Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/67—General methods for enhancing the expression
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
  • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the invention is in the field of biotechnology, especially microbial
• Protein synthesis More particularly, the invention relates to a method of producing proteins by genetically modified microorganisms and further proposing microorganisms used in such methods. The invention further relates to uses of such microorganisms for protein production.
• 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.
• 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.
• biotechnological production of the relevant microorganisms are cultured in fermenters, which are designed according to the metabolic properties of the microorganisms.
• the microorganisms metabolize the substrate offered 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.
• complex protein rich raw materials are typically used as a substrate besides a carbon source (typically glucose).
• 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.
• Microorganisms are focused on the desired product.
• the object of the present invention is to increase the product yield, in particular of a protein, in a microbial fermentation.
• the invention relates to a method for producing a protein by a
• Microorganism comprising the process steps
• a method according to the invention optionally further comprises the further method step
• the introduction and expression of the auxiliary protease into the microorganism unexpectedly does not result in a decrease in the product yield of protein (target protein), for example due to the proteolytic activity of the expressed auxiliary protease that could degrade synthesized target protein. Rather, the expression of the auxiliary protease causes the product yield of protein to be increased.
• the substrate specificity of the background proteases and of the auxiliary protease thus advantageously complements the substrate digestion by the microorganism.
• auxiliary protease it is not necessary for the auxiliary protease to have a significant portion of the fermentation product.
• Auxiliary protease has a beneficial effect on the expression of the protein and consequently on the product yield of protein, but its content in the fermentation product is preferably small or not detectable.
• their proportion in the fermentation product is less than 25% and increasingly preferably less than 20% by weight, 15% by weight, 10% by weight, 8% by weight, 7% by weight, 6% by weight. , 5 wt .-%, 2.5 wt .-%, 2 wt .-%, 1, 5 wt .-%, 1 wt .-%, 0.5 wt .-%. 0.1% by weight and 0.05% by weight.
• the fermentation product in this respect is the composition in which the protein is contained after a process according to the invention has been carried out, the microorganism has been cultured in a culture medium and optionally the microorganism has been digested to release the protein from it, if it has not been secreted by it.
• the microorganisms or their fragments were separated from the fermentation product.
• 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 compared with a similar method, which differs from a method according to the invention only by dispensing with the method step b), whereby in process step c) consequently no auxiliary protease is expressed in the microorganism.
• An expression construct is a nucleic acid sequence that causes the protein or the auxiliary protease to be expressed in the microorganism. It includes the genetic information, that is, the nucleic acid sequence (gene) that is responsible for the protein or for the
• RNA ribonucleic acid
• expression comprises transcription, that is to say the synthesis of a messenger ribonucleic acid (mRNA) based on the DNA (deoxyribonucleic acid) sequence of the gene and its translation into the corresponding polypeptide chain, the
• 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).
• a gene regulatory sequence here is any nucleic acid sequence whose presence in the respective
• Microorganism the transcriptional frequency of that nucleic acid sequence influenced, preferably increased, which codes for the protein or the auxiliary protease. It is preferably a promoter sequence, since such a sequence is essential for the expression of a nucleic acid sequence.
• 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.
• a promoter is understood as meaning a DNA sequence which enables the regulated expression of a gene.
• a promoter is understood as meaning a DNA sequence which enables the regulated expression of a gene.
• Promoter sequence is part of a gene and is often at its 5 'end, and thus before the RNA coding region.
• the promoter sequence in an expression construct according to the invention 5 ' is located downstream of that for the protein or the auxiliary protease
• a promoter is therefore preferably a DNA sequence with promoter activity, i. a DNA sequence to which at least one
• Transcription factor binds to initiate transcription of a gene, at least transiently.
• 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. Such a promoter sequence is thus naturally present in the microorganism. Alternatively, a promoter of a
• Expression construct also be 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 first expression construct encodes a protein. It thus comprises a nucleic acid sequence encoding this protein.
• any nucleic acid sequence which can be translated into a protein can be used for this purpose.
• This is the protein which is to be produced by means of a method according to the invention (target protein).
• target protein Preferably, it is an enzyme, more preferably an enzyme as described below.
• the second expression construct encodes the auxiliary protease.
• the auxiliary protease differs from the protein, i. they and the protein have different amino acid sequences.
• the auxiliary protease comprises an amino acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 1 to at least 50% and more preferably at least 55%, 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84%. , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and most preferably to 100% identical.
