DE102017215015A1 - Method for improved expression of enzymes - Google Patents

Abstract

The invention relates to a recombinant bacterium, the use of a bacterium according to the invention for the expression of a protein, a method for the expression of a protein, a bacterial culture and a vector.

Description

The invention is in the field of biotechnology, in particular microbial protein synthesis. The invention particularly relates to a recombinant bacterium, the use of a bacterium according to the invention for the expression of a protein, a method for the expression of a protein, a bacterial culture and a vector.

For the production of recyclables microorganisms can be used. Recyclable materials are, for example, low molecular weight compounds, such as food supplements or pharmaceutically active compounds, or proteins, for which, due to their diversity, in turn, there is a large technical field of application. In the first case, the metabolic properties of the microorganisms in question are utilized and / or modified for the production of valuable substances; in the second case, preferably microorganisms are used which express the genes of the proteins of interest.

For large-scale, biotechnological production of the relevant microorganisms are cultured in fermenters, which are designed according to the metabolic properties of the microorganisms. During culturing, the microorganisms metabolize the substrate provided and form the desired product which, after completion of the fermentation, is usually separated from the production organisms and purified and / or concentrated from the fermenter slurry and / or the fermentation medium. In the fermentative production of proteins, complex protein rich raw materials are typically used as a substrate besides a carbon source (typically glucose). The protein production thus corresponds to a biotransformation of substrate protein to the target protein. This requires complete hydrolysis of the substrate protein into the individual amino acids, which are then available for biosynthesis of the target protein.

For the fermentation of microorganisms, therefore, there is a rich state of the art, ranging from the optimization of the strains concerned, for example, in terms of rate of formation and nutrient utilization, on the technical design of the fermenter to the recovery of valuable materials from the microorganisms and / or the fermentation medium ,

The use of bacteria in microbial fermentations is basically desirable. Bacteria are characterized by short generation times and low demands on cultivation conditions. As a result, inexpensive cultivation methods or production methods can be established. In addition, the expert has a wealth of experience in bacteria in fermentation technology. Grampositive bacteria are preferably used because they secrete the protein to be produced (target protein) into the surrounding medium.

Usually, the highest possible product yields in microbial fermentation are desirable. For example, the international patent application discloses WO 91/02792 the improved fermentative production of an alkaline protease from Bacillus lentus in an optimized Bacillus licheniformis strain under the control of gene regulatory sequences from Bacillus licheniformis, in particular the Bacillus licheniformis promoter.

To Bacillus licheniformis alternative production organisms, with which comparably high or even improved product yields can be achieved, are not available in the state of the art in a satisfactory manner. There continues to be a high demand for microbial fermentation processes which enable a high product yield.

The object of the present invention is to achieve a high product yield, in particular of a protein, in a microbial fermentation. The invention relates to a recombinant bacterium comprising a nucleic acid sequence which comprises a) a first nucleic acid sequence comprising a sequence according to SEQ ID NO: 1 or a sequence which is at least 80% identical to the nucleic acid sequence given in SEQ ID NO: 1; and b) a second nucleic acid sequence encoding a (target) protein.

In a further aspect, the present invention relates to the use of the recombinant bacterium according to the invention for the expression of a (target) protein.

Furthermore, the invention relates to a method for the expression of a (target) protein, wherein in at least one method step, a recombinant bacterium according to the invention is used.

Furthermore, the invention relates to a bacterial culture comprising a) a recombinant bacterium according to any one of claims 1-5; and b) culture medium.

A further aspect of the invention relates to a vector comprising a) a first nucleic acid sequence comprising a sequence according to SEQ ID NO: 1 or a sequence which is at least 80% identical to the nucleic acid sequence given in SEQ ID NO: 1; and b) a second nucleic acid sequence encoding a (target) protein. The vector may be a plasmid. The recombinant bacteria described herein may be the indicated nucleic acid sequence in the form of the vector described herein, in particular plasmid.

In preferred embodiments, the recombinant bacterium is Bacillus licheniformis or Bacillus aeolius, for example Bacillus aeolius DSM 15084. In further preferred embodiments, the first nucleic acid sequence comprises or consists of one of a sequence of SEQ ID NO: 2 or a sequence corresponding to that shown in SEQ ID NO : 2 specified nucleic acid sequence is at least 80% identical.

In preferred embodiments, the recombinant bacterium of the invention comprises a vector / plasmid comprising the first and second nucleic acid sequences.

In preferred embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked.

In preferred embodiments, the second nucleic acid sequence encodes an enzyme, more preferably a protease, and even more preferably a protease according to SEQ ID NO: 3.

In preferred embodiments of the bacterial culture according to the invention, the culture medium is full medium with a nitrogen source.

The terms "culture medium" and "nutrient medium" as used synonymously herein refer to liquid and solid media (nutrient media) that are used to grow microorganisms. The culture medium contains all the nutrients required for the growth of certain microorganisms. The present invention encompasses chemically defined culture media containing exact amounts of highly purified chemicals and undefined or complex culture media consisting of hydrolyzates of casein, beef, soybeans, yeast cells, and the like. Since the nutrient requirements of each microorganism are very different, the culture medium must contain all the essential nutrients in the right form and in the right amounts. These are generally known to the person skilled in the art. In preferred embodiments of the invention, the culture medium comprises proteins which serve as nitrogen source. For the purposes of the present invention, a "complete medium" is a nutrient medium (nutrient medium) which contains not only the essential but also many or all compounds which the organism itself can synthesize.

In preferred embodiments of the vector according to the invention, the first nucleic acid sequence and the second nucleic acid sequence are functionally coupled. In further preferred embodiments of the vector according to the invention, the second nucleic acid sequence encodes an enzyme, preferably a protease and more preferably a protease according to SEQ ID NO: 3. In further likewise preferred embodiments of the vector according to the invention, the first nucleic acid sequence comprises or consists of a sequence according to SEQ ID NO: 2 or a sequence which is at least 80% identical to the nucleic acid sequence given in SEQ ID NO: 2.

In preferred embodiments according to the invention, a process according to the invention is a fermentation process.

Surprisingly, it has been found that the use of a bacterium of the species Bacillus licheniformis or Bacillus aeolius in a method according to the invention enables a high product yield and is therefore advantageous.
In a preferred embodiment, the method according to the invention is consequently a method for increasing the expression of a protein in a microorganism. An increased expression of the protein is present when a larger amount of protein is obtained by a method according to the invention in comparison with a similar method, which differs from a method according to the invention only in that another production organism is used. Both methods to be compared are carried out in this respect under the same conditions as optimal as possible for the microorganisms and for the same duration.

An expression construct is a nucleic acid sequence that causes the protein to be expressed in the microorganism. It comprises the genetic information, ie the nucleic acid sequence (gene) which codes for the protein. Expression of a nucleic acid sequence is its translation into the gene product (s) encoded by this sequence, ie into a polypeptide (protein) or into a plurality of polypeptides (proteins). The terms polypeptide and protein are used synonymously in the present application. For the purposes of the present invention, expression thus refers to the biosynthesis of ribonucleic acid (RNA) and proteins from the genetic information. In general, the expression comprises the transcription, ie the synthesis of messenger ribonucleic acid (mRNA) on the basis of the DNA (deoxyribonucleic acid) sequence of the gene and its translation into the corresponding polypeptide chain, which may be post-translationally modified can. The expression of a protein thus describes its biosynthesis from the genetic information present in the microorganism according to the invention.

