Bacillus Licheniformis Host Cell

The present invention relates to Bacillus licheniformis host cells producing heterologous polypeptide of interest, wherein at least one gene in the lan gene cluster inactivated and methods for producing the polypeptide of interest by cultivating said cells.

REFERENCE TO SEQUENCE LISTING

FIELD OF THE INVENTION

The present invention relates toBacillus licheniformishost cells producing a heterologous polypeptide of interest, wherein at least one gene in the lan gene cluster is inactivated and methods for producing the polypeptide of interest by cultivating said cells.

BACKGROUND OF THE INVENTION

The complete genome sequences of severalBacillusspecies are in the public domain, see, e.g., Kunst et al., 1997,The complete genome sequence of the Gram-positive bacterium Bacillus subtilis,Nature 390, 249-256; Rey et al, 2004,Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species,Genome Biol. 2004; 5(10):R77; and Veith et al, 2004, The complete genome sequence ofBacillus licheniformisDSM13, an organism with great industrial potential, J. Mol. Microbiol. Biotechnol. 7 (4), 204-211.

One of the preferred workhorses in the recombinant production of polypeptides, especially of enzymes, is the prokaryotic bacteriumBacillus licheniformis.The industrial production of polypeptides is a competitive business, where even small incremental improvements in yield are highly desirable and where intense research activities are directed towards achieving this goal.

SUMMARY OF THE INVENTION

In the examples provided herein it was demonstrated that inactivation of a gene in the putative lantibiotic biosynthesis gene cluster or inactivation of the entire lan cluster in aB. licheniformishost cell surprisingly resulted in a significant increase in the yield of a heterologous polypeptide enzyme of interest produced by said cell.

Accordingly, in a first aspect the invention provides aBacillus licheniformishost cell producing a heterologous polypeptide of interest, wherein at least one gene in the lan gene cluster is inactivated.

In a second aspect, the invention provides a method for producing a polypeptide of interest, said method comprising a) cultivating aBacillus licheniformishost cell as defined in any of the previous claims in a medium and under conditions conducive for the production of said polypeptide; and optionally b) recovering said polypeptide.

DEFINITIONS

Expression: The term “expression” includes 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: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

Isolated: The term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

DETAILED DESCRIPTION OF THE INVENTION

Host Cells

In a first aspect, the invention relates to aBacillus licheniformishost cell producing a heterologous polypeptide of interest, wherein at least one gene in the lan gene cluster is inactivated.

In a preferred embodiment, the polypeptide of interest is expressed with or without a secretion signal peptide; more preferably the polypeptide of interest is secreted, non-secreted or intracellular. Expression inBacillusof natively non-secreted polypeptides and natively secreted enzymes without a secretion signal peptide is disclosed, for example, in WO 2014/206829 or WO 2014/202793.

Stable expression of heterologous polypeptides inB. licheniformishost cells may be achieved through the integration of one or more copies of an expression construct in the chromosome of the host cell, for example, by transiently expressed phage-integrase mediated site-specific simultaneous integration in several loci as disclosed in WO 2006/042548.

Accordingly, in a preferred embodiment of the invention, the heterologous polypeptide of interest is encoded by an exogenous polynucleotide integrated into the chromosome of the host cell in at least one copy; preferably in at least two copies; more preferably in at least three copies; still more preferably in at least four copies; yet more preferably in at least five copies and most preferably in at least six copies.

There are many well-known ways to inactivate a gene, for example by mutating the gene through the introduction of a non-sense mutation or a frameshift mutation, or by partial or full deletion of the open reading frame, or by manipulation of one or more control sequence.

Accordingly, in a preferred embodiment of the invention, the at least one gene in the lan gene cluster is inactivated by a non-sense mutation in said at least one gene, a partial deletion of said at least one gene or open reading frame or a full deletion of said at least one gene or open reading frame.

It is well-known thatBacillus licheniformisspecies are very similar, so it is expected that other strains of that species will probably also have the lan gene cluster in their chromosome and it is expected that inactivation of one or more lan gene will have yield benefits as was demonstrated in theBacillus licheniformisspecies employed in the examples herein. Even though the differentBacillus licheniformisspecies are similar, the DNA sequences of the lan genes may differ to some extent due to genetic variation or silent mutations.