• the auxiliary protease has an amino acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 1 at least 50% identical and more preferably at least 55%, 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and most preferably is 100% identical.
• 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 based on the BLAST algorithm established and commonly used in the prior art (see, for example, Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool. "J. Mol. Biol. 215: 403-410, and Altschul, Stephan F. Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J.
• 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.
• 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 default default parameters.
• 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.
• the auxiliary protease also has a proteolytic activity. It is thus catalytically active, i. it is an active enzyme.
• the determination of the enzyme activity can in this regard - matched to the particular type of enzyme - carried out in the usual way. Methods for determining activity are familiar to the expert in the field of enzyme technology and are routinely used by him. Method for the determination of
• protease activity are disclosed, for example, in Tenside, Vol. 7 (1970), pp. 125-132.
• the proteolytic activity can be further determined by the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA).
• 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 the enzymatic activity is (see Del Mar et al., 1979).
• the measurement is carried out at a temperature of 25 ° C, at pH 8.6 and a wavelength of 410 nm.
• the measuring time is 5 min. at a measuring interval of 20s to 60s.
• the protease activity is preferably indicated in PE (protease units).
• 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 art of biotechnology Examples of such methods are the chemical synthesis or the polymerase chain reaction (PCR), optionally in combination with other standard molecular biological and / or chemical or biochemical methods.
• PCR polymerase chain reaction
• the present invention is particularly suitable for the recombinant production of
• the first and the second expression construct are introduced into a microorganism, preferably by transformation.
• the introduction of the respective expression construct or parts thereof preferably takes place via vectors, in particular expression vectors.
• only parts of the expression construct preferably at least the nucleic acid coding for the protein or the auxiliary protease, are introduced into the microorganism in such a way that the finished expression construct only arises in the microorganism.
• Hilfsprotease is used.
• the gene coding for the auxiliary protease can be functionally linked in this manner with an endogenous promoter of the microorganism and then represents the second expression construct.
• the concept of introduction thus includes the possibility that an expression construct is introduced completely into the microorganism, preferably transformed, but also the possibility that only part of the
• Expressionskonstruktes particularly preferably the nucleic acid encoding the Hilfsprotease introduced into the microorganism, preferably transformed, and the complete
• Expression construct only arises in the microorganism. At least part of the
• Vectors are known to those skilled in the biotechnology field. Especially when used in bacteria, they are special plasmids, ie circular genetic elements.
• the expression constructs are preferably cloned into a vector.
• the vectors may include, for example, those that are different from bacterial Derived plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins. With the other genetic elements present in each case, vectors are able to survive in the microorganisms over several
• 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- ⁇ -D-thiogalactopyranoside (IPTG), which is used as the activator of the bacterial lactose operon (lac operon).
• a method according to the invention is characterized in that the protein is not naturally present in the microorganism and / or the auxiliary protease is not naturally present in the microorganism.
• the protein or the auxiliary protease is not a separate protein or enzyme of the microorganism. Consequently, the protein or auxiliary protease can 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 or the auxiliary protease 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.
• a protein that is not naturally present in the microorganism is not
• auxiliary protease present in the microorganism or the respective 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 with the protein or the auxiliary protease or the respective nucleic acid sequence coding therefor.
• one protein or the auxiliary protease may quite naturally be present in another microorganism - relevant for the consideration is exclusively the microorganism used in the process.
• Both the protein and the auxiliary protease may not be naturally present in the
• Microorganism be present.
• the protein may naturally be present in the microorganism and the auxiliary protease may not be naturally present in the microorganism.
• the protein is not naturally present in the microorganism and the auxiliary protease is naturally present in the microorganism.
• a method according to the invention is characterized in that the auxiliary protease replaces at least one chromosomally encoded protease in the microorganism.
• at least one present chromosomal protease is replaced by a heterologous protease which functions as auxiliary protease according to the invention.
• the nucleic acid sequence coding for the auxiliary protease is transformed into a
• the chromosomally encoded protease is functionally inactivated.
• the microorganism expresses the auxiliary protease.
• the original promoter used for the chromosomally encoded protease can be used, such that the second expression construct in this case, in addition to the nucleic acid sequence coding for the helper protease, the promoter sequence of the chromosomally encoded protease, i. a natural promoter sequence of the microorganism.
• a promotor specifically provided for the auxiliary protease can be used so that the second expression construct in this case does not comprise the promoter sequence of the chromosomally encoded protease in addition to the nucleic acid sequence coding for the auxiliary protease.