An expression construct further comprises at least one nucleic acid sequence, preferably DNA, with a control function for the expression of the nucleic acid sequence coding for the protein or the auxiliary protease (so-called gene regulatory sequence). When the nucleotide sequence encoding the promoter and the nucleotide sequence encoding the target protein are brought into close proximity, the promoter may take control of the nucleotide sequence encoding the target protein. This is a "functional coupling" in the sense of the present application. A spatial proximity between promoter and target protein coding sequence can be achieved in that both sequences are close to each other (for example, immediately consecutive) on a nucleotide molecule. This is called a "cis-regulation". However, the sequence coding for the promoter and the target protein can also be located at a greater distance or both sequences can be located on different nucleotide molecules. By folding the nucleotide molecule or the various nucleotide molecules, the promoter and the nucleotide sequence coding for the target protein can be brought into close proximity. If, in such a case, a regulation of the promoter on the target protein coding sequence before, one speaks of a so-called "trans-regulation". A gene regulatory sequence here is any nucleic acid sequence whose presence in the microorganism influences, preferably increases, the transcription frequency of the nucleic acid sequence which codes for the protein. It is preferably a promoter sequence, since such a sequence is essential for the expression of a nucleic acid sequence. In the present invention, such a gene regulatory sequence is a sequence comprising or consisting of a sequence according to SEQ ID NO: 1, preferably according to SEQ ID NO: 2. However, an expression construct according to the invention may also comprise further gene regulatory sequences, for example one or more enhancer sequences. An expression construct in the context of the invention thus comprises at least one functional unit of gene and promoter. It can, but does not necessarily have to, exist as a physical entity. The presence of at least one promoter is essential to an expression construct of the invention. Accordingly, a promoter is understood as meaning a DNA sequence which enables the regulated expression of a gene. Of course, a promoter sequence is part of a gene and is often at its 5 'end, and thus before the RNA coding region. Preferably, the promoter sequence in an expression construct of the invention is 5'-downstream of the nucleic acid sequence encoding the protein (in the present invention, the second nucleic acid sequence encoding a (target) protein). The most important property of a promoter is the specific interaction with at least one DNA-binding protein or polypeptide, which mediates the start of transcription of the gene by an RNA polymerase and is referred to as a transcription factor. Often, several transcription factors and / or other proteins are involved in the initiation of transcription by an RNA polymerase. A promoter is therefore preferably a DNA sequence with promoter activity, ie a DNA sequence to which at least one transcription factor binds at least transiently to initiate the transcription of a gene. The strength of a promoter is measurable via the transcription frequency of the expressed gene, ie via the number of RNA molecules generated per unit time, in particular mRNA molecules. A promoter of an expression construct according to the invention may be a promoter of the microorganism in addition to the first nucleic acid sequence. Such a promoter sequence is thus naturally present in the microorganism. Alternatively, a promoter of an expression construct according to the invention may also have been recombinantly introduced into the microorganism. The same applies to all other gene regulatory sequences which an expression construct according to the invention may have. The promoter in an expression construct as used in a method according to the invention causes the expression of the nucleic acid sequence coding for the protein (target protein) in the expression construct.

1. (a) Nukleinsäuresequenz, die zu der in SEQ ID NO:1 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,2%, 99,3%, 99,4%, 99,5% und ganz besonders bevorzugt zu 100% identisch ist; oder
2. (b) Nukleinsäuresequenz, die zu der in SEQ ID NO:2 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99,2%, 99,3%, 99,4%, 99,5% und ganz besonders bevorzugt zu 100% identisch ist.

In a preferred embodiment, a bacterium / method according to the invention is characterized in that the promoter comprises a nucleic acid sequence which is selected from

1. (a) Nucleic acid sequence corresponding to the nucleic acid sequence given in SEQ ID NO: 1 to at least 80% and increasingly preferably to at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5 % and most preferably 100% identical; or
2. (b) Nucleic acid sequence corresponding to the nucleic acid sequence given in SEQ ID NO: 2 to at least 80% and increasingly preferably to at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5 % and most preferably 100% identical.

In an alternative embodiment, the promoter has a nucleic acid sequence as described above.

It has been shown that particularly high product yields of protein can be achieved with such promoter sequences in a method according to the invention. Preferably, the promoter is characterized in that it contains at least 80%, and more preferably at least 81.0%, 82.0%, 83.0%, 84.0 of the nucleic acid sequence corresponding to the nucleic acid sequence given in SEQ ID NO: 1 %, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0 %, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5%, and most preferably is 100% identical, and the promoter causes a transcription frequency of the gene expressed by it, which corresponds at least to that of a promoter according to SEQ ID NO: 1. Alternatively, the promoter is characterized by having a nucleic acid sequence corresponding to the nucleic acid sequence set forth in SEQ ID NO: 2 of at least 80%, and more preferably at least 81.0%, 82.0%, 83.0%, 84.0 %, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0 %, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5%, and most preferably is 100% identical, and the promoter causes a transcription frequency of the gene expressed by it, which corresponds at least to that of a promoter according to SEQ ID NO: 2.

The identity of nucleic acid or amino acid sequences is determined by a sequence comparison. Such a comparison is made by assigning similar sequences in the nucleotide sequences or amino acid sequences to each other. This sequence comparison is preferably carried out based on the BLAST algorithm established in the prior art and commonly used (cf., for example, Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ. Biol. 215: 403-410, and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, pp.3389-3402) and, in principle, happens by assigning similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences to one another. A tabular assignment of the respective positions is referred to as alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, are usually created using computer programs. Frequently used, for example, the Clustal series (see, for example Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500) , T-Coffee (cf., for example Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217 ) or programs based on these programs or algorithms. In the context of the present invention, sequence comparisons and alignments are preferably created using the Vector NTI® Suite 10.3 computer program (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the default default parameters specified.

Such a comparison allows a statement about the similarity of the compared sequences to each other. It is usually given in percent identity, that is to say the proportion of identical nucleotides or amino acid residues at the same positions or in an alignment with one another. The broader concept of homology involves conserved amino acid substitutions in the consideration of amino acid sequences, that is, amino acids with similar properties, since these usually perform similar activities or functions within the protein. Therefore, the similarity of the sequences compared may also be stated as percent homology or percent similarity. Identity and / or homology information can be made about whole polypeptides or genes or only over individual regions. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by matches in the sequences. They often have the same or similar functions. They can be small and comprise only a few nucleotides or amino acids. Often, such small regions exert essential functions for the overall activity of the protein. It may therefore be useful to relate sequence matches only to individual, possibly small areas. Unless stated otherwise, identity or homology information in the present application, however, refers to the total length of the respectively indicated nucleic acid or amino acid sequence. In the case of proteins, in particular enzymes and, in particular, proteases, the data also refer to the mature protein, unless stated otherwise. Unless otherwise indicated, a sequence consideration for a protein is thus always directed to the mature, ready-processed protein, even when encoded by the associated gene is an immature form that is still processed to the mature form upon translation.

The expression construct to be introduced into the microorganism in a method according to the invention further codes for a protein. It thus comprises a nucleic acid sequence encoding this protein. In principle, any nucleic acid sequence which can be translated into a protein can be used for this purpose. This is the protein that is produced with the help of a protein according to the invention to be produced (target protein). Preferably, it is an enzyme, more preferably an enzyme as described below.

Nucleic acids and expression constructs according to the invention can be produced by methods known per se for the modification of nucleic acids. Such are illustrated, for example, in the relevant handbooks such as those by Fritsch, Sambrook and Maniatis, "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989, and are familiar to those skilled in the biotechnology field. Examples of such methods are the chemical synthesis or the polymerase chain reaction (PCR), optionally in conjunction with other molecular biological and / or chemical or biochemical standard methods.

The present invention is particularly suitable for the recombinant production of proteins, in particular enzymes. For this purpose, the expression construct is introduced into the microorganism, preferably by transformation. In this regard, the introduction of the respective expression construct or parts thereof preferably takes place via vectors, in particular expression vectors. However, it is also possible that only parts of the expression construct, preferably at least the nucleic acid which codes for the protein, are introduced into the microorganism in such a way that the finished expression construct only arises in the microorganism. This can be done, for example, by a vector which causes the gene for the protein in the host cell to be inserted into an already existing genetic element such as the chromosome, the chromosomal DNA or other vectors such that, for example, an endogenous promoter for the expression of the Gene is used for the protein. The concept of introduction thus includes the possibility that an expression construct is completely introduced into the microorganism, preferably transformed, but also the possibility that only a part of the expression construct, particularly preferably the nucleic acid coding for the protein, is introduced into the microorganism , preferably transformed, and the complete expression construct is formed only in the microorganism. However, at least part of the expression construct is always introduced into the microorganism within the scope of the invention.

Vectors are known to those skilled in the biotechnology field. Especially when used in bacteria, they are special plasmids, ie circular genetic elements. In the context of the present invention, the expression construct is preferably cloned into a vector. The vectors may include, for example, those derived from bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins. With the other genetic elements in each case, vectors are able to establish themselves as stable units in the microorganisms over several generations. For the purposes of the invention, it is irrelevant whether they establish themselves as extrachomosomal units or integrate into the chromosomal DNA. Which of the numerous systems is chosen depends on the individual case. Decisive factors can be, for example, the achievable copy number, the available selection systems, including, in particular, antibiotic resistances, or the cultivability of the microorganisms capable of accepting the vectors.

Expression vectors may also be regulatable by changes in culture conditions, such as cell density or the addition of certain compounds. An example of such a compound is the galactose derivative isopropyl-β-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (Iac operon).

In a further embodiment of the invention, a bacterium / method according to the invention is characterized in that the protein is not naturally present in the microorganism. Not naturally present in this context means that the protein is not a separate protein or enzyme of the microorganism. The protein can thus not be expressed in the microorganism by a nucleic acid sequence which is part of the chromosomal DNA of the microorganism in its wild-type form. The protein and / or the respective nucleic acid sequence coding therefor is therefore not present in the wild-type form of the microorganism and / or can not be isolated from the wild-type form of the microorganism. Preferably, a protein not naturally present in the microorganism or the nucleic acid sequence coding therefor has been deliberately introduced into the microorganism with the aid of genetic engineering, so that the microorganism has been enriched by the protein or the nucleic acid sequence coding therefor. However, one protein may quite naturally be present in another microorganism - relevant for the consideration is exclusively the microorganism used in the process.

