyme activity. Such extended polypeptides are intended to be encompassed by the present invention.

Fragment: The term “fragment” as used in the context of a polypeptide means a polypeptide having one or more amino acids absent from its amino and/or carboxyl terminus, wherein the fragment has mannanase activity. The fragment may be produced naturally during expression and/or purification of the polypeptide, or may be the result of expression of a modified nucleotide sequence expressing the fragment or of targeted removal of amino acids from the amino and/or carboxy terminus.

Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus and/or the C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Non-limiting examples for fusion polypeptides are the polypeptides with SEQ ID NO:10 orSEQ ID NO: 12, comprising the signal peptide of the invention and mannanase variant 1 (v1) or mannanase variant 2 (v2), respectively. Techniques for producing fusion polypeptides are known in the art, and include ligating or SOE/POE-PCR of DNA fragments of the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art. Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide expressed in a host cell.

Mannanase: The term “mannanase” means a beta-mannanase having an endo-1 ,4-beta- mannosidase activity (EC 3.2.1.78) that catalyzes the hydrolysis of (1 ->4)-beta-D-mannosidic linkages in mannans, galactomannans and glucomannans. Synonyms for beta-mannanases include but are not limited to mannan endo-1 , 4-beta-mannosidase, GH53 endo-1 , 4-beta- mannanase, endo-1 , 4-beta-mannanase, endo-beta-1 , 4-mannase, beta-mannanase B, beta-1 , 4- mannan 4-mannanohydrolase, endo-beta-mannanase, beta-D-mannanase, and 1 ,4-beta-D- mannan mannanohydrolase.

Mannanase Activity: Mannanase activity may be determined according to the method disclosed in the examples described under the section ’’Mannanase activity assay”.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following translation and any post-translational modifications such as N-terminal processing (e.g. removal of signal peptide), C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (/.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g. having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide. Mature polypeptides of the invention may therefore have slight differences at the N- and/or C-terminal due to such differentiated expression by the host cell. A mature polypeptide having one or more amino acids absent from the N- and/or C-terminal may be considered to be a “fragment” of the full-length polypeptide. In some aspects, the mature polypeptide is amino acids 1 to 491 of SEQ ID NO:4 and amino acids -26 to -1 of SEQ ID NO:4 are a signal peptide. In some aspects, the mature polypeptide is amino acids 1 to 491 of SEQ ID NO:6 and amino acids -26 to -1 of SEQ ID NO:6 are a signal peptide.

Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having mannanase activity.

In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 1473 of SEQ ID NO: 3.

In one aspect, the mature polypeptide coding sequence is nucleotides 79 to 1551 of SEQ ID NO: 9 and nucleotides 1 to 78 of SEQ ID NO: 9 encode a signal peptide. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 1473 of SEQ ID NO: 5.

In one aspect, the mature polypeptide coding sequence is nucleotides 79 to 1551 of SEQ ID NO: 11 and nucleotides 1 to 78 of SEQ ID NO: 11 encode a signal peptide.

Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.

Obtained polypeptide/peptide/polynucleotide: The term “obtained” or “derived” when used in reference to a polynucleotide sequence, polypeptide sequence, mannanase sequence, variant sequence or signal peptide sequence, means that the molecule originally has been isolated from the given source and that the molecule can either be utilized in its native sequence or that the molecule is modified by methods known to the skilled person.

Operably linked: The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.

Parent: With respect to a signal peptide, the term “parent” means a polypeptide functioning as a signal peptide, to which an alteration is made to produce variants of the signal peptide of the present invention. With respect to a polypeptide having mannanase activity, the term “parent” means a polypeptide having mannanase activity, to which an alteration is made to produce variants of the parent polypeptide having mannanase activity. The parent signal peptide and/or parent polypeptide having mannanase activity may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.

With respect to a host cell, the term “parent” or ’’parental” means a host cell expressing a polypeptide having mannanase activity, lacking the first polynucleotide encoding the signal peptide of the invention, and instead encodes no signal peptide, or a different signal peptide for the expression of the polypeptide having mannanase activity.

Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process. Subsequence: The term “subsequence” means a polynucleotide having one or more nucleotides absent from the 5' and/or 3' end of a polypeptide coding sequence or mature polypeptide coding sequence; wherein the subsequence encodes a fragment of a signal peptide or wherein the subsequence encodes a fragment having mannanase activity, respectively.

Variant: The term “variant” means a polypeptide having mannanase activity or a polypeptide functioning as signal peptide, comprising a man-made mutation, i.e., a substitution, insertion (including extension), and/or deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

Detailed Description of the Invention

The present invention is based on the surprising and inventive finding that expression of mannanases with a signal peptide from a bacterial galactanase polypeptide provides an improved yield of the mannanase compared to expression of the same mannanase with other signal peptides.

As can be seen from the Examples disclosed herein, use of the galactanase signal peptide (SEQ ID NO:2) from a B. licheniformis galactanase (SEQ ID NO:21) provides an improved yield of various mannanase variants. Based on this observation, the present inventors expect a similar improvement for other mannanases, in particular other mannanase variants, and/or other signal peptides obtained or derived from bacterial galactanases, in particular signal peptides having a high sequence similarity to the signal peptide with amino acid sequence of SEQ ID NO:2.