Accordingly, in a preferred embodiment of the invention, the at least one gene in the lan gene cluster is selected from the group consisting of a lanI gene having a nucleotide sequence at least 70% identical to the lanI shown in SEQ ID NO:1, a lanH gene having a nucleotide sequence at least 70% identical to the lanH shown in SEQ ID NO:2, a lanE gene having a nucleotide sequence at least 70% identical to the lanE shown in SEQ ID NO:3, a lanG gene having a nucleotide sequence at least 70% identical to the lanG shown in SEQ ID NO:4, a lanF gene having a nucleotide sequence at least 70% identical to the lanF shown in SEQ ID NO:5, a lanY gene having a nucleotide sequence at least 70% identical to the lanY shown in SEQ ID NO:6, a lanR gene having a nucleotide sequence at least 70% identical to the lanR shown in SEQ ID NO:7, a lanX gene having a nucleotide sequence at least 70% identical to the lanX shown in SEQ ID NO:8, a lanP gene having a nucleotide sequence at least 70% identical to the lanP shown in SEQ ID NO:9, a lanT gene having a nucleotide sequence at least 70% identical to the lanT shown in SEQ ID NO:10, a lanM2 gene having a nucleotide sequence at least 70% identical to the lanM2 shown in SEQ ID NO:11, a lanA2 gene having a nucleotide sequence at least 70% identical to the lanA2 shown in SEQ ID NO:12, a lanA1 gene having a nucleotide sequence at least 70% identical to the lanA1 shown in SEQ ID NO:13 and a lanM1 gene having a nucleotide sequence at least 70% identical to the lanM1 shown in SEQ ID NO:14.

It is preferred that the at least one gene in the lan gene cluster is selected from the group consisting of a lanI gene having the nucleotide sequence shown in SEQ ID NO:1, a lanH gene having the nucleotide sequence shown in SEQ ID NO:2, a lanE gene having the nucleotide sequence shown in SEQ ID NO:3, a lanG gene having the nucleotide sequence shown in SEQ ID NO:4, a lanF gene having the nucleotide sequence shown in SEQ ID NO:5, a lanY gene having the nucleotide sequence shown in SEQ ID NO:6, a lanR gene having the nucleotide sequence shown in SEQ ID NO:7, a lanX gene having the nucleotide sequence shown in SEQ ID NO:8, a lanP gene having the nucleotide sequence shown in SEQ ID NO:9, a lanT gene having the nucleotide sequence shown in SEQ ID NO:10, a lanM2 gene having the nucleotide sequence shown in SEQ ID NO:11, a lanA2 gene having the nucleotide sequence shown in SEQ ID NO:12, a lanA1 gene having the nucleotide sequence shown in SEQ ID NO:13 and a lanM1 gene having the nucleotide sequence shown in SEQ ID NO:14.

In a preferred embodiment of the invention, two or more genes in the lan gene cluster are inactivated; preferably three or more genes in the lan gene cluster are inactivated; even more preferably four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen or more genes in the lan gene cluster are inactivated.

Preferably, the genes in the lan gene cluster are inactivated by a non-sense mutation, a partial deletion or a full deletion of said genes, or by a combination thereof. It is preferred that the entire lan gene cluster is deleted.

Methods of Production

The polypeptide may be detected using methods known in the art that are specific for the polypeptides. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered.

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

In a second aspect, the invention relates to a method for producing a polypeptide of interest, said method comprising:a) cultivating aBacillus licheniformishost cell as defined in the first aspect in a medium and under conditions conducive for the production of said polypeptide; and optionallyb) recovering said polypeptide.

Sources of Polypeptides

The heterologous polypeptide of interest to be produced according to the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

The present invention also relates to the expression of heterologous polynucleotides encoding the heterologous polypeptide of interest.

The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990,PCR: A Guide to Methods and Application,Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.

Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for synthesizing polypeptides substantially similar to the polypeptide. The term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. The variants may be constructed, e.g., by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, 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 Purification2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid expression constructs comprising a polynucleotide of the present invention 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 to produce the heterologous polypeptide according to the invention.

The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

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.

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 are obtained from the genes forBacillus clausfialkaline protease (aprH),Bacillus licheniformisalpha-amylase (amyL), andEscherichia coliribosomal RNA (rrnB).