In a further embodiment of the invention, the method or other objects of the invention is characterized in that the protein is an enzyme, in particular an acid cellulase, alpha-amylase, alpha-acetodecarboxylase, Aminopetidase, amylase, arabanase, beta-glucanase, beta-glucosidase, beta-mannosidase, carageenase, carbohydrase, catalase, cellobiose oxidase, cellulase, chymosin, endo-1,3-beta-glucanase, endo-1,3,3 (4) Β-glucanase, endo-1,4-beta-xylanase, endopeptidase, esterase, exopeptidase, G4-amylase, glucoamylase, glucose oxidase, glucosidase, glycolipase, hemicellulase, laccase, lipase, lysophospholipase, maltogenic amylase, mannanase, neutral protease, nuclease , Oxidase, oxidoreductase, pectate lyase, pectinase, pectinesterase, pentosanase, perhydrolase, phospholipase, phytase, polygalacturonase, protease, proteinase, pullulanase, Rennet enzyme, rhamnogalacturonase, subtilisin, tannase, transferase, transglutaminase, xanthanase, xylanase, xyloglucanase or mixtures thereof. Most preferably, the protein is a protease. In particularly advantageous embodiments of a method according to the invention, the protease to be produced (= target protease) is simultaneously involved in the hydrolysis of the protein substrate for the microorganism and can advantageously bring about a further improved digestion of substrate protein. Consequently, the microorganism then has improved nutrient conditions available.

For example, with a method according to the invention, the enzymes mentioned below can be advantageously prepared. Among the proteases, subtilisins are preferred. Examples thereof are the subtilisins BPN 'and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, subtilisin DY and the enzymes thermitase, proteinase K and the subtilases, but not the subtilisins in the narrower sense Proteases TW3 and TW7. Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase® from Novozymes A / S, Bagsvaerd, Denmark. The subtilisins 147 and 309 are sold under the trade names Esperase®, and Savinase® by the company Novozymes.

From the protease from Bacillus lentus DSM 5483 derived under the name BLAP® protease variants derived. Further preferred proteases are, for example, the enzymes known as PUR. Other proteases include those under the trade names Durazym®, Relase®, Everlase®, Nafizym®, Natalase®, Kannase® and Ovozyme® from Novozymes under the trade names, Purafect®, Purafect® OxP, Purafect® Prime, Excellase ® and Properase® from Genencor / Danisco, sold under the trade name Protosol® by Advanced Biochemicals Ltd., Thane, India, under the trade name Wuxi® by Wuxi Snyder Bioproducts Ltd., China, under the trade names Proleather® and Protease P® from Amano Pharmaceuticals Ltd., Nagoya, Japan, and the enzyme available under the name Proteinase K-16 from Kao Corp., Tokyo, Japan. Also preferred are the proteases from Bacillus gibsonii and Bacillus pumilus, which are disclosed in the international patent applications WO2008 / 086916 and WO2007 / 131656 , Furthermore, the protease according to SEQ ID NO: 3 is preferred, whose nucleotide sequence including SP and PP sequences is shown in SEQ ID NO: 4.

Examples of amylases are the alpha-amylases from Bacillus licheniformis, from Bacillus amyloliquefaciens or from Bacillus stearothermophilus and in particular also their improved for use in detergents or cleaners further developments. The enzyme from Bacillus licheniformis is available from the company Novozymes under the name Termamyl® and from the company Danisco / Genencor under the name Purastar®ST. Further development products of this α-amylase are available from the company Novozymes under the trade name Duramyl® and Termamy Dultra, from the company Danisco / Genencor under the name Purastar®OxAm and from the company Daiwa Seiko Inc., Tokyo, Japan, as Keistase®. The Bacillus amyloliquefaciens α-amylase is sold by the company Novozymes under the name BAN®, and variants derived from the Bacillus stearothermophilus α-amylase under the names BSG® and Novamyl®, also from the company Novozymes. Furthermore, the α-amylase from Bacillus sp. A 7-7 (DSM 12368) and cyclodextrin glucanotransferase (CGTase) from Bacillus agaradherens (DSM 9948). Likewise, fusion products of all the molecules mentioned can be used. In addition, the further developments of the α-amylase from Aspergillus niger and A. oryzae available under the trade name Fungamyl® from the company Novozymes are suitable. Further advantageous commercial products are, for example, the Amylase Powerase® from the company Danisco / Genencor and the amylases Amylase-LT®, Stainzyme® and Stainzyme Plus®, the latter from the company Novozymes. Also variants of these enzymes obtainable by point mutations can be prepared according to the invention. Further preferred amylases are disclosed in International Publications WO 00/60060 . WO 03/00271 . WO 03/054177 and WO07 / 079938 , whose disclosure is therefore expressly referred to, or whose disclosure in this regard is therefore expressly incorporated into the present patent application. Amylases to be prepared according to the invention are furthermore preferably α-amylases.

Examples of lipases or cutinases are those originally available from Humicola lanuginosa (Thermomyces lanuginosus), respectively further developed lipases, especially those with the amino acid exchange D96L. They are sold for example by the company Novozymes under the trade names Lipolase®, Lipolase®Ultra, LipoPrime®, Lipozyme® and Lipex®. Furthermore, for example, the cutinases can be produced, which were originally isolated from Fusarium solani pisi and Humicola insolens. From the company Danisco / Genencor, for example, the lipases or cutinases can be produced, the initial enzymes were originally isolated from Pseudomonas mendocina and Fusarium solanii. Further important commercial products are the preparations M1 Lipase® and Lipomax® which were originally sold by Gist-Brocades (now Danisco / Genencor) and those from Meito Sangyo KK, Japan, under the name Lipase MY-30®, Lipase OF® and Lipase PL® distributed enzymes, also the product Lumafast® from Danisco / Genencor.

Examples of cellulases (endoglucanases, EG) include sequences of the fungal, endoglucanase (EG) -rich cellulase preparation or their further developments, which is offered by the company Novozymes under the trade name Celluzyme®. Endolase® and Carezyme®, also available from Novozymes, are based on the 50 kD EG or 43 kD EG from Humicola insolens DSM 1800. Further manufacturable commercial products of this company are Cellusoft®, Renozyme® and Celluclean®. Also manufacturable are, for example, cellulases available from AB Enzymes, Finland, under the tradenames Ecostone® and Biotouch®, which are based, at least in part, on the 20 kD EG of melanocarpus. Other cellulases from AB Enzymes are Econase® and Ecopulp®. Other suitable cellulases are from Bacillus sp. CBS 670.93 and CBS 669.93, those derived from Bacillus sp. CBS 670.93 is available from the company Danisco / Genencor under the trade name Puradax®. Other manufacturable commercial products of the company Danisco / Genencor are "Genencor detergent cellulase L" and IndiAge®Neutra.

Also variants of these enzymes obtainable by point mutations can be prepared according to the invention. Particularly preferred cellulases are Thielavia terrestris cellulase variants described in International Publication WO 98/12307 Cellulases from Melanocarpus, in particular Melanocarpus albomyces, disclosed in the international publication WO 97/14804 Cellulases of the EGIII type from Trichoderma reesei disclosed in the European patent application EP 1305432 and variations obtainable therefrom, in particular those disclosed in the European patent applications EP 1240525 and EP 1305432 , and cellulases disclosed in International Publication WO 1992006165, WO 96/29397 and WO 02/099091 , The respective disclosure is therefore expressly referred to, or the disclosure content of which in this respect is therefore expressly included in the present patent application.

Furthermore, other enzymes can be prepared, which are summarized by the term hemicellulases. These include, for example, mannanases, xanthan lyases, xanthanases, pectin lyases (= pectinases), pectin esterases, pectate lyases, xyloglucanases, xylanases, pullulanases and β-glucanases. Suitable enzymes for this purpose are, for example, under the name Gamanase®, Pektinex AR® and Pectaway® from Novozymes, under the name Rohapec® B1 L from AB Enzymes and under the name Pyrolase® from Diversa Corp., San Diego, CA, USA available. The β-glucanase obtained from Bacillus subtilis is available under the name Cereflo® from Novozymes. Hemicellulases which are particularly preferred according to the invention are mannanases which are sold, for example, under the trade names Mannaway® by the company Novozymes or Purabrite® by the company Genencor.

Furthermore, oxidoreductases, for example oxidases, oxygenases, catalases, peroxidases, such as halo-, chloro-, bromo-, lignin, glucose or manganese peroxidases, dioxygenases or laccases (phenol oxidases, polyphenol oxidases) can also be prepared. Suitable commercial products are Denilite® 1 and 2 from Novozymes. Other enzymes are in international patent applications WO 98/45398 . WO 2005/056782 . WO 2004/058961 such as WO 2005/124012 disclosed.