• the second expression construct in this case comprises a non-natural promoter sequence of the microorganism. If a promoter intended for the auxiliary protease is used, it was preferably used together with the
• Nucleic acid sequence coding for the auxiliary protease inserted into the chromosomal nucleic acid sequence coding for a protease of the microorganism can be used for the expression of the auxiliary protease.
• a method according to the invention is characterized in that the auxiliary protease is expressed in the microorganism in addition to the chromosomally encoded proteases.
• an additional proteolytic activity is provided in the microorganism by a heterologous protease which functions as auxiliary protease according to the invention.
• the second expression construct thus preferably comprises at least the nucleic acid sequence coding for the auxiliary protease and a promoter intended for the expression of the auxiliary protease.
• the second expression construct in this case comprises a non-natural promoter sequence of the microorganism.
• a method according to the invention is characterized in that the auxiliary protease replaces at least one chromosomally encoded protease in the microorganism comprising an amino acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 2 at least 80% and more preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, of the amino acid sequence 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% identical.
• the chromosomally encoded protease has an amino acid sequence which corresponds to that shown in SEQ ID NO.
• a method according to the invention is characterized in that the expression of the protein is enhanced by the expression of the auxiliary protease.
• the expression of the auxiliary protease in preferred embodiments according to the invention causes the product yield of protein to be increased. This is due to increased expression, i. an increased synthesis of the protein by the microorganism achieved.
• the auxiliary protease is involved in the hydrolysis of substrate protein and, optionally together with other, chromosomally encoded proteases of the
• Microorganism an improved digestion of substrate protein, which is provided to the microorganism during its cultivation.
• more of the necessary precursor molecules, in particular amino acids, are available for the synthesis of the protein.
• This situation is determined by the frequency of transcription of the nucleic acid sequence coding for the protein and its translation into the desired protein, ie by the number of proteins produced per unit of time.
• the translation step is included in the determination of expression.
• a method is used, which differs from the method according to the invention by the absence of the auxiliary protease. Such a comparison is made according to the invention in microorganisms of the same type and under the same conditions in order to ensure the comparability of the measurements.
• the method is characterized in that the protein is an enzyme, in particular a protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, ⁇ -glucosidase, pectinase, carrageenase, perhydrolase, oxidase , Oxidoreductase or a lipase.
• the protein is a protease.
• 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 further developed form under the trade name Alcalase® from Novozymes A / S, Bagsvasrd, Denmark.
• 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.
• 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, sold under the tradename Protosol® by Advanced Biochemicals Ltd., Thane, India, under the name
• 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.
• proteases from Bacillus gibsonii and Bacillus pumilus, which are disclosed in international patent applications WO2008 / 086916 and WO2007 / 131656.
• amylases are the Bacillus licheniformis ⁇ -amylases, from Bacillus
• amyloliquefaciens or from Bacillus stearothermophilus and in particular 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.
• this ⁇ -amylase is available from the company Novozymes under the trade name Duramyl® and Termamyl®ultra, from the company Danisco / Genencor under the name Purastar®OxAm and from the company Daiwa Seiko Inc., Tokyo, Japan, as Keistase®.
• the ⁇ -amylase from Bacillus amyloliquefaciens is sold by the company Novozymes under the name BAN®, and derived variants of the Bacillus stearothermophilus ⁇ -amylase under the names BSG® and Novamyl®, also from the company
• Amylase Powerase® from the company Danisco / Genencor and the amylases Amylase-LT®, Stainzyme® and Stainzyme plus®, the latter from the company Novozymes.
• variants of these enzymes obtainable by point mutations can be prepared according to the invention.
• Further preferred amylases are disclosed in International Publication WO 00/60060, WO 03/00271 1, WO 03/054177 and WO07 / 079938, the disclosure of which is therefore expressly referred to or their
• Amylases to be prepared according to the invention are also preferably ⁇ -amylases.
• lipases or cutinases are those originally from Humicola lanuginosa
• 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®.
• 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.
• 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.
• cellulases examples include fungal,
• cellulases from AB Enzymes are Econase® and Ecopulp®.
• 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 Danisco / Genencor are "Genencor detergent cellulase L" and lndiAge®Neutra. Also variants of these enzymes obtainable by point mutations can be prepared according to the invention.