Polynucleotides

The present invention also relates to polynucleotides encoding a signal peptide of the present invention, as described herein. The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof. The polynucleotide may be cloned from a strain of Bacillus, or a related organism and thus, for example, may be a polynucleotide sequence encoding a variant of the signal peptide of the invention.

In an embodiment, the polynucleotide is a subsequence encoding a fragment having signal peptide functionality of the present invention. In an aspect, the subsequence contains at least 69 nucleotides (e.g., nucleotides 1 to 69 of SEQ ID NO: 1 , or nucleotides 10 to 78 of SEQ ID NO:1), at least 75 nucleotides (e.g., nucleotides 4 to 78 of SEQ ID NO: 1 , or nucleotides 1 to 75 of SEQ ID NO:1), or at least 72 nucleotides (e.g., nucleotides 7 to 78 of SEQ ID NO: 1 , or nucleotides 1 to 72 of SEQ ID NO:1).

In one embodiment the polynucleotide encoding the signal peptide of the present invention is isolated from a Bacillus cell.

In one embodiment the polynucleotide encoding the signal peptide of the present invention is isolated from a Bacillus licheniformis cell.

The polynucleotide may also be mutated by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the signal peptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.

In an aspect, the polynucleotide is isolated.

In another aspect, the polynucleotide is purified.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

In a first aspect, the present invention relates to a nucleic acid construct comprising: a) a first polynucleotide encoding a signal peptide from a bacterial galactanase; and b) a second polynucleotide encoding a polypeptide having mannanase activity; wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion. The second polynucleotide is located downstream from the first polynucleotide. In one embodiment, the signal peptide is a naturally occurring signal peptide, or a functional fragment or functional variant of a naturally occurring signal peptide.

The signal peptide may be from any bacterial galactanase. In one embodiment, the signal peptide is from a galactanase expressed by a Bacillus species; preferably the signal peptide is derived from a galactanase, such as a GH53 galactanase, expressed by a Bacillus species selected from the group consisting of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells; more preferably the signal peptide is derived from a galactanase expressed by Bacillus licheniformis, Bacillus subtilis or Bacillus pumilus', most preferably the signal peptide is from a galactanase expressed by Bacillus licheniformis.

In one embodiment the signal peptide is derived from a bacterial galactanase having a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:21 ; preferably the bacterial galactanase comprises, consists essentially of, or consists of SEQ ID NO:21. More preferably the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:2. Most preferably the signal peptide comprises, consists essentially of, or consists of the amino acid sequence with SEQ ID NO:2.

In one embodiment, the signal peptide comprises or consists of amino acids 1 to 26 of SEQ ID NO:2 (MKNVLAVFVVLIFVLGAFGTSGPAEA).

In one embodiment, the first polynucleotide encoding the signal peptide is nucleotides 1 to 78 of SEQ ID NO: 1.

In some embodiments, the signal peptide has an additional Ala (Ala = A = alanine) at the C-terminus of SEQ ID NO:2, such as the signal peptide of MKNVLAVFVVLIFVLGAFGTSGPAEAA (the additional alanine is underlined). In like manner, in some embodiments, the first polynucleotide encoding the signal peptide has an additional GCG codon at the 3’ end of the signal peptide coding region compared to SEQ ID NO:1.

It is expected that the invention will be just as effective when employing a signal peptide that is highly similar to the signal peptide disclosed in SEQ ID NO:2 and encoded by SEQ ID NO:1. One or more non-essential amino acids may, for example, be altered. Non-essential amino acids in a signal peptide can be identified according to procedures known in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for signal peptide activity to identify amino acid residues that are critical to the activity of the molecule and residues that are non-essential. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The identity of essential and non- essential amino acids can also be inferred from an alignment with one or more related signal peptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g. Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a/., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

Thus, in a preferred embodiment, the signal peptide comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:2; most preferably the signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:2.

In a preferred embodiment, the first polynucleotide encoding the signal peptide comprises or consists of a polynucleotide having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the polynucleotide sequence of SEQ ID NO:1 ; most preferably the polynucleotide comprises, consists essentially of, or consists of the polynucleotide sequence of SEQ ID NO:1.

In one aspect, the signal peptide is a variant (/.e., functional variant) or fragment (/.e., functional fragment) of the signal peptide of SEQ ID NO:2. In one aspect, the number of alterations in the signal peptide variant of the present invention is 1-10, e.g., 1-5, such as 1 , 2, 3, 4, or 5 alterations. Alterations includes substitutions, insertions, and/or deletions at one or more (e.g., several) positions compared to the parent. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

In a preferred embodiment, the signal peptide is a variant of the mature polypeptide of SEQ ID NO:2 comprising 1-10 alterations, e.g., 1-5, such as 1 , 2, 3, 4, or 5 alterations, compared to SEQ ID NO:2.

The polypeptide having mannanase activity may be any such polypeptide or fragment or variant thereof. In one embodiment, the polypeptide having mannanase activity is a microbial polypeptide; preferably a bacterial polypeptide. Other, non-limiting examples of available mannnases are disclosed in patent applications WO9425576, WO9964619, WO15040159, WO17021515, WO17021514, WO17021518, W018206302, W018206300, WO19068715, WO19068713, WO19185726, WO21152123, and WO21152120, or are any functional variants thereof.