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensiscryIIIA gene (WO 94/25612) and aBacillus subtilisSP82 gene (Hue et al., 1995,Journal of Bacteriology177: 3465-3471).

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.

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.

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.

Expression Vectors

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication inE. coli,and pUB110, pE194, pTA1060, and pAMR1 permitting replication inBacillus.

EXAMPLES

Materials and Methods

Media

To screen for protease phenotypes agar plates were supplemented with 1% skim milk to allow halos to form around the colonies that produces protease.

To screen for amylase phenotypes agar plates were supplemented with 1% starch to allow halos to form around the colonies that produces amylase.

Spizizen II medium consists of Spizizen I medium supplemented with 0.5 mM CaCl2, and 2.5 mM MgCl2.

Strains

E. coliTG1. Commercial strain used for cloning purposes (Stratagene).B. subtilisPP3724. This strain is donor strain for conjugation ofB. licheniformisas described in several patents (U.S. Pat. Nos. 5,695,976A, 5,733,753A, 5,843,720A, 5,882,888A, WO2006042548A1).B. licheniformisSJ1904: This strain is aB. licheniformisstrain described in WO 08066931 A2. The gene encoding the alkaline protease (aprL) is inactivated.B. subtilisBKQ1707: This strain is PP3724 with pBKQ1697 for deletion of lanA1.B. subtilisBKQ1754: This strain is PP3724 with pBKQ1751 for deletion of lan gene cluster.B. licheniformisSJ12713: This strain is an alkaline protease AprH producing strain.B. licheniformisBKQ1944: This strain corresponds to SJ12713 with deleted lanA1.B. licheniformisBKQ1946: This strain corresponds to SJ12713 with deleted lan gene cluster.

Primers

Plasmids

pSJ3372: pUC derived plasmid with chloramphenicol marker from pC194 (U.S. Pat. No. 5,882,888)pC194: Plasmid isolated fromStaphylococcus aureus(Horinouchi and Weisblum, 1982, Nucleotide Sequence and Functional Map of pE194, a Plasmid That Specifies Inducible Resistance to Macrolide, Lincosamide, and Streptogramin Type B Antibiotics, J Bacteriol 150(2):804-814).pPP3932 (SEQ ID NO:35): Temperature sensitive plasmid to be used for chromosomal replacement, mutation or deletion ofB. licheniformis.pBKQ1697 (SEQ ID NO:36): Plasmid pPP3932 with insertion of flanking regions of lanA1 fromB. licheniformisSJ1904 in MluI and SacI site. The plasmid can be used for deletion of lanA1 inB. lichenformisSJ1904 derivatives.pBKQ1699 (SEQ ID NO:37): Plasmid pPP3932 with insertion of flanking regions of lan gene cluster fromB. licheniformisSJ1904 in MluI site.pBKQ1751 (SEQ ID NO:38): Plasmid pBKQ1699 with insertion of res-cat-res region from pSJ3372 in between flanking regions of the lan gene cluster. The plasmid can be used for deletion of the entire lan gene cluster inB. lichenformisSJ1904 derivatives.

Molecular Biological Methods

DNA manipulations and transformations were performed by standard molecular biology methods as described in: Sambrook et al. (1989): Molecular cloning: A laboratory manual. Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. Ausubel et al. (eds) (1995): Current protocols in Molecular Biology. John Wiley and Sons. Harwood and Cutting (eds) (1990): Molecular Biological Methods forBacillus.John Wiley and Sons.

Enzymes for DNA manipulation were obtained from New England Biolabs, Inc. and used essentially as recommended by the supplier.

Competent cells and transformation ofB. subtiliswas obtained as described in Yasbin et al. (1975): Transformation and transfection in lysogenic strains ofBacillus subtilis:evidence for selective induction of prophage in competent cells.J. Bacteriol.121, 296-304.

Standard Cultivation Procedure

All growth media were sterilized by methods known in the art. Unless otherwise described, tap water was used. The ingredient concentrations referred to in the below recipes are before any inoculation.

Inoculum steps: First the strain was grown on SSB-4 agar slants 1 day at 37° C. The agar was then washed with M-9 buffer, and the optical density (OD) at 650 nm of the resulting cell suspension was measured. The inoculum shake flask (PRK-50) was inoculated with an inoculum of OD (650 nm)×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 (fermentation tank) was started by inoculating the main fermentor with the growing culture from the shake flask. The inoculated volume was 11% of the make-up medium (80 ml for 720 ml make-up 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.