In a further embodiment of the invention, the method is characterized in that the microorganism is Bacillus licheniformis or Bacillus aeolius, in particular strain DSM 15084.

In a further embodiment of the invention, the strain to be used in a method according to the invention is genetically modified. The genetic modification advantageously further increases the product yield or advantageously alters a property of the product. The product here is the expressed protein which is present in the fermentation medium. For example, its odor is reduced, its color is weakened, and / or the product is clarified (ie less turbid) or its density is reduced. The genetic modification in this regard does not mean the introduction of the expression construct as described above. Rather, the one in the inventive method to be used microorganism of the species Bacillus licheniformis or aeolius already genetically modified, and indeed before the expression construct according to the invention is introduced into the microorganism. Preferably, the presence of the genetic modification is determined in comparison with the corresponding Bacillus wild-type, in particular Bacillus licheniformis or aeolius. As a genetic modification, substitutions, insertions and / or deletions are possible in this regard. Advantageously, the genetic modification causes the functional change, for example the functional inactivation, of a gene in the microorganism. The functional change, for example the functional inactivation, of the gene then in turn results in an increased and / or improved production of the protein and thus an improved product yield and / or the obtaining of a product with one or more improved properties. By functional inactivation is meant that the gene product (s) encoded by this gene are no longer formed or formed in a biologically inactive form so that it no longer has its function (s) in the microorganism can / can exercise. In this regard, a functional inactivation of a gene can in particular also take place in that it is completely or partially replaced by an alternative gene. This alternative gene can then be expressed in place of the original gene. Thus, the originally present gene was functionally inactivated, instead the alternative gene is expressed, thereby causing the functional change. The alternative gene may be a gene related to the original gene (more than 50% identity to the original gene) or a gene unrelated to the original gene (50% or less identity to the original gene). For example, the alternative gene can be inserted by insertion into the coding sequence of the original gene. This functionally inactivates the original gene and instead expresses the alternative gene. The genetic modification may be present both in the gene product coding sequence and in a gene regulatory sequence belonging to the gene.

Microorganisms to be used according to the invention can be modified, for example, with regard to their requirements of the culture conditions, changed with regard to their movement behavior, altered with regard to their sporulation ability, altered with regard to a specific metabolic pathway - for example to prevent the formation of bad odors during the fermentation - or else other or have additional selection markers.

• Agrobacterium radiobacter K84, Agrobacterium tumefaciens str. C58, Agrobacterium vitis S4, Arcobacter butzleri ED-1, Arcobacter nitrofigilis DSM 7299, Arcobacter sp. L, Aromatoleum aromaticum EbN1 , Arthrobacter aurescens TC1, Arthrobacter chlorophenolicus A6, Arthrobacter phenanthrenivorans Sphe3, Arthrobacter sp. FB24, Bacillus amyloliquefaciens DSM 7, Bacillus amyloliquefaciens FZB42, Bacillus anthracis str. Arnes, Bacillus atrophaeus 1942, Bacillus cellulosilyticus DSM 2522, Bacillus cereus ATCC 10987, Bacillus cereus ATCC 14579, Bacillus cereus B4264, Bacillus clausii KSM-K16, Bacillus coagulans 36D1, Bacillus cytotoxicus NVH 391 - 98, Bacillus halodurans C-125, Bacillus licheniformis ATCC 14580, Bacillus megaterium DSM 319, Bacillus pseudofirmus OF4, Bacillus pseudomycoides DSM 12442 (305 parts in a CON entry), Bacillus pumilus SAFR-032, Bacillus selenitireducens MLS10, Bacillus subtilis BSn5, Bacillus subtilis subsp. spizizenii str. W23, Bacillus subtilis subsp. spizizenii TU-B-10, Bacillus subtilis subsp. subtilis RO-NN-1, Bacillus subtilis subsp. subtilis str. 168, Bacillus thuringiensis BMB1713, Bacillus thuringiensis serovar konkukian str. 97-27T, Bacillus thuringiensis serovar thuringiensis str. T01001, Bacillus tusciae DSM 2912, Bacillus weihenstephanensis KBAB4, Bifidobacterium adolescentis ATCC 15703, Bifidobacterium animalis subsp. lactis V9, Bifidobacterium bifidum PRL2010, Bifidobacterium breve UCC2003, Bifidobacterium dentium Bd1, Bifidobacterium longum DJO10A, Bifidobacterium longum NCC27051, Bifidobacterium longum subsp. infantis ATCC 15697, Bradyrhizobium sp. ORS 278, Brevibacillus brevis NBRC 100599, Corynebacterium aurimucosum ATCC 700975, Corynebacterium diphtheriae NCTC 13129, Corynebacterium efficiens YS-314, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum R, Corynebacterium jeikeium K41 1, Corynebacterium kroppenstedtii DSM 44385, Corynebacterium pseudotuberculosis FRC41, Corynebacterium resistens DSM 45100, Corynebacterium ulcerans BR-AD22, Corynebacterium urealyticum DSM 7109, Corynebacterium variabile DSM 44702, Desulfovibrio aespoeensis Aspo-2, Desulfovibrio alaskensis G20, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Desulfovibrio magneticus RS-1, Desulfovibrio salexigens DSM 2638, Desulfovibrio vulgaris RCH1, Desulfovibrio vulgaris str. Miyazaki F, Desulfurobacterium thermolithotrophum DSM 1 1699, Enterobacter aerogenes KCTC 2190, Enterobacter asburiae LF7a, Enterobacter cloacae subsp. cloacae ATCC 13047, Enterobacter sp. 638, Escherichia coli 536, Escherichia coli APEC 01, Escherichia coli CFT073, Escherichia coli O103:H2 str. 12009, Escherichia coli SE1 1, Escherichia coli SE15-, Escherichia fergusonii ATCC 35469T chromosome, Ethanoligenens harbinense YUAN-3, Eubacterium cylindroides T2-87 draft, Eubacterium eligens ATCC 27750, Eubacterium limosum KIST612, Eubacterium rectale M104/1 draft, Eubacterium siraeum 70/3 draft, Exiguobacterium sibiricum 255-15, Exiguobacterium sp. AT1 b, Flavobacteriaceae bacterium 3519-10, Flavobacterium branchiophilum FL-15, Flavobacterium johnsoniae UW101, Flavobacterium psychrophilum JIP02/86, Geobacillus kaustophilus HTA426, Geobacillus sp. C56-T3, Geobacillus sp. WCH70, Geobacillus sp. Y4.1 MC1, Geobacillus sp. Y412MC52, Geobacillus sp. Y412MC61; plasmid pGYMC6101, Geobacillus thermodenitrificans NG80-2, Geobacillus thermoglucosidasius C56-YS93, Geobacter bemidjiensis Bern, Geobacter lovleyi SZ, Geobacter metallireducens GS-15, Geobacter sp. FRC-32, Geobacter sp. M18, Geobacter sp. M21, Geobacter sulfurreducens PCA, Geobacter uraniireducens Rf4, Gloeobacter violaceus PCC 74212, Gluconacetobacter diazotrophicus PAI 5; ATCC 49037, Gluconacetobacter xylinus NBRC 3288, Gluconobacter oxydans 621 H, Hydrogenobaculum sp. Y04AAS1, Lactobacillus acidophilus 30SC, Lactobacillus amylovorus GRL 1 1 12, Lactobacillus brevis ATCC 367, Lactobacillus buchneri NRRL B-30929, Lactobacillus casei ATCC 334, Lactobacillus casei BD-II, Lactobacillus casei BL23, Lactobacillus casei LC2W, Lactobacillus crispatus ST10, Lactobacillus delbrueckii subsp. bulgaricus ND02, Lactobacillus fermentum CECT 5716, Lactobacillus gasseri ATCC 33323, Lactobacillus helveticus H10, Lactobacillus johnsonii NCC 533, Lactobacillus kefiranofaciens ZW3, Lactobacillus plantarum WCFS1, Lactobacillus reuteri SD21 12, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus GG ATCC 53103, Lactobacillus ruminis ATCC 27782, Lactobacillus sakei subsp. sakei 23K, Lactobacillus salivarius CECT 5713, Lactobacillus sanfranciscensis TMW 1.1304, Mannheimia succiniciproducens MBEL55E, Mycobacterium abscessus chromosome, Mycobacterium africanum GM041 182, Mycobacterium avium 104, Mycobacterium bovis BCG str. Tokyo 172, Mycobacterium canettii CIPT 140010059, Mycobacterium gilvum PYR-GCK, Mycobacterium marinum M, Mycobacterium smegmatis str. MC2 155, Mycobacterium sp. JDM601, Mycobacterium sp. JLS, Mycobacterium sp. KMS, Mycobacterium sp. MCS, Mycobacterium sp. Spyrl, Mycobacterium vanbaalenii PYR-1, Pseudomonas aeruginosa NCGM2.S17, Pseudomonas brassicacearum subsp. brassicacearum NFM421, Pseudomonas entomophila L48 chromosome, Pseudomonas fluorescens Pf-5, Pseudomonas fulva 12-X, Pseudomonas mendocina NK-01, Pseudomonas putida KT2440+, Pseudomonas syringae pv. phaseolicola 1448A, Pseudomonas stutzeri DSM 4166, Pseudomonas syringae pv. syringae B728a+, Pseudomonas syringae pv. tomato str. DC3000/, Stenotrophomonas maltophilia K279a strain K279a, Streptobacillus moniliformis DSM 121 12, Streptomyces avermitilis MA-4680, Streptomyces bingchenggensis BCW-1, Streptomyces cattleya NRRL 8057 main chromosome, Streptomyces clavuligerus ATCC 27064, Streptomyces coelicolor, Streptomyces flavogriseus ATCC 33331, Streptomyces griseus subsp. griseus NBRC 13350, Streptomyces scabiei 87.22, Streptomyces sp. SirexAA-E, Streptomyces venezuelae ATCC 10712, Streptomyces violaceusniger Tu 41 13, Sulfobacillus acidophilus TPY, Thermobifida fusca YX, Thermotoga lettingae TMO, Thermotoga maritima MSB8, Thermotoga naphthophila RKU-10, Thermotoga neapolitana DSM 4359, Thermotoga petrophila RKU-1, Thermotoga sp. RQ2, Thermovibrio ammonificans HB-1, Thermus thermophilus HB27, Xanthomonas albilineans, Xanthomonas axonopodis pv. citrumelo F1, Xanthomonas axonopodis pv. citri str. 306/, Xanthomonas campestris pv. campestris str. 8004, Xanthomonas euvesicatoria, Xanthomonas oryzae pv. Oryzae.