Similar and as described above in relation to the signal peptide, it is expected that the invention will be just as effective when employing other polypeptides having mannanase activity that is highly similar to the mature polypeptide of SEQ ID NO:4 (encoded by SEQ ID NO:3) or to the mature polypeptide of SEQ ID NO:6 (encoded by SEQ ID NO:5).

In one embodiment, the polypeptide having mannanase activity is obtained from Paenibacillus species.

In one embodiment, the polypeptide having mannanase activity is obtained from Paenibacillus illinoisensis.

Thus, in a preferred embodiment, the polypeptide having mannanase activity comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide of SEQ ID NO:4, preferably the polypeptide having mannanase activity comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:4.

In another preferred embodiment, the polypeptide having mannanase activity comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide of SEQ ID NO:6, preferably the polypeptide having mannanase activity comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:6.

In one aspect, the polypeptide having mannanase activity is a variant (/.e., functional variant) or fragment (/.e., functional fragment) of the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:6. In one aspect, the number of alterations in the variants of the present invention is 1-20, e.g. 1-10 and 1-5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations. Alterations includes substitutions, insertions, and/or deletions at one or more e.g. several) positions compared to the parent. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

In a preferred embodiment, the polypeptide having mannanase activity is a variant of the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:6 comprising 1-20 alterations, e.g. 1-10 and 1-5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations, compared to SEQ ID NO:4 or SEQ ID NO:6, respectively.

Most preferably, the polypeptide having mannanase activity comprises or consists of the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:6. In one embodiment, the polypeptide having mannanase activity comprises or consists of the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:6 with an additional N-terminal Ala.

Due to the degeneracy of the genetic code, different polynucleotides can encode the same polypeptide. Thus, in a preferred embodiment, the polynucleotide encoding the polypeptide having mannanase activity comprises or consists of a polynculeotide sequence having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:3 or SEQ ID NO:5; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:3 or SEQ ID NO:5.

The first and second polynucleotide are operably linked in translational fusion. In the context of the present invention, the term “operably linked in translation fusion” means that the signal peptide encoded by the first polynucleotide and the polypeptide having mannanase activity encoded by the second polynucleotide are encoded in frame and translated together as a single polypeptide. Preferably, following translation, the signal peptide is removed to provide the mature polypeptide having mannanase activity. Alternatively, the signal peptide is not removed, or only removed partly to provide the mature polypeptide having mannanase activity and comprising at least a fragment of the signal peptide. The first and second polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a nucleic acid construct or expression vector may be desirable or necessary depending on the construct or vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

Besides a signal peptide, the nucleic acid constructs of the invention may be operably linked to one or more further control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

Promoters

The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are described in “Molecular Cloning: A laboratory manual” (2001 , J. Sambrook and D.V. Russel) and by Y. Song et al. (2016) PLoS ONE 11 (7) :e0158447.

In one embodiment, the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, and the second polynucleotide are operably linked. The promoter is orientated upstream of the first polynucleotide.

In an embodiment, the promoter is a heterologous promoter. Preferably, the promoter is a tandem promoter. More preferably, the promoter is a P3 promoter or a P3-based promoter. In one embodiment the promoter is the P3-promoter comprising or consisting of a polynculeotide sequence having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the polynucleotide sequence of SEQ ID NO: 17; most preferably the polynucleotide comprises, consists essentially of, or consists of the polynucleotide sequence of SEQ ID NO: 17. mRNA Stabilizers

The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene. Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471). Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce// 5(11): 1838-1846.

In one embodiment, the promoter is a promoter, such as a P3 promoter, operably linked to an mRNA stabilizer region. Preferably, the mRNA stabilizer region is the crylllA mRNA stabilizer region, preferably the crylllA mRNA stabilizer region of SEQ ID NO: 18.

Terminators

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention. Preferred terminators for bacterial host cells may be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

Propeptides

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. Additionally or alternatively, when both signal peptide and propeptide sequences are present, the polypeptide may comprise only a part of the signal peptide sequence and/or only a part of the propeptide sequence. Alternatively, the final or isolated polypeptide may comprise a mixture of mature polypeptides and polypeptides which comprise, either partly or in full length, a propeptide sequence and/or a signal peptide sequence. Regulatory Sequences

It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. Other examples of regulatory sequences are those that allow for gene amplification.

Leader Sequences

The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059, and by Kaberdin and Blasi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.

Transcription Factors

The control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. The transcription factor may function alone and/or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor. The transcription factor may regulate the expression of a protein of interest either directly, i.e. by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, i.e. by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., Subcell Biochem 2011 ; 52:7-23, as well in Balleza et al., FEMS Microbiol Rev 2009, 33(1): 133-151 .

Polyadenylation Sequences

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

Expression Vectors

In a second aspect, the present invention also relates to recombinant expression vectors comprising a nucleic acid construct according to the first aspect. The expression vectors comprise a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, /.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, erythromycin, or tetracycline resistance.