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.

AB. licheniformishost strain expressing six site-specific chromosomally integrated copies of an AprH expression construct was constructed using standard methods, for example as described in U.S. Pat. Nos. 5,695,976, 5,733,753, 5,843,720, 5,882,888 and/or WO2006042548. The expression construct encoded the aprL signal peptide fromBacillus licheniformisin translational fusion with the aprH pro-peptide and mature peptide fromBacillus clausii(shown in SEQ ID NO:39) The recipient host was aB. licheniformisSJ1904 derivative (WO2008066931). The resulting six-copy AprH expression host was denoted SJ12713.

Plasmid pBKQ1697 was designed to delete the structural lanA1 gene within theB. licheniformislan gene cluster.

Colony PCR was performed onB. licheniformisSJ1904. A first 1.1 kb fragment of theB. licheniformisSJ1904 chromosome, containing the upstream region of lanA1, was amplified by PCR using primers pr535 and pr536 by standard PCR. A cleavage site for restriction enzyme MluI was incorporated into primer pr535. A cleavage site for restriction enzyme BamHI was incorporated into primer pr536.

A second 1.1 kb fragment of theB. licheniformisSJ1904 chromosome, containing the flanking region immediate downstream of lanA1, was PCR amplified using primers pr537 and pr538. A cleavage site for the BamHI restriction enzyme (bold) was incorporated into primer pr537. A cleavage site for the SacII restriction enzyme (bold) was incorporated into primer pr538.

The resulting two DNA fragments were amplified by PCR using the PHUSION HOT START® II DNA polymerase (Thermo Fisher Scientific). The PCR amplification reaction mixture containedB. licheniformisSJ1904 genomic DNA (10 μl template solution (colony solution cooked at 99 C for 10 minutes in H2O), 1 μl of sense primer (20 pmol/μl), 1 μl of anti-sense primer (20 pmol/μl), 10 μl of 5× PCR buffer, 8 μl of dNTP mix (5 mM each), 18.5 μl H2O, and 0.5 μl (2 U/μl) DNA polymerase mix. An Eppendorf Mastercycler thermocycler was used to amplify the fragment with the following settings: One cycle at 94° C. for 2 minutes; 25 cycles each at 94° C. for 30 seconds, 54° C. for 45 seconds, 72° C. for 60 seconds; one cycle at 72° C. for 5 minutes; and 10° C. hold. The PCR products were purified from a 1% agarose SYBR® Safe DNA gel stain gel (Life Technologies) with 0.5× TBE buffer using the Qiagen QIAquick Gel Extraction Kit (Qiagen, Inc., Valencia, Calif.) according to the manufacturer's instructions.

The two purified PCR products were digested with restriction enzyme BamHI as follows: 45 μl purified PCR, 5 μl NEB2 buffer, 1 μl BamHI and incubated for 1 hour at 37° C. The digested DNA was subsequently purified using Qiagen PCR purification kit according to manufacturer's instructions. The two PCR products were mixed and ligated as follows: 4.25 μl of each digested PCR product, 1 μl 10× Ligation buffer and 0.5 μl T4 DNA ligase. Ligation mixture was incubated at room temperature for 1 hour.

A subsequent PCR amplification using the ligated PCR fragments as template DNA was performed to create a single fragment using the PHUSION HOT START® II DNA polymerase (Thermo Fisher Scientific) as follows: The PCR amplification reaction mixture contained 10 μl of a 100 times diluted ligation mixture described above, 1 μl of primer pr535 (20 pmol/μl), 1 μl of primer pr538 (20 pmol/μl), 10 μl of 5× PCR buffer, 8 μl of dNTP mix (5 mM each), 18.5 μl H2O, and 0.5 μl (2 U/μl) PHUSION HOT START® II DNA polymerase (Thermo Fisher Scientific). An Eppendorf Mastercycler thermocycler was used to amplify the fragment with the following settings: One cycle at 94° C. for 2 minutes; 25 cycles each at 94° C. for 30 seconds, 54° C. for 45 seconds, 72° C. for 3 minutes; one cycle at 72° C. for 5 minutes; and 10° C. hold, resulting in a 2.2 kb PCR fragment.