In this regard, all genes are genetically modifiable in the Bacillus licheniformis or aeolius strain to be used in a method according to the invention, to which a correspondence exists in one or more of the genomes of the following microorganisms:

• Agrobacterium radiobacter K84, Agrobacterium tumefaciens str. C58, Agrobacterium vitis S4, Arcobacter butzleri ED-1, Arcobacter nitrofigilis DSM 7299, Arcobacter sp. L, Aromatoleum aromaticum EbN1, Arthrobacter aurescens TC1, Arthrobacter chlorophenolicus A6, Arthrobacter phenanthrenivorans Sphe3, Arthrobacter sp. FB24, Bacillus amyloliquefaciens DSM 7, Bacillus amyloliquefaciens FZB42, Bacillus anthracis str. Arnes, Bacillus atrophaeus 1942, Bacillus cellulosilyticus DSM 2522, Bacillus cereus ATCC 10987, Bacillus cereus ATCC 14579, Bacillus cereus B4264, Bacillus clausii KSM-K16, Bacillus coagulans 36D1, Bacillus cytotoxic NVH 391-98, Bacillus halodurans C-125, Bacillus licheniformis ATCC 14580, Bacillus megaterium DSM 319, Bacillus pseudofirmus OF4, Bacillus pseudomycoides DSM 12442 (305 parts in a CON entry), Bacillus pumilus SAFR-032, Bacillus selenitireducens MLS10, Bacillus subtilis BSn5, Bacillus subtilis subsp. spizizenii str. W23, Bacillus subtilis subsp. spizizenii TU-B-10, Bacillus subtilis subsp. subtilis RO-NN-1, Bacillus subtilis subsp. subtilis st. 168, Bacillus thuringiensis BMB1713, Bacillus thuringiensis serovar konkukian str. 97-27T, Bacillus thuringiensis serovar thuringiensis str. T01001, Bacillus tusciae DSM 2912, Bacillus weihenstephanensis KBAB4, Bifidobacterium adolescentis ATCC 15703, Bifidobacterium animalis subsp. lactis V9, Bifidobacterium bifidum PRL2010, Bifidobacterium breve UCC2003, Bifidobacterium dentium Bd1, Bifidobacterium longum DJO10A, Bifidobacterium longum NCC27051, Bifidobacterium longum subsp. infantis ATCC 15697, Bradyrhizobium sp. ORS 278, Brevibacillus brevis NBRC 100599, Corynebacterium aurimucosum ATCC 700975, Corynebacterium diphtheriae NCTC 13129, Corynebacterium efficiens YS-314, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum R, Corynebacterium jeikeium K41 1, Corynebacterium kroppenstedtii DSM 44385, Corynebacterium pseudotuberculosis FRC41, Corynebacterium resistens DSM 45100, Corynebacterium ulcerans BR-AD22, Corynebacterium urealyticum DSM 7109, Corynebacterium variabile DSM 44702, Desulfovibrio aespoeensis Aspo-2, Desulfovibrio alaskensis G20, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Desulfovibrio magneticus RS-1, Desulfovibrio salexigen DSM 2638, Desulfovibrio vulgaris RCH1, Desulfovibrio vulgaris str. Miyazaki F, Desulfurobacterium thermolithotrophum DSM 1 1699, Enterobacter aerogenes KCTC 2190, Enterobacter asburiae LF7a, Enterobacter cloacae subsp. cloacae ATCC 13047, Enterobacter sp. 638, Escherichia coli 536, Escherichia coli APEC 01, Escherichia coli CFT073, Escherichia coli O103: H2 str. Escherichia coli SE1 1, Escherichia coli SE15, Escherichia fergusonii ATCC 35469T chromosomes, ethanoligenic harbinense YUAN-3, Eubacterium cylindroides T2-87 draft, Eubacterium eligens ATCC 27750, Eubacterium limosum KIST612, Eubacterium rectale M104 / 1 draft, Eubacterium siraeum 70 / 3 draft, Exiguobacterium sibiricum 255-15, Exiguobacterium sp. AT1b, Flavobacteriaceae bacterium 3519-10, Flavobacterium branchiophilum FL-15, Flavobacterium johnsoniae UW101, Flavobacterium psychrophilum JIP02 / 86, Geobacillus kaustophilus HTA426, Geobacillus sp. C56-T3, Geobacillus sp. WCH70, Geobacillus sp. Y4.1 MC1, Geobacillus sp. Y412MC52, Geobacillus sp. Y412MC61; plasmid pGYMC6101, Geobacillus thermodenitrificans NG80-2, Geobacillus thermoglucosidase C56-YS93, Geobacter bemidjiensis Bern, Geobacter lovleyi SZ, Geobacter metallireducens GS-15, Geobacter sp. FRC-32, Geobacter sp. M18, Geobacter sp. M21, Geobacter sulfurreducens PCA, Geobacter uraniireducens Rf4, Gloeobacter violaceus PCC 74212, Gluconacetobacter diazotrophicus PAI 5; ATCC 49037, Gluconacetobacter xylinus NBRC 3288, Gluconobacter oxydans 621H, Hydrogenobaculum sp. Y04AAS1, Lactobacillus acidophilus 30SC, Lactobacillus amylovorus GRL 1 1 12, Lactobacillus brevis ATCC 367, Lactobacillus buchneri NRRL B-30929, Lactobacillus casei ATCC 334, Lactobacillus casei BD-II, Lactobacillus casei BL23, Lactobacillus casei LC2W, Lactobacillus crispatus ST10, Lactobacillus delbrueckii subsp. bulgaricus ND02, Lactobacillus fermentum CECT 5716, Lactobacillus gasseri ATCC 33323, Lactobacillus helveticus H10, Lactobacillus johnsonii NCC 533, Lactobacillus kefiranofaciens ZW3, Lactobacillus plantarum WCFS1, Lactobacillus reuteri SD21 12, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus GG ATCC 53103, Lactobacillus ruminis ATCC 27782, Lactobacillus sakei subsp. sakei 23K, Lactobacillus salivarius CECT 5713, Lactobacillus sanfranciscensis TMW 1.1304, Mannheimia succiniciproducens MBEL55E, Mycobacterium abscessus chromosomes, Mycobacterium africanum GM041 182, Mycobacterium avium 104, Mycobacterium bovis BCG str. Tokyo 172, Mycobacterium canettii CIPT 140010059, Mycobacterium gilvum PYR-GCK, Mycobacterium marinum M, Mycobacterium smegmatis str. MC2 155, Mycobacterium sp. JDM601, Mycobacterium sp. JLS, Mycobacterium sp. KMS, Mycobacterium sp. MCS, Mycobacterium sp. Spyrl, Mycobacterium vanbaalenii PYR-1, Pseudomonas aeruginosa NCGM2.S17, Pseudomonas brassicacearum subsp. brassicacearum NFM421, Pseudomonas entomophila L48 chromosomes, Pseudomonas fluorescens Pf-5, Pseudomonas fulva 12-X, Pseudomonas mendocina NK-01, Pseudomonas putida KT2440 +, Pseudomonas syringae pv. phaseolicola 1448A, Pseudomonas stutzeri DSM 4166, Pseudomonas syringae pv. syringae B728a +, Pseudomonas syringae pv. tomato st. DC3000 /, Stenotrophomonas maltophilia K279a strain K279a, Streptobacillus moniliformis DSM 121 12, Streptomyces avermitilis MA-4680, Streptomyces bingchenggensis BCW-1, Streptomyces cattleya NRRL 8057 main chromosomes, Streptomyces clavuligerus ATCC 27064, Streptomyces coelicolor, Streptomyces flavogriseus ATCC 33331, Streptomyces griseus subsp , griseus NBRC 13350, Streptomyces scabiei 87.22, Streptomyces sp. SirexAA-E, Streptomyces venezuelae ATCC 10712, Streptomyces violaceus Tu 41 13, Sulfobacillus acidophilus TPY, Thermobifida fusca YX, Thermotoga lettingae TMO, Thermotoga maritima MSB8, Thermotoga naphthophila RKU-10, Thermotoga neapolitana DSM 4359, Thermotoga petrophila RKU-1, Thermotoga sp , RQ2, Thermovibrio ammonificans HB-1, Thermus thermophilus HB27, Xanthomonas albilineans, Xanthomonas axonopodis pv. Citrumelo F1, Xanthomonas axonopodis pv. Citri str. 306 /, Xanthomonas campestris pv. Campestris str. 8004, Xanthomonas euvesicatoria, Xanthomonas oryzae pv. Oryzae.