The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

Host Cells

In a third aspect, the invention relates to bacterial host cells comprising in it's genome: a) a nucleic acid construct according to the first aspect; and/or b) an expression vector according to the second aspect.

A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The polypeptide encoded by the introduced polynucleotide can be native or heterologous to the recombinant host cell. Also, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell may comprise a single copy, or at least two copies, e.g. three, four, five or more copies of the polynucleotide of the present invention.

In one embodiment, the host cell comprises two or more copies of the nucleic acid construct and/or the expression vector.

The host cell may be any bacterial cell useful in the recombinant production of a polypeptide of the present invention, e.g., Gram-positive or a Gram-negative bacterium.

In a preferred embodiment the host cell is a Gram-positive host cell. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

In one embodiment the host cell is a Bacillus cell; preferably a Bacillus cell selected from the group consisting of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cell; most preferably a Bacillus licheniformis cell.

For purposes of this invention, Bacillus classes/genera/species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.

The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending e.g. on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods 64: 391-397, and Donald, Guedon and Renault, 2013, Journal of Bacteriology, 195:11(2612-2620).

In an aspect, the host cell is isolated.

In another aspect, the host cell is purified.

Methods of Production

In a fourth aspect, the present invention also relates to methods of producing a polypeptide having mannanase activity, the method comprising: a) cultivating a bacterial host cell according to the third aspect under conditions conducive for production of the polypeptide having mannanase activity; and optionally b) recovering the polypeptide having mannanase activity. The host cell is cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state, and/or microcarrier-based fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that are specific for the polypeptide, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an assay determining the relative or specific activity of the polypeptide.

The polypeptide may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the polypeptide is recovered. In another aspect, a cell-free fermentation broth comprising the polypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and/or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science’, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

In an alternative aspect, the polypeptide having mannanase activity is not recovered. In one aspect the polypeptide having mannanase activity is not recovered, but rather a host cell of the present invention expressing the polypeptide having mannanase activity is used as a source of the variant.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Examples

Materials and methods

Chemicals used as buffers and substrates were commercial products of at least reagent grade. PCR amplifications were performed using standard textbook procedures, employing a commercial thermocycler and either Ready-To-Go PCR beads, Phusion polymerase, or RED- TAQ polymerase from commercial suppliers.

LB agar: See EP0506780, p.9.

LBPSG agar plates contain LB agar supplemented with phosphate (0.01 M K3PO4), glucose (0.4 %), and starch (0.5 %); See EP0805867, paragraph [0137],

TY: liquid broth medium; See WO94/14968, p. 16.

Oligonucleotide primers were obtained from Integrated DNA Technologies, Leuven, Belgium. DNA manipulations (plasmid and genomic DNA preparation, restriction digestion, purification, ligation, DNA sequencing) were performed following the standard manufacturer’s instructions with commercially available kits and reagents.

All the constructions described in the examples were assembled from synthetic DNA fragments ordered from Geneart (Thermo Fisher Scientific). The fragments were assembled by Prolonged Overlap Extension PCR (POE-PCR), which generates multimeric plasmids, as previously described (You et al. 2012, Applied and Environmental Microbiology 78, 1593-1595). The POE-PCR products were used for direct transformation of PCR product (DNA multimer) to Bacillus licheniformis made competent as described in patent US 2019/0185847 A1.

Direct transformation into B. licheniformis was one as previously described in patent US 2019/0185847 A1. Genomic DNA was prepared by using the commercially available QIAamp DNA Blood Kit from Qiagen. The respective DNA fragments were amplified by PCR using the Phusion Hot Start DNA Polymerase system (Thermo Scientific). PCR amplification reaction mixtures contained 1 pL (0, 1 pg) of template DNA, 1 pL of sense primer (20pmol/pL), 1 pL of antisense primer (20pmol/pL), 10pL of 5X PCR buffer with 7,5mM MgCh, 8pL of dNTP mix (1 ,25mM each), 39pL water, and 0.5pL (2 U/) DNA polymerase. A thermocycler was used to amplify the fragment. The PCR products were purified from a 1.2% agarose gel with 1x TBE buffer using the Qiagen QIAquick Gel Extraction Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's instructions.

The condition for POE-PCR is as follows: purified PCR products were used in a subsequent PCR reaction to create a single fragment using Prolonged Overlap Extension PCR (POE) using the Phusion Hot Start DNA Polymerase system (Thermo Scientific). The very 5’ end fragment and the very 3’ end fragment have complementary end, which will allow the PCR fragments to concatemer into the POE PCR product. The PCR amplification reaction mixture contained 50 ng of each of the three gel purified PCR products. POE PCR was performed as described in (You et al. 2012, Applied and Environmental Microbiology 78, 1593-1595). Fed-batch cultivation procedure

All growth media were sterilized by methods known in the art.

Inoculum steps: First the strain was grown on agar slants 1 day at 37 °C. The agar was then washed with buffer, and the optical density (OD) at 650 nm of the resulting cell suspension was measured. The inoculum shake flask was inoculated with an inoculum of OD (650 nm) x ml cell suspension = 0.1. The shake flask was incubated at 37 °C at 300 rpm for 20 hr. The fermentation in the main fermentor was started by inoculating the main fermentor with the growing culture from the shake flask. The inoculated volume was 11 % of the medium (inorganic salts, protein hydrolysate, trace metals, and vitamins) (/.e. 80 ml for 720 ml media).