The resulting PCR product (lig-PCR lanA1 flanks; SEQ ID NO:40) containing the flanking upstream and downstream region of lanA1 ligated in the BamHI site was run on a 1% agarose TBE gel and purified on Qiagen QIAquick Gel Extraction Kit according to manufacturer's instructions. The purified PCR product was subsequently digested with MluI and SacII as follows: 45 μl purified PCR product, 5 μl NEB2 buffer, 1 μl MluI, and 1 μl SacII and incubated at 37° C., resulting in a 2.2 kb fragment. In another tube, plasmid vector pPP3932 was digested with MluI and SacII according to manufacturer's instructions, resulting in a 5.7 kb fragment.

The digested PCR product and plasmid were subsequently run on a 1% agarose gel by electrophoresis using TBE buffer followed by purification using the Qiagen QIAquick Gel Extraction Kit (Qiagen, Inc.) according to manufacturer's instructions. The purified DNA fragments were then ligated using T4 DNA ligase as follows: 1 μl pPP3932 fragment, 1 μl PCR product, 6.5 μl H2O, 1 μl ×10 T4 DNA ligase buffer, 0.5 μl T4 DNA ligase. The ligase reaction was incubated at room temperature for 2 hours. The 10 μl aliquot of the ligation was used to transformE. coliTG1 cells according to the manufacturer's instructions.

Plasmid DNA was prepared fromE. colitransformants and confirmed by restriction analysis and subsequent sequencing with primers: pr535, pr536, pr537, pr538, pr539, pr540, pr541, and pr542.

The verified plasmid was then used to transform donor strainB. subtilisPP3724 as described previously in Materials and Methods, resulting inB. subtilisBKQ1707. Donor strainB. subtilisBKQ1707 was subsequently used for conjugation ofB. licheniformisSJ1904 derivatives according to method described above in order to introduce the temperature sensitive plasmid to the relevant strains.

Plasmid pBKQ1751 was designed to delete the entire lan gene cluster (SEQ ID NO:41) inB. licheniformis.Colony PCR was performed onB. licheniformisSJ1904. A 1.05 kb fragment of theB. licheniformisSJ1904 chromosome, containing the upstream region of the lan gene cluster, was amplified by PCR using primers pr547 and pr548 by standard PCR. A cleavage site for restriction enzyme MluI was incorporated into primer pr547. A cleavage site for restriction enzyme BamHI was incorporated into primer pr548.

A second 1.05 kb fragment of theB. licheniformisSJ1904 chromosome, containing the flanking region immediate downstream to the lan gene cluster, was amplified by PCR by standard PCR technique using primers pr549 and pr550. A cleavage site for the BamHI restriction enzyme (bold) was incorporated into primer pr549. A cleavage site for the MluI restriction enzyme (bold) was incorporated into primer pr550.

An Eppendorf Mastercycler thermocycler was used to amplify the fragment with the following settings: One cycle at 94° C. for 2 minutes; 25 cycles each at 94° C. for 30 seconds, 54° C. for 45 seconds, 72° C. for 60 seconds; one cycle at 72° C. for 5 minutes; and 10° C. hold. The PCR products were purified from a 1% agarose SYBR® Safe DNA gel stain gel (Life Technologies) with 0.5× TBE buffer using the Qiagen QIAquick Gel Extraction Kit (Qiagen, Inc., Valencia, Calif.) according to the manufacturer's instructions.

The two purified PCR products were digested with restriction enzyme BamHI as follows: 45 μl purified PCR, 5 μl NEB2 buffer, 1 μl BamHI and incubated for 1 hour at 37 C. The digested DNA was subsequently purified using Qiagen PCR purification kit according to manufacturer's instructions.