The modifiable genes, in particular their sequences, are available in publicly available databases, for example in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database at http://www.genome.jp/kegg or in the databases of the NCBI (National Center for Biotechnology Information, US National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, USA) at http://www.ncbi.nlm.nih.gov. The KEGG database has been used since 1995 by the laboratories of Kanehisa et al. developed by the Kyoto University Bioinformatics Center and the Human Genome Center of the University of Tokyo. In addition to individual genes, these databases also contain information and sequences of whole genomes or large parts of the genome of different microorganisms.

A genetic correspondence in the sense of the present patent application is distinguished, on the one hand, by the highest possible sequence homology between the gene of the Bacillus licheniformis or aeolius strain to be used according to the invention and the gene of the abovementioned microorganism. On the other hand, a genetic correspondence is characterized by a similar function, ie the corresponding genes of the Bacillus to be used according to the invention licheniformis or aeolius strain and the above-mentioned microorganism have a similar function in the respective microorganism.

With this gene and / or genome information, it is possible to identify the respective gene in the Bacillus licheniformis or aeolius strain to be used in a method according to the invention by means of sequence comparisons. Based on the genetic information, in particular the sequence information from the genome of a microorganism mentioned above, the expert can determine the nucleic acid sequence with the highest sequence identity in the genome of the Bacillus licheniformis or aeolius strain by genetic comparison and / or standard molecular biology, which genetically modified and then to be used in the method according to the invention. The confirmation of a similar function, i. a functional correspondence, can provide comparative experiments with the respective microorganisms, in each of which the gene compared based on the sequence comparison is changed (preferably functionally inactivated) in the same way and it is observed whether similar changes occur in both microorganisms, in particular phenotypic changes , If, for example, the change, in particular the functional inactivation, of the gene in question in the abovementioned microorganism is accompanied by a change in the metabolic activity, the movement or the sporulation behavior, and a corresponding change is observed in the Bacillus licheniformis or aeolius strain to be modified and used according to the invention, so here is a confirmation of the correct assignment to see. Appropriate methods are standard in the field of genetics, in particular the genetics of microorganisms, and are well known to those skilled in the art.

In a particularly preferred embodiment, the microorganism is sporulation inhibited. This is preferably achieved by functionally inactivating its gene spolV (yqfD) or its genetic equivalent, in particular by deleting the gene spolV (yqfD) or its genetic equivalent or parts thereof. It has been found that with such a sporulation-inhibited Bacillus licheniformis or aeolius strain a particularly high product yield is achieved in a method according to the invention.

The microorganisms used in the process according to the invention can be cultured and fermented in the usual way, for example in discontinuous or continuous systems. In the first case, a suitable nutrient medium is inoculated with the microorganisms and the protein harvested from the medium after a period to be determined experimentally. Continuous fermentations are characterized by achieving a flow equilibrium, in which over a relatively long period of time cells partly die out but also regrow and at the same time the protein formed can be removed from the medium.

Processes according to the invention are preferably fermentation processes. Fermentation processes are known per se from the prior art and represent the actual large-scale production step, usually followed by a suitable purification method of the protein produced. All fermentation processes which are based on a process according to the invention for producing a protein represent embodiments of a process according to the invention.

Fermentation processes, which are characterized in that the fermentation is carried out via a feed strategy, come in particular into consideration. Here, the media components consumed by the ongoing cultivation are fed.

As a result, considerable increases in both the cell density and in the cell mass or dry matter can be achieved. Furthermore, the fermentation can also be designed so that undesired metabolic products are filtered out or neutralized by the addition of buffer or suitable counterions. The produced protein can be harvested from the fermentation medium. Such a fermentation process is resistant to isolation of the protein from the microorganism, i. a product preparation from the cell mass (dry matter) is preferred. This can be achieved, for example, by the provision of suitable secretion markers or mechanisms and / or transport systems for the microorganisms to secrete the protein into the fermentation medium. Alternatively, without secretion, the isolation of the protein from the host cell, i. a purification of the same from the cell mass, carried out, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.

A further subject of the invention is a recombinant bacterium comprising a nucleic acid sequence which is a) a first nucleic acid sequence comprising a sequence according to SEQ ID NO: 1 or a sequence which is at least 80% identical to the nucleic acid sequence given in SEQ ID NO: 1; and b) a second nucleic acid sequence encoding a (target) protein.

In preferred embodiments, the recombinant bacterium is Bacillus licheniformis or Bacillus aeolius, more preferably Bacillus aeolius DSM 15084.

These are therefore all microorganisms that may be the subject of a method according to the invention. All facts, subjects and embodiments described for inventive method are also applicable to this subject of the invention. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to the microorganisms of the invention.

1. (a) der Promotor eine Nukleinsäuresequenz umfasst, die ausgewählt ist aus
   1. (i) Nukleinsäuresequenz, die zu der in SEQ ID NO:1 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81,0%, 82,0%, 83,0%, 84,0%, 85,0%, 86,0%, 87,0%, 88,0%, 89,0%, 90,0%, 91,0%, 92,0%, 93,0%, 94,0%, 95,0%, 96,0%, 97,0%, 98,0%, 99,0%, 99,2%, 99,3%, 99,4%, 99,5% und ganz besonders bevorzugt zu 100% identisch ist; oder
   2. (ii) Nukleinsäuresequenz, die zu der in SEQ ID NO:2 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81,0%, 82,0%, 83,0%, 84,0%, 85,0%, 86,0%, 87,0%, 88,0%, 89,0%, 90,0%, 91,0%, 92,0%, 93,0%, 94,0%, 95,0%, 96,0%, 97,0%, 98,0%, 99,0%, 99,2%, 99,3%, 99,4%, 99,5 % und ganz besonders bevorzugt zu 100% identisch ist, und/oder
2. (b) das Protein nicht natürlicherweise in dem Mikroorganismus vorhanden ist, und/oder
3. (c) das Protein ein Enzym ist, insbesondere eine saure Cellulase, Alpha-Amylase, Alpha-Acetodecarboxylase, Aminopetidase, Amylase, Arabanase, Beta-Glucanase, Beta-Glucosidase, Beta-Mannosidase, Carageenase, Carbohydrase, Catalase, Cellobiose-Oxidase, Cellulase, Chymosin, Endo-1,3-Beta-Glucanase, Endo-1,3(4)-Beta-Glucanase, Endo-1,4-Beta-Xylanase, Endopeptidase, Esterase, Exopeptidase, G4-Amylase, Glucoamylase, Glucoseoxidase, Glucosidase, Glycolipase, Hemicellulase, Laccase, Lipase, Lyspohospholipase, maltogene Amylase, Mannanase, neutrale Protease, Nuklease, Oxidase, Oxidoreduktase, Pectatlyase, Pectinase, Pectinesterase, Pentosanase, Perhydrolase, Phospholipase, Phytase, Polygalacturonase, Protease, Proteinase, Pullulanase, Rennet-Enzym, Rhamnogalacturonase, Subtilisin, Tannase, Transferase, Transglutaminase, Xanthanase, Xylanase, Xyloglucanase, bevorzugt Protease oder Alpha-Amylase, oder Mischungen hieraus, und/oder
4. (d) es sich bei dem Mikroorganismus um Bacillus licheniformis oder Bacillus aeolius, insbesondere den Stamm DSM 15084, handelt, und/oder
5. (e) der Mikroorganismus sporulationsgehemmt ist, vorzugsweise durch eine Veränderung des Gens spolV (yqfD), insbesondere durch eine Deletion des Gens spolV (yqfD) oder Teilen davon, und/oder
6. (f) der Mikroorganismus genetisch modifiziert ist.