Standard lab fermentors were used equipped with a temperature control system, pH control with ammonia water and phosphoric acid, dissolved oxygen electrode to measure oxygen saturation through the entire fermentation. Feed medium: Sucrose 708 g/l.

Fermentation parameters: Temperature: 30 - 42 °C; The pH was controlled using ammonia water and phosphoric acid.

Aeration: 1.5 liter/min/kg broth weight.

Agitation: 1500 rpm.

Experimental setup: The cultivation was run for five days with constant agitation, and the oxygen tension was followed on-line in this period. The different strains were compared side by side.

Mannanase activity assay

Culture broths were used to measure Mannanase activity. The diluted samples and standards (20 l) were incubated with lOOpI of a 1 % solution of Galactomannan (Carob). The reaction was performed at pH 8.0 at 50 degrees C for 45 minutes. After which time the reaction was stopped by addition of lOOpI of an alkaline reagent containing PAHBAH and Bismuth and further incubated for 20 minutes at 50 degrees Celsius. The samples were centrifuged, and supernatant transferred to a new plate and measured in a plate reader as an endpoint reading (molecular devices) and the sample absorbance (405 nm) measured simultaneously with the assay specific standard. The produced colour was proportional to Mannanase activity and was measured relative to a Mannanase enzyme standard. Preparation of samples for activity measurement was performed on a Biomek FXp liquid handler (Beckman Coulter) and samples were diluted and assayed in 96 well microtitre plates using two concentrations (60000x and 120000x) within a standard curve. Strains

MOL3330: A derivative of B. licheniformis Ca63 with deletions in amyL, aprL, bgIC, cypX, gntP, lacA2, mprL, sacB, spollAC, xylA. Furthermore, insertions forD::prsA and ara::dNuclease- sgRNA::mecA-ERM (see US 2019/0185847 A1 for insertion of a dead nuclease = dNuclease)

BT12171: MOL3330 with deletion of ydhT (native mannanase)

BT12221: BT12171with rpoB A478D mutation (see W02003/055996).

BT18109: BT12171 with one copy of the expression cassette encoding aprH signal peptide (SPapr/7) fused to the mannanase_v1 and an erythromycin resistance gene in the ara locus.

BT18111 : BT12171 with one copy of the expression cassette encoding galactanase signal peptide (galactanaseSP) fused to the mannanase_v1 and an erythromycin resistance gene in the ara locus.

BT12227: BT12221 with one copy of the expression cassette encoding aprH signal peptide (SPapr/7) fused to the mannanase_v2 and an erythromycin resistance gene in the ara locus.

BT12236: BT12221 with one copy of the expression cassette encoding galactanase signal peptide (galactanaseSP) fused to the mannanase_v2 and an erythromycin resistance gene in the ara locus.

Example 1. Construction of Bacillus licheniformis strain BT18109 expressing mannanase_v1 with the aprH signal peptide

Construction of a strain containing a 1C (one copy) SPapr/7-mannanase_v1 in the ara locus of B. licheniformis was performed by transformation of B. licheniformis BT12171 with multimerized linear DNA of SEQ ID NO: 24 (see also Fig. 3), followed by selection on LBPGS+1 pg/mL erythromycin and overnight incubation at 37°C.

The linear DNA of SEQ ID NO: 24 (Fig. 3) comprises a DNA fragment coding for the upstream flanking region of the ara gene, a triple promoter (P3, SEQ ID NO: 17) followed by the crylllA mRNA stabilizer region (SEQ ID NO:18, as described in WO 99/43835), a DNA fragment coding for the aprH signal peptide (SPapr/7) and mannanase_v1 gene, a DNA fragment coding for erythromycin resistance gene and downstream flanking region of the ara gene (SEQ ID NO: 19). Transformation of B. licheniformis BT12171 was carried out as described in patent application US 2019/0185847 A1. Selection was done on LBPGS plates supplemented with 1 pg/mL erythromycin. Chromosomal integration of the SPapr/7-mannanase_v1 gene was confirmed by PCR analysis. A confirmed B. licheniformis integrant was named BT18109. A map of ara-P3-SPapr/7-mannanase_v1-erm-ara is shown in Fig. 3, the DNA sequence encoding mannanase_v1 with the aprH signal peptide is shown in SEQ ID NO: 13 (comprising SEQ ID NO:7 encoding SPapr/7 and SEQ ID NO:3 encoding mannanase_v1), and the corresponding amino acid sequence is shown in SEQ ID NO: 14 (SPapr/7-mannanase_v1 : comprising SPaprH with SEQ ID NO:8 and mannanase_v1 with SEQ ID NO:4).

Example 2. Construction of Bacillus licheniformis strain BT18111 expressing mannanase_v1 with the galactanase signal peptide

Construction of a strain containing a 1C galactanaseSP-mannanase_v1 in the ara locus of B. licheniformis was performed by transformation of B. licheniformis BT12171 with multimerized linear DNA of SEQ ID NO: 22 (see also Fig. 1), followed by selection on LBPGS+1 pg/mL erythromycin and overnight incubation at 37°C.