• cellulases are Thielavia terrestris cellulase variants disclosed in International Publication WO 98/12307, cellulases from Melanocarpus, in particular Melanocarpus albomyces, which are described in International
• WO 97/14804 discloses cellulases of the EGIII type from Trichoderma reesei which are disclosed in European patent application EP 1 305 432 or variants obtainable therefrom, in particular those disclosed in the European patent applications
• 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.
• hemicellulases include, for example, mannanases, xanthanlyases,
• 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 ⁇ -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.
• oxidoreductases for example oxidases, oxygenases, catalases, peroxidases, such as halo, chloro, bromo, lignin, glucose or manganese peroxidases,
• Dioxygenases or laccases phenol oxidases, polyphenol oxidases
• Suitable commercial products are Denilite® 1 and 2 from Novozymes.
• Further enzymes are disclosed in international patent applications WO 98/45398, WO 2005/056782, WO 2004/058961 and WO 2005/124012.
• the method is characterized in that the microorganism is a bacterium.
• Bacteria are preferred microorganisms in methods of the invention. 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 bacteria in the fermentation technology rich experience. Both Gram-negative and Gram-positive bacteria may be suitable.
• Gram-negative bacteria such as Escherichia coli
• Gram-negative bacteria can also be designed such that they eject the expressed proteins not only into the periplasmic space but into the medium surrounding the bacterium.
• gram-positive bacteria such as Bacilli or microorganisms such as Actinomycetes or other representatives of Actinomycetales have no outer membrane, so that secreted proteins are released immediately into the medium surrounding the bacteria, usually the nutrient medium, from which the expressed proteins can be purified. They can be isolated directly from the medium or further processed.
• a preferred bacterium according to the invention is selected from the genera of Escherichia, Klebsiella, Burkholderia, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas, Pseudomonas and Vibrio.
• Bacillus licheniformis is one selected from the group of Escherichia coli, Klebsiella planticola, Burkholderia glumae, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor, Stenotrophomonas maltophilia and Vibrio metschnikovii. Very particularly preferred is Bacillus licheniformis.
• the microorganism may also be a eukaryotic cell which is characterized by having a nucleus.
• eukaryotic cells are capable of post-translationally modifying the protein formed. Examples thereof are fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This may be particularly advantageous, for example, if the proteins are to undergo specific modifications in the context of their synthesis that enable such systems. Among the modifications that eukaryotic systems particularly related to the
• Protein synthesis include, for example, the binding of low molecular weight
• oligosaccharide modifications may be desirable, for example, to reduce the allergenicity of an expressed protein.
• coexpression with the enzymes naturally produced by such cells, such as cellulases or lipases, may be advantageous. Furthermore, they can be advantageous.
• thermophilic fungal expression systems especially for expression
• microorganisms to be used according to the invention may be modified with regard to their requirements of the culture conditions, have different or additional selection markers or express other or additional proteins.
• it may also be those microorganisms which express several target proteins transgene. Preferably, they secrete the protein (target protein) into the medium surrounding the microorganism.
• microorganisms used in the process according to the invention can be conventionally cultivated and fermented, for example in discontinuous or continuous systems.
• Inoculated microorganisms and the protein harvested from the medium after a period to be determined experimentally 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 based on a method of producing a protein according to the invention provide embodiments thereof
• Fermentation processes which are characterized in that the fermentation is carried out via a feed strategy, come in particular into consideration.
• the fermentation is carried out via a feed strategy
• 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 preferred over isolation of the protein from the microorganism, ie product preparation from the cell mass (dry matter). This can be accomplished, for example, by the provision of suitable microorganisms or by one or more suitable secretion markers or mechanisms and / or transport systems for the microorganisms to secrete the protein into the fermentation medium.
• the isolation of the protein from the host cell ie, a purification of the same from the cell mass, carried out, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.
• Another object of the invention is a microorganism obtained by a process comprising the process steps
• the protein is not naturally present in the microorganism and / or
• auxiliary protease is not naturally present in the microorganism and / or
• the auxiliary protease replaces at least one chromosomally encoded protease in the microorganism, or that the auxiliary protease is expressed in the microorganism in addition to the chromosomally encoded proteases, and / or
• the microorganism is a bacterium, preferably one selected from the genera Escherichia, Klebsiella, Burkholderia, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas, Pseudomonas and Vibrio, more preferably one selected from the group from the group of Escherichia coli, Klebsiella planticola, Burkholderia glumae, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor, Stenotrophomonas mal
• Microorganisms according to the invention are advantageously used in processes according to the invention for producing a protein. Consequently, therefore, is another object
• the invention relates to the use of a microorganism according to the invention for producing a protein, in particular an enzyme.