The two PCR products were mixed and ligated as follows: 4.25 μl of each digested PCR product, 1 μl 10× Ligation buffer and 0.5 μl T4 DNA ligase. Ligation mixture was incubated at room temperature for 1 hour. A subsequent PCR amplification using the ligated PCR fragments as template DNA was performed to create a single fragment using the PHUSION HOT START® II DNA polymerase (Thermo Fisher Scientific) as follows: The PCR amplification reaction mixture contained 10 μl of a 100 times diluted ligation mixture described above, 1 μl of primer pr535 (20 pmol/μl), 1 μl of primer pr538 (20 pmol/μl), 10 μl of 5× PCR buffer, 8 μl of dNTP mix (5 mM each), 18.5 μl H2O, and 0.5 μl (2 U/μl) PHUSION HOT START® II DNA polymerase (Thermo Fisher Scientific).

An Eppendorf Mastercycler thermocycler was used to amplify the fragment with the following settings: One cycle at 94° C. for 2 minutes; 25 cycles each at 94° C. for 30 seconds, 54° C. for 45 seconds, 72° C. for 3 minutes; one cycle at 72° C. for 5 minutes; and 10° C. hold, resulting in a 2.1 kb PCR fragment.

The resulting PCR product (lig-PCR lan gene cluster flanks; SEQ ID NO:42) containing the flanking upstream and downstream region of the entire lan gene cluster was run on a 1% agarose TBE gel and purified on Qiagen QIAquick Gel Extraction Kit according to manufacturer's instructions. The purified PCR product was subsequently digested with MluI as follows: 45 μl purified PCR product, 5 μl NEB3 buffer and 1 μl MluI and incubated at 37° C., resulting in a 2.1 kb fragment.

In another tube, plasmid vector pPP3932 was digested with MluI and treated with Calf Intestine Phosphatase according to manufacturer's instructions, resulting in a 5.8 kb fragment.

The digested PCR product and plasmid were subsequently run on a 1% agarose gel by electrophoresis using TBE buffer followed by purification using the Qiagen QIAquick Gel Extraction Kit (Qiagen, Inc.) according to manufacturer's instructions.

The purified DNA fragments were then ligated using T4 DNA ligase as follows: 2 μl pPP3932 fragment, 0.5 μl PCR product, 5 μl H2O, 1 μl ×10 T4 DNA ligase buffer, 0.5 μl T4 DNA ligase. The ligase reaction was incubated at room temperature for 2 hours. The 10 μl aliquot of the ligation was used to transform 50 μlE. coliTG1 cells according to the manufacturer's instructions. Plasmid DNA was prepared fromE. colitransformants and confirmed by restriction analysis and subsequent sequencing with primer pr547, pr548, pr549, pr550, pr551, pr552, pr553 and pr554.

In order to enable deletion of the entire lan gene cluster (approximately 15.2 kb), a chloramphenicol resistance gene surrounded by resolvase recognizable regions (res-sites) was inserted between the upstream and downstream flanking regions of the lan gene cluster present in pBKQ1699 as follows: Plasmid pSJ3372, which contains a res-cat-res region (see U.S. Pat. No. 5,882,888) surrounded by a BclI and a BamHI site, was digested with BclI and BamHI according to manufacturer's instructions, resulting in a 1.2 kb fragment containing the res-cat-res region.

Plasmid pBKQ1699 was digested with BamHI and treated with Calf Intestine Phosphatase by standard technique, resulting in a 7.9 kb fragment. The digestion mixtures were run on 1% agarose gel by electrophoresis using TBE buffer followed by purification using the Qiagen QIAquick Gel Extraction Kit (Qiagen, Inc.) according to manufacturer's instructions.

The purified DNA fragments were then ligated using T4 DNA ligase as follows: 3 μl pBKQ1699 fragment (plasmid vector), 0.5 μl pSJ3372 fragment (res-cat-res), 5 μl H2O, 1 μl ×10 T4 DNA ligase buffer, 0.5 μl T4 DNA ligase. The ligase reaction was incubated overnight at 16° C. The 10 μl aliquot of the ligation was used to transform 50 μlE. coliTG1 cells according to the manufacturer's instructions. Plasmid DNA was prepared fromE. colitransformants and confirmed by restriction analysis, resulting in pBKQ1751 in which the res-cat-res region was inserted in between the flanking regions of the lan gene cluster in pBKQ1699.

The verified plasmid pBKQ1751 was then used to transform donor strainB. subtilisPP3724 as described previously in Materials and Methods, resultingB. subtilisBKQ1754. Donor strainB. subtilisBKQ1754 was subsequently used for conjugation ofB. licheniformisSJ1904 derivatives according to method described above in order to introduce the temperature sensitive plasmid to the relevant strains.