Particularly preferred embodiments of microorganisms according to the invention are characterized in that

1. (a) the promoter comprises a nucleic acid sequence selected from
   1. (i) Nucleic acid sequence corresponding to the nucleic acid sequence given in SEQ ID NO: 1 to at least 80% and increasingly preferably to at least 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5% and most preferably 100% identical; or
   2. (ii) Nucleic acid sequence corresponding to at least 80% and more preferably at least 81.0%, 82.0%, 83.0%, 84.0%, 85.0% of the nucleic acid sequence given in SEQ ID NO: 2, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5% and most preferably 100% identical, and /or
2. (b) the protein is not naturally present in the microorganism, and / or
3. (c) the protein is an enzyme, in particular an acid cellulase, alpha-amylase, alpha-acetodecarboxylase, aminopetidase, amylase, arabanase, beta-glucanase, beta-glucosidase, beta-mannosidase, carageenase, carbohydrase, catalase, cellobiose oxidase, Cellulase, chymosin, endo-1,3-beta-glucanase, endo-1,3 (4) -beta-glucanase, endo-1,4-beta-xylanase, endopeptidase, esterase, exopeptidase, G4-amylase, glucoamylase, glucose oxidase , Glucosidase, glycolipase, hemicellulase, laccase, lipase, lysophospholipase, maltogenic amylase, mannanase, neutral protease, nuclease, oxidase, oxidoreductase, pectate lyase, pectinase, pectinesterase, pentosanase, perhydrolase, phospholipase, phytase, polygalacturonase, protease, proteinase, pullulanase, Rennet Enzyme, rhamnogalacturonase, subtilisin, tannase, transferase, transglutaminase, xanthanase, xylanase, xyloglucanase, preferably protease or alpha-amylase, or mixtures thereof, and / or
4. (d) the microorganism is Bacillus licheniformis or Bacillus aeolius, in particular strain DSM 15084, and / or
5. (e) the microorganism is inhibited by sporulation, preferably by a modification of the gene spolV (yqfD), in particular by a deletion of the gene spolV (yqfD) or parts thereof, and / or
6. (F) the microorganism is genetically modified.

1. (a) der Promotor eine Nukleinsäuresequenz umfasst, die ausgewählt ist aus
   1. (i) Nukleinsäuresequenz, die zu der in SEQ ID NO:1 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81,0%, 82,0%, 83,0%, 84,0%, 85,0%, 86,0%, 87,0%, 88,0%, 89,0%, 90,0%, 91,0%, 92,0%, 93,0%, 94,0%, 95,0%, 96,0%, 97,0%, 98,0%, 99,0%, 99,2%, 99,3%, 99,4%, 99,5% und ganz besonders bevorzugt zu 100% identisch ist; (ii) Nukleinsäuresequenz, die zu der in SEQ ID NO:2 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81,0%, 82,0%, 83,0%, 84,0%, 85,0%, 86,0%, 87,0%, 88,0%, 89,0%, 90,0%, 91,0%, 92,0%, 93,0%, 94,0%, 95,0%, 96,0%, 97,0%, 98,0%, 99,0%, 99,2%, 99,3%, 99,4%, 99,5% und ganz besonders bevorzugt zu 100% identisch ist, und/oder
2. (b) das Protein nicht natürlicherweise in dem Mikroorganismus vorhanden ist, und/oder
3. (c) das Protein eine Protease, bevorzugt ein Subtilisin, oder eine Alpha-Amylase ist, und/oder
4. (d) es sich bei dem Mikroorganismus um Bacillus licheniformis oder Bacillus aeolius, insbesondere den Stamm DSM 15084, handelt, und/oder
5. (e) der Mikroorganismus sporulationsgehemmt ist, vorzugsweise durch eine Veränderung des Gens spolV (yqtD), insbesondere durch eine Deletion des Gens spolV (yqfD) oder Teilen davon, und/oder
6. (f) der Mikroorganismus genetisch modifiziert ist.

Very particularly preferred embodiments of microorganisms according to the invention are characterized in that

1. (a) the promoter comprises a nucleic acid sequence selected from
   1. (i) Nucleic acid sequence corresponding to the nucleic acid sequence given in SEQ ID NO: 1 to at least 80% and increasingly preferably to at least 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5% and most preferably 100% identical; (ii) Nucleic acid sequence corresponding to at least 80% and more preferably at least 81.0%, 82.0%, 83.0%, 84.0%, 85.0% of the nucleic acid sequence given in SEQ ID NO: 2, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5% and most preferably 100% identical, and /or
2. (b) the protein is not naturally present in the microorganism, and / or
3. (c) the protein is a protease, preferably a subtilisin, or an alpha-amylase, and / or
4. (d) the microorganism is Bacillus licheniformis or Bacillus aeolius, in particular strain DSM 15084, and / or
5. (e) the microorganism is inhibited by sporulation, preferably by a modification of the gene spolV (yqtD), in particular by a deletion of the gene spolV (yqfD) or parts thereof, and / or
6. (F) the microorganism is genetically modified.

1. (a) der Promotor eine Nukleinsäuresequenz umfasst, die ausgewählt ist aus
   1. (i) Nukleinsäuresequenz, die zu der in SEQ ID NO:1 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81,0%, 82,0%, 83,0%, 84,0%, 85,0%, 86,0%, 87,0%, 88,0%, 89,0%, 90,0%, 91,0%, 92,0%, 93,0%, 94,0%, 95,0%, 96,0%, 97,0%, 98,0%, 99,0%, 99,2%, 99,3%, 99,4%, 99,5% und ganz besonders bevorzugt zu 100% identisch ist;
   2. (ii) Nukleinsäuresequenz, die zu der in SEQ ID NO:2 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81,0%, 82,0%, 83,0%, 84,0%, 85,0%, 86,0%, 87,0%, 88,0%, 89,0%, 90,0%, 91,0%, 92,0%, 93,0%, 94,0%, 95,0%, 96,0%, 97,0%, 98,0%, 99,0%, 99,2%, 99,3%, 99,4%, 99,5% und ganz besonders bevorzugt zu 100% identisch ist, und/oder
2. (b) das Protein nicht natürlicherweise in dem Mikroorganismus vorhanden ist, und/oder
3. (c) das Protein eine Protease gemäß SEQ ID NO:3 ist, und/oder
4. (d) es sich bei dem Mikroorganismus um Bacillus licheniformis oder Bacillus aeolius, insbesondere den Stamm DSM 15084, handelt, und/oder
5. (e) der Mikroorganismus sporulationsgehemmt ist, vorzugsweise durch eine Veränderung des Gens spolV (yqfD), insbesondere durch eine Deletion des Gens spolV (yqfD) oder Teilen davon, und/oder
6. (f) der Mikroorganismus genetisch modifiziert ist.

Further particularly preferred embodiments of microorganisms according to the invention are characterized in that

1. (a) the promoter comprises a nucleic acid sequence selected from
   1. (i) Nucleic acid sequence corresponding to the nucleic acid sequence given in SEQ ID NO: 1 to at least 80% and increasingly preferably to at least 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5% and most preferably 100% identical;
   2. (ii) Nucleic acid sequence corresponding to at least 80% and more preferably at least 81.0%, 82.0%, 83.0%, 84.0%, 85.0% of the nucleic acid sequence given in SEQ ID NO: 2, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5% and most preferably 100% identical, and /or
2. (b) the protein is not naturally present in the microorganism, and / or
3. (c) the protein is a protease according to SEQ ID NO: 3, and / or
4. (d) the microorganism is Bacillus licheniformis or Bacillus aeolius, in particular strain DSM 15084, and / or
5. (e) the microorganism is inhibited by sporulation, preferably by a modification of the gene spolV (yqfD), in particular by a deletion of the gene spolV (yqfD) or parts thereof, and / or
6. (F) the microorganism is genetically modified.