The linear DNA of SEQ ID NO: 22 (Fig. 1) comprises a DNA fragment coding for the upstream flanking region of the ara gene, a triple promoter (P3, SEQ ID NO: 17) followed by the crylllA mRNA stabilizer region (SEQ ID NO: 18, as described in WO 99/43835), a DNA fragment containing the identical upstream flank of the mannanase gene as in BT18109, the coding sequence for the galactanase signal peptide (galactanaseSP, gaISP) and mannanase_v1 gene, the identical downstream flanking region as for BT18109, a DNA fragment coding for erythromycin resistance gene and downstream flanking region of the ara gene (SEQ ID NO: 19). Transformation of B. licheniformis BT12171 was carried out as described in patent application US 2019/0185847 A1. Transformants were selected on LBPGS plates supplemented with 1 pg/mL erythromycin. Chromosomal integration of the galactanaseSP-mannanase_v1 gene was finally confirmed by PCR analysis followed by sequencing. A confirmed B. licheniformis integrant with the expected sequence was named BT18111. A map of ara-P3-galactanaseSP- mannanase_v1-erm-ara is shown in Fig. 1 , the DNA sequence encoding mannanase_v1 with the galactanase signal peptide is shown in SEQ ID NO:9 (comprising SEQ ID NO:1 encoding galactanaseSP and SEQ ID NO:3 encoding mannanase_v1), and the corresponding amino acid sequence is shown in SEQ ID NO: 10 (galactanaseSP-mannanase_v1 : comprising galactanaseSP with SEQ ID NO:2 and mannanase_v1 with SEQ ID NO:4).

Example 3. Construction of Bacillus licheniformis strain BT12227 expressing mannanase_v2 with the aprH signal peptide

Construction of a strain containing a 1C SPaprH-mannanase_v2 in the ara locus of B. licheniformis was performed by transformation of B. licheniformis BT12221 with multimerized linear DNA of SEQ ID NO: 25 (see also Fig. 4), followed by selection on LBPGS+1 pg/mL erythromycin and overnight incubation at 37°C.

The linear DNA of SEQ ID NO: 25 (Fig. 4) comprises a DNA fragment coding for the upstream flanking region of the ara gene, a triple promoter (P3) followed by the crylllA mRNA stabilizer region (as described in WO 99/43835), the coding sequence for the aprH signal peptide and mannanase_v2 gene, a DNA fragment coding for erythromycin resistance gene and a downstream flanking region (SEQ ID NO: 27) of the ara gene.

Transformation of B. licheniformis BT12221 was carried out as described in patent US 2019/0185847 A1. Selection took place on plates supplemented with 1 pg/mL erythromycin. Chromosomal integration of the SPapr/7-mannanase_v2 gene was confirmed by PCR analysis. A confirmed B. licheniformis integrant was named BT12227. A map of ara-P3-SP a prH- mannanase_v2-erm-ara is shown in Fig. 4, the DNA sequence encoding mannanase_v2 with the aprH signal peptide is shown in SEQ ID NO:15 (comprising SEQ ID NO:26 encoding SPaprH and SEQ ID NO:5 encoding mannanase_v2), and the corresponding amino acid sequence is shown in SEQ ID NO:16 (SPapr/7-mannanase_v2: comprising SPaprH with SEQ ID NO:8 and mannanase_v2 with SEQ ID NO:6).

Example 4. Construction of Bacillus licheniformis strain BT12236 expressing mannanase_v2 with the galactanase signal peptide.

Construction of a strain containing a 1C galactanaseSP-mannanase_v2 in the ara locus of B. licheniformis was performed by transformation of B. licheniformis BT12221 with multimerized linear DNA of SEQ ID NO: 23 (see also Fig. 2), followed by selection on LBPGS+1 pg/mL erythromycin and overnight incubation at 37°C.

The linear DNA of SEQ ID NO: 23 (Fig. 2) comprises a DNA fragment coding for the upstream flanking region of the ara gene, a triple promoter (P3) followed by the crylllA mRNA stabilizer region (as described in WO 99/43835), a coding sequence for the galactanase signal peptide (galactanaseSP), a coding sequence for the mature mannanase_v2, a DNA fragment coding for erythromycin resistance gene and downstream flanking region (SEQ ID NO: 27) of the ara gene. Transformation of B. licheniformis BT12221 was carried out as described in patent application US 2019/0185847 A1. Selection was done on LBPGS supplemented with 1 pg/mL erythromycin. Chromosomal integration of the galactanaseSP-mannanase_v2 gene was confirmed by PCR analysis. A confirmed B. licheniformis integrant was named BT12236. A map of ara-P3-galactanaseSP-mannanase_v2-erm-ara is shown in Fig. 2, the DNA sequence encoding mannanase_v2 with the galactanase signal peptide is shown in SEQ ID NO:11 (comprising SEQ ID NO:1 encoding galactanaseSP and SEQ ID NO:5 encoding mannanase_v2), and the corresponding amino acid sequence is shown in SEQ ID NO:12 (galactanaseSP- mannanase_v2: comprising galactanaseSP with SEQ ID NO:2 and mannanase_v2 with SEQ ID NO:6). Example 5. Galactanase signal peptide results in increased mannanase_v1 expression.