Donor strainB. subtilisBKQ1707 was used for conjugation ofB. licheniformisrecipients as previously described (U.S. Pat. No. 5,843,720) in order to introduce the temperature sensitive plasmid pBKQ1697 to the relevant strains.

B. licheniformisconjugants containing plasmid pBKQ1697 were then grown on LB PGS selective medium at 50° C. to force integration of the vector. Selection of strains with chromosomal integration of the plasmid was performed based on their ability to grow on LB PGS+5 microgram/ml of erythromycin at 50° C. These strains were then grown without selection on LB PGS plates at 34° C. to allow excision of the integrated plasmid.

A streak of culture was inoculated in 10 ml LB medium and incubated for 6 hours at 34° C. Dilution series were made in LB medium and the diluted cell cultures were plated on LB PGS plates and incubated overnight at 37° C. Next day, replica plating was performed on LB PGS and LB PGS+5 microgram/ml of erythromycin. The plates were incubated overnight at 34° C.

Next day, erythromycin sensitive colonies were identified. Colony PCR on a series of erythromycin sensitive colonies was performed with primer pr601 and primer pr602 in order to identify strains in which lanA1 has been deleted.

Using temperature sensitive plasmid pBKQ1697 for deletion of lanA1 inB. licheniformisSJ1904 derivatives by homologeous recombination, the following strain was isolated:B. licheniformisBKQ1944 (AprH producing).

Example 5: Deletion of the Entire lan Gene Cluster inB. licheniformis

Donor strainB. subtilisBKQ1754 was used for conjugation ofB. licheniformisrecipients as previously described (U.S. Pat. No. 5,843,720) in order to introduce the temperature sensitive plasmid pBKQ1751.B. licheniformisconjugants containing plasmid pBKQ1751 were then grown on LB PGS plates supplemented with 6 microgram/ml of chloramphenicol and incubated at 50° C. to force integration of the plasmid.

Strains with chromosomal integrated plasmids were selected based on their ability to grow on LB PGS+6 microgram/ml of chloramphenicol at 50° C. The selected strains were then re-streaked on LB PGS plates supplemented with 6 microgram/ml of chloramphenicol and incubated at 34° C. to allow excision of the integrated plasmid.

Next day, a streak of culture was inoculated in 10 ml LB medium supplemented with 6 microgram/ml of chloramphenicol and incubated for 6 hours at 34° C. Dilution series were made in LB medium and the diluted cell cultures were plated on LB PGS+6 microgram/ml chloramphenicol and incubated overnight at 37° C.

Next day, replica plating was performed on LB PGS+6 microgram/ml chloramphenicol and LB PGS+5 microgram/ml erythromycin. The plates were incubated overnight at 34° C. Next day, erythromycin sensitive colonies were identified. Colony PCR on a series of erythromycin sensitive colonies was performed with primer pr555 and primer pr556 in order to identify strains in which the entire lan gene cluster (approximately 15.2 kb has been deleted and replaced by a res-cat-res region.

Using temperature sensitive plasmid pBKQ751 for deletion of the entire lan gene cluster inB. licheniformisSJ1904 derivatives by homologeous recombination, the following strain was isolated:B. licheniformisBKQ1946 (AprH producing).

Example 6. AprH inB. licheniformisStrains with lanA1 or lan Gene Cluster Deleted

Four independent cultures of each of AprH-producingB. licheniformisSJ12713 (reference),B. licheniformisBKQ1944 (ΔlanA1) andB. licheniformisBKQ1946 (Δlan gene cluster) were cultivated. Samples were regularly taken once a day for a period of five days. The titer and yield of AprH were then measured. After day 5, significantly increased AprH titers and yields were found in both the strain with a deleted lanA1 and in the strain with a deleted lan gene cluster, when compared with the reference strainB. licheniformisSJ12713. The results are listed in table 2 below. The data clearly show that deletion of lanA1 or the entire lan gene cluster results in significantly increased yields of AprH when compared to the control reference strain.

A similar expression study of the AmyL amylase in a 4 gene copy lan gene cluster deleted host strain was carried out which demonstrated yield improvements in a lan gene cluster deleted host strain of about 2% compared with the control reference strain (data not shown).