1. (a) der Promotor eine Nukleinsäuresequenz umfasst, die zu der in SEQ ID NO:2 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81,0%, 82,0%, 83,0%, 84,0%, 85,0%, 86,0%, 87,0%, 88,0%, 89,0%, 90,0%, 91,0%, 92,0%, 93,0%, 94,0%, 95,0%, 96,0%, 97,0%, 98,0%, 99,0%, 99,2%, 99,3%, 99,4%, 99,5% und ganz besonders bevorzugt zu 100% identisch ist; und/oder
2. (b) das Protein nicht natürlicherweise in dem Mikroorganismus vorhanden ist, und/oder
3. (c) das Protein eine Protease, bevorzugt eine Protease gemäß SEQ ID NO:3 ist, und/oder
4. (d) es sich bei dem Mikroorganismus um Bacillus licheniformis oder Bacillus aeolius, insbesondere den Stamm DSM 15084, handelt, und/oder
5. (e) der Mikroorganismus sporulationsgehemmt ist, vorzugsweise durch eine Veränderung des Gens spolV (yqfD), insbesondere durch eine Deletion des Gens spolV (yqfD) oder Teilen davon, und/oder
6. (f) der Mikroorganismus genetisch modifiziert ist.

Highly preferred embodiments of microorganisms according to the invention are characterized in that

1. (a) the promoter comprises a nucleic acid sequence which is at least 80% and more preferably at least 81.0%, 82.0%, 83.0%, 84.0%, 85% of the nucleic acid sequence given in SEQ ID NO: 2 , 0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95% , 0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5% and most preferably 100% is identical; and or
2. (b) the protein is not naturally present in the microorganism, and / or
3. (c) the protein is a protease, preferably a protease according to SEQ ID NO: 3, and / or
4. (d) the microorganism is Bacillus licheniformis or Bacillus aeolius, in particular strain DSM 15084, and / or
5. (e) the microorganism is inhibited by sporulation, preferably by a modification of the gene spolV (yqfD), in particular by a deletion of the gene spolV (yqfD) or parts thereof, and / or
6. (F) the microorganism is genetically modified.

1. (a) der Promotor eine Nukleinsäuresequenz umfasst, die zu der in SEQ ID NO:1 angegebenen Nukleinsäuresequenz zu mindestens 80% und zunehmend bevorzugt zu mindestens 81,0%, 82,0%, 83,0%, 84,0%, 85,0%, 86,0%, 87,0%, 88,0%, 89,0%, 90,0%, 91,0%, 92,0%, 93,0%, 94,0%, 95,0%, 96,0%, 97,0%, 98,0%, 99,0%, 99,2%, 99,3%, 99,4%, 99,5% und ganz besonders bevorzugt zu 100% identisch ist; und/oder
2. (b) das Protein nicht natürlicherweise in dem Mikroorganismus vorhanden ist, und/oder
3. (c) das Protein eine Protease, bevorzugt eine Protease gemäß SEQ ID NO:3 ist, und/oder
4. (d) es sich bei dem Mikroorganismus um Bacillus licheniformis oder Bacillus aeolius, insbesondere den Stamm DSM 15084, handelt, und/oder
5. (e) der Mikroorganismus sporulationsgehemmt ist, vorzugsweise durch eine Veränderung des Gens spolV (yqtD), insbesondere durch eine Deletion des Gens spolV (yqfD) oder Teilen davon, und/oder
6. (f) der Mikroorganismus genetisch modifiziert ist.

Further highly preferred embodiments of microorganisms according to the invention are characterized in that

1. (a) the promoter comprises a nucleic acid sequence which is at least 80% and more preferably at least 81.0%, 82.0%, 83.0%, 84.0%, 85% of the nucleic acid sequence given in SEQ ID NO: 1 , 0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95% , 0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.2%, 99.3%, 99.4%, 99.5% and most preferably 100% is identical; and or
2. (b) the protein is not naturally present in the microorganism, and / or
3. (c) the protein is a protease, preferably a protease according to SEQ ID NO: 3, and / or
4. (d) the microorganism is Bacillus licheniformis or Bacillus aeolius, in particular strain DSM 15084, and / or
5. (e) the microorganism is inhibited by sporulation, preferably by a modification of the gene spolV (yqtD), in particular by a deletion of the gene spolV (yqfD) or parts thereof, and / or
6. (F) the microorganism is genetically modified.

Microorganisms according to the invention are advantageously used in processes according to the invention for producing a protein. Consequently, a further subject of the invention is therefore the use of a microorganism according to the invention for the production of a protein, in particular of an enzyme.

Further objects of the present application are a bacterial culture comprising a recombinant bacterium according to the invention, and a vector comprising nucleic acid sequences as described herein.

All facts, objects and embodiments described for inventive method or microorganisms are also applicable to these subjects of the invention. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to the uses of the invention.

Examples

All molecular biology procedures are followed by standard methods such as those described in the Handbook of Fritsch, Sambrook and Maniatis "Molecular Cloning: a Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1989, or similar pertinent works. Enzymes, kits and devices were used according to the specifications of the respective manufacturers.

Example 1: Protease production

Comparison of the production of a protease (target protein, protein sequence according to SEQ ID NO: 3) in a shake flask with Bacillus aeolius DSM 15084 and Bacillus licheniformis in a complete medium with complex nitrogen source.

The two abovementioned Bacillus strains each contain an expression plasmid which in each case encodes a gene for a protease (target protein) and comprises the functional constitutive promoter P43. In both Bacillus aeolius DSM 15084 and Bacillus licheniformis, the gene spolV was functionally inactivated by deletion so that the strains no longer sporulate.

Both strains were cultured in 250 ml shake flasks in a complete medium with complex nitrogen source. Cultivation conditions: 37 ° C, 48 h, filling volumes 10 ml, 350 rpm.

After 48 h, the protease activity contained in the cell supernatant was determined via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF). The protease cleaves the substrate and releases pNA. The release of pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of enzymatic activity (see Del Mar et al., 1979). The measurement was carried out at a temperature of 25 ° C, at pH 8.6, and a wavelength of 410 nm. The measuring time was 5 min and the measuring interval 20s to 60s.

Compared to Bacillus licheniformis, the yield increased significantly with Bacillus aeolius (see Table 1). The protease activity from the AAPF assay in U / ml is given. Table 1: tribe Protease activity (%) Bacillus aeolius DSM15084 141% Bacillus licheniformis 100%

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 91/02792 [0006]
• WO 2008/086916 [0037]
• WO 2007/131656 [0037]
• WO 00/60060 [0038]
• WO 0300271 [0038]
• WO 03/054177 [0038]
• WO 07/079938 [0038]
• WO 98/12307 [0041]
• WO 97/14804 [0041]
• EP 1305432 [0041]
• EP 1240525 [0041]
• WO 96/29397 [0041]
• WO 02/099091 [0041]
• WO 98/45398 [0043]
• WO 2005/056782 [0043]
• WO 2004/058961 [0043]
• WO 2005/124012 [0043]

Cited non-patent literature

• S. F., Gish, W., Miller, W., Myers, E.W. & Lipman, DJ. Biol. 215: 403-410, and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, pp.3389-3402). [0027]
• Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500). [0027]
• Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. Biol. 302, 205-217 [0027]

Claims (10)

A recombinant bacterium comprising a nucleic acid sequence which a) a first nucleic acid sequence comprising a sequence according to SEQ ID NO: 1 or a sequence which is at least 80% identical to the nucleic acid sequence given in SEQ ID NO: 1; and b) a second nucleic acid sequence coding for a (target) protein comprises. The recombinant bacterium after Claim 1 where a) is the recombinant bacterium Bacillus licheniformis or aeolius, preferably Bacillus aeolius DSM 15084; and / or b) the first nucleic acid sequence comprises or consists of a sequence according to SEQ ID NO: 2 or a sequence which is at least 80% identical to the nucleic acid sequence given in SEQ ID NO: 2. The recombinant bacterium after Claim 1 or 2 wherein the first and second nucleic acid sequences are contained in a vector, in particular a plasmid. The recombinant bacterium after one of the Claims 1 - 3 wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked. The recombinant bacterium after one of the Claims 1 - 4 , wherein the second nucleic acid sequence encodes an enzyme, preferably a protease and more preferably a protease according to SEQ ID NO: 3. Use of the recombinant bacterium according to any one of Claims 1 - 5 for the expression of a (target) protein. A method for the expression of a (target) protein, wherein in at least one method step, a recombinant bacterium according to one of Claims 1 - 5 is used. A bacterial culture comprising a) a recombinant bacterium according to any one of Claims 1 - 5 ; and b) culture medium, wherein the culture medium is preferably full medium with a nitrogen source. Including a vector a) a first nucleic acid sequence comprising a sequence according to SEQ ID NO: 1 or a sequence which is at least 80% identical to the nucleic acid sequence given in SEQ ID NO: 1; and b) a second nucleic acid sequence encoding a (target) protein. The vector after Claim 9 wherein a) the first nucleic acid sequence and the second nucleic acid sequence are functionally coupled; and / or b) the second nucleic acid sequence encodes an enzyme, preferably a protease and more preferably a protease according to SEQ ID NO: 3; and / or c) the first nucleic acid sequence comprises or consists of a sequence according to SEQ ID NO: 2 or a sequence which is at least 80% identical to the nucleic acid sequence given in SEQ ID NO: 2; and / or d) the vector is a plasmid.