This example describes the comparison of mannanase fed-batch production by B. licheniformis integrants expressing mannanase_v1 with either the aprH signal peptide (strain BT18109) or with the galactanase signal peptide (strain BT18111). B. licheniformis strains BT18109 and BT18111 were tested with respect to mannanase productivity in fed-batch cultivations as described above. Mannanase production by the strains was compared using mannanase activity assay. Relative total mannanase products are shown in Table 1 . The amount of mannanase product was 2.1-fold increased in BT18111 with the galactanase signal peptide relative to BT18109 with the aprH signal peptide. Expression of the mannanase_v1 has increased using the galactanase signal peptide. Mannanase_v1 expression was increased more than 2- fold relative to the control. The significant 2-fold increase was absolutely surprising and unexpected, in particular as the gaISP sequence has not previously been reported as a good SP to express recombinant proteins, and has never been associated with increased expression of mannanase enzymes.

Table 1. Relative total mannanase product for B. licheniformis strains expressing mannanase_v1.

Example 6. Galactanase signal peptide results in increased mannanase_v2 expression.

This example describes the comparison of mannanase fed-batch production by B. licheniformis integrants expressing mannanase_v2 with either the aprH signal peptide (strain BT12227) or with the galactanase signal peptide (strain BT12236). B. licheniformis strains BT12227 and BT12236 were tested with respect to mannanase productivity in fed-batch cultivations as described above. Mannanase production by the strains was compared using mannanase activity assay. Relative total mannanase products are shown in Table 2. The amount of mannanase product was 3.3-fold increased in BT12236 with the galactanase signal peptide relative to BT12227 with the aprH signal peptide. Expression of the mannanase_v2 was increased using the galactanase signal peptide. Mannanase_v2 expression was increased more than 3-fold relative to the control, which significant increase was absolutely surprising and unexpected. Compared to the 2-fold increased expression of mannanase_v1 from example 5, the 3-fold increased expression of example 6 further highlights that galactanase signal peptides (galSPs), and in particular the signal peptide of SEQ ID NO:2, are truly capable of increasing expression of mannanase enzymes.

Table 2. Relative total mannanase product for B. licheniformis strains expressing mannanase_v2.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

LIST OF EMBODIMENTS

The invention is further defined by the following numbered embodiments:

[1] A nucleic acid construct comprising: a) a first polynucleotide encoding a signal peptide from a bacterial galactanase; and b) a second polynucleotide encoding a polypeptide having mannanase activity; wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion.

[2] The nucleic acid construct according to embodiment 1 , wherein the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, and the second polynucleotide are operably linked.

[3] The nucleic acid construct according to embodiment 2, wherein the promoter is a P3 promoter or a P3-based promoter, preferably the heterologous promoter is a tandem promoter comprising the P3 promoter or is a tandem promoter derived from the P3 promoter.

[4] The nucleic acid construct according to any of embodiments 2 to 3, wherein the promoter is operably linked to an mRNA stabilizer region; preferably the mRNA stabilizer region is the crylllA mRNA stabilizer region, more preferably the crylllA mRNA stabilizer region is the crylllA mRNA stabilizer region of SEQ ID NO:18. [5] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is a naturally occurring signal peptide, or a functional fragment or functional variant of a naturally occurring signal peptide.

[6] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a endo-beta-1 , 4-galactanase, such as a GH53 endo-beta-1 , 4- galactanase, expressed by a Gram-positive bacterium, preferably by a Bacillus species, more preferably by a Bacillus licheniformis.

[7] The nucleic acid construct according to any of the preceding embodiments, wherein the first polynucleotide encoding the signal peptide has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:1 ; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ I D NO: 1.

[8] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from an endo-beta-1 , 4-galactanase expressed by a Bacillus species selected from the group consisting of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[9] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a endo-beta-1 , 4-galactanase expressed by Bacillus licheniformis, Bacillus subtilis or Bacillus pumilus.

[10] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from an endo-beta-1 , 4-galactanase expressed by Bacillus licheniformis.

[11] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a bacterial endo-beta-1 , 4-galactanase having a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:21.

[12] The nucleic acid construct according to any of the preceding embodiments, wherein the bacterial endo-beta-1 , 4-galactanase comprises, consists essentially of, or consists of SEQ ID NO:21. [13] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:2 ’’MKNVLAVFVVLIFVLGAFGTSGPAEA”.

[14] The nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises, consists essentially of, or consists of SEQ ID NO:2 ’’MKNVLAVFVVLIFVLGAFGTSGPAEA”.

[15] The nucleic acid construct according to any of the preceding embodiments, wherein the N- and/or C-terminal end of the signal peptide has been extended by addition of one or more amino acids.

[16] The nucleic acid construct according to any of embodiments 1 to 14, wherein the signal peptide is a fragment of the signal peptides of any of embodiments 1 to 14.

[17] The nucleic acid construct according to any of the preceding embodiments, wherein polynucleotide encoding the polypeptide having mannanase activity has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:3 or SEQ ID NO:5; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:3 or SEQ ID NO:5.

[18] The nucleic acid construct according to any of the preceding embodiments, wherein the mannanase activity of the polypeptide having mannanase activity is measured by the method of the examples listed as ’’Mannanase activity assay”.

[19] The nucleic acid construct according to any of the preceding embodiments, wherein the polypeptide having mannanase activity is a bacterial polypeptide.

[20] The nucleic acid construct according to any one of embodiments 18 to 19, wherein the polypeptide having mannanase activity is obtained from Bacillus circulans group 6.

[21] The nucleic acid construct according to any one of embodiments 18 to 20, wherein the polypeptide having mannanase activity is obtained from Paenibacillus illinoisensis.

[22] The nucleic acid construct according to any preceding embodiments, wherein the polypeptide having mannanase activity has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:6.

[23] The nucleic acid construct according to embodiment 22, wherein the polypeptide having mannanase activity comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:6.

[24] The nucleic acid construct according to any of the preceding embodiments, wherein the N- and/or C-terminal end of the polypeptide having mannanase activity has been extended by addition of one or more amino acids.

[25] The nucleic acid construct according to any of embodiments 1 to 24, wherein the signal peptide is a fragment of the signal peptides of any of embodiments 1 to 24.

[26] The nucleic acid construct according to any preceding embodiment, wherein a third polynucleotide comprising the first and the second polynucleotide has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the polypeptide coding sequence of SEQ ID NO:9 or SEQ ID NO:11 ; most preferably the third polynucleotide comprises, consists essentially of, or consists of the polypeptide coding sequence of SEQ ID NO:9 or SEQ ID NO:11 .

[27] The nucleic acid construct according to any preceding embodiments, wherein a fusion polypeptide comprising the signal peptide and the polypeptide having mannanase activity has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide of SEQ ID NO: 10 or SEQ ID NO: 12, most preferably the fusion polypeptide comprises, consists essentially of, or consists of SEQ ID NQ:10 or SEQ ID NO:12.

[28] An expression vector comprising a nucleic acid construct according to any of embodiments 1 to 35.

[29] A bacterial host cell comprising in it's genome: a) a nucleic acid construct according to any of embodiments 1 to 27; and/or b) an expression vector according to embodiment 28.

[30] The bacterial host cell of embodiment 29, wherein the bacterial host cell is a Gram-positive host cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[31] The bacterial host cell of any of embodiments 29 to 30, wherein the bacterial host cell is a Bacillus cell.

[32] The bacterial host cell of any of embodiments 29 to 31 , wherein the bacterial host cell is a Bacillus cell selected from the group consisting of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cell.

[33] The bacterial host cell of any of embodiments 29 to 32, wherein the bacterial host cell is a Bacillus licheniformis cell.

[34] The bacterial host cell of any of embodiments 29 to 33, wherein the host cell comprises at least two copies of the nucleic acid construct and/or the expression vector, such as two copies, three copies, four copies or more than four copies.

[35] The bacterial host cell according to any preceding embodiments, wherein the host cell shows increased expression of the polypeptide having mannanase activity, relative to a parental host cell lacking the first polynucleotide and being otherwise isogenic to the host cell, when cultivated under the same conditions.

[36] The bacterial host cell according to embodiment 35, wherein the host cell and the parental host cell are cultivated in a fed-batch mode.

[37] The bacterial host cell according to any of embodiments 35 to 36, wherein the host cell and the parental host cell are cultivated at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, or at least 168 hours.

[38] The bacterial host cell according to any of embodiments 35 to 37, wherein the host cell has at least 1.1 -fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6- fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1 -fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.1 -fold, at least 3.2-fold, or at least 3.3- fold increased expression of the polypeptide having mannanase activity, relative to the parental host cell lacking the first polynucleotide, when cultivated under the same conditions.

[39] The bacterial host cell according to any of embedments 35 to 38, wherein the parental host cell comprises a fourth polynucleotide encoding an control signal peptide, said fourth polynucleotide being operably linked to the second polynucleotide encoding the polypeptide having mannanase activity, preferably the control signal peptide is a aprH signal peptide.

[40] The bacterial host cell according to embodiment 39, wherein the fourth polynucleotide is encoding the aprH signal peptide and has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:7 or SEQ ID NO:26; most preferably the fourth polynucleotide comprises, consists essentially of, or consists of the polypeptide coding sequence of SEQ ID NO:7 or SEQ ID NO:26.

[41] The bacterial host cell according to any of embodiments 39 to 40, wherein the aprH signal peptide has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:8.

[42] The bacterial host cell according to any of embodiments 35 to 41 , wherein the first polynucleotide is endogenous to the host cell.

[43] The bacterial host cell according to any of embodiments 35 to 42, wherein the polypeptide having mannanase activity is heterologous to the host cell.

[44] A method of producing a polypeptide having mannanase activity, the method comprising: a) cultivating a bacterial host cell according to any of embodiments 35 to 43 under conditions conducive for production of the polypeptide having mannanase activity; and optionally b) recovering the polypeptide having mannanase activity.