• oligonucleotides were used as primers (listed in 5 '- 3' orientation) were used: aprE1 AAACTGCAGCGTACCGGCTCCTTGAAAGG (SEQ ID NO. 3)
• aprE2 (SEQ ID NO: 4) GAGAAGACTAAGCTTCCCGAGGATCCCGAATGACAGG
• aprE3 (SEQ ID NO: 5) TTCGGGATCCTCGGGAAGCTTAGTCTTCTCATTCTGATGAAGGTTG
• aprE4 (SEQ ID NO: 6) AAATCTAGATAGACCTCGAAGAGGAGAAGC
• tet5 (SEQ ID NO. 7) GCGGATCCAAACGGGCCATATTGTTGTATAAG
• tet6 (SEQ ID NO: 8) CCAAGCTTCTCTCGTATCTTTTATTCAGCAATCGC
• aprE7 (SEQ ID NO.9) CGAAAGTATGAATAGACCGCTTCAGCCTGGCAGGGAAAGAGGTCC
• aprE8 (SEQ ID NO 10) CTGCCGCTCAATAACATATTCTAACAAATAGCATATAGAAAAGCTA
• target9 (SEQ ID NO: 1 1) CTGCCAGGCTGAAGCGGTCTATTCATACTTTCGAACTGAACATTTTT
• Sequence sections of 1033 bp and 1015 bp upstream and downstream of the aprE gene amplified (flanks A and B). Due to corresponding overhangs of the primers aprE2 and aprE3, the two flanks were fused by fusion polymerase chain reaction (PCR) using the outer primers aprE1 and aprE4.
• PCR fusion polymerase chain reaction
• the 2018 bp fusion PCR product was cloned into the PstI and XbaI interfaces of the vector pE194 (Horinouchi & Weisblum, J. Bacteriol. (150), p. 804ff (1982)).
• the tet gene was amplified by means of the primers tet5 and tet6 from the plasmid pBC16 (Bernhard et al., J. Bacteriol. (133), p. 897ff, (1978)) and into the central interfaces BamHI and HindIII of the fused aprE Flanks Moniert (see Figure 1).
• the resulting plasmid pRK25 was transformed into Bacillus licheniformis. Selection was made with 5 ⁇ g / ml erythromycin as well as with 15 ⁇ g / ml tetracycline at 30 ° C.
• clones with integrated vector were identified by growth to 0.3 ⁇ g / ml erythromycin. After renewed cultivation at 42 ° C. but without the use of erythromycin, clones were obtained which were screened for tetracycline resistance and by PCR for the successful exchange of the aprE gene for the tet gene (Bacillus licheniformis AaprE :: tet, compare FIG 2).
• sequence sections of 1061 bp and 1058 bp upstream and downstream of the aprE gene were amplified by means of the primers aprE1 and aprE7 and the primers aprE8 and aprE4 (flanks C and D).
• the gene coding for an auxiliary protease according to SEQ ID NO. 1 was amplified from the plasmid pCB76R49CK by means of the primers ziel9 and zieH0. Due to corresponding overhangs of the primers aprE7 and ziel9 or aprE8 and pull 0 were by fusion PCR using the outer primers aprE1 and aprE4, the two flanks with the
• helper protease (two bands), AP217 is the target protease). Compared to the target protease, only a very small amount of auxiliary protease is present in the culture supernatant.
• FIG. 1 Schematic representation of the construction of the vectors pRK25 and pRK19 for the chromosomal exchange of the gene which is required for a protease according to SEQ ID NO. 2 encoded (aprE) against the tetracycline resistance gene tet or the gene which is suitable for an auxiliary protease according to SEQ ID NO. 1 coded.
• Arrows indicate the reading frames of the genes ydeD, aprE and yhfN, ⁇ mark primers, bars indicate PCR products.
• Figure 2 Genort of the aprE gene in Bacillus licheniformis.
• the wild-type situation (above), the pRK19-mediated exchange of the aprE gene for the tetracycline resistance gene tet (center) and the pRK25-mediated exchange of the tetracycline resistance gene tet for the gene described for an auxiliary protease according to SEQ ID NO. 1 coded (below).
• FIG. 3 Relative yields of the production strains Bacillus licheniformis / target protease and Bacillus licheniformis AaprE :: auxiliary protease / target protease. The tribes were in one
• FIG. 4 Secretion analysis of the fermentation supernatant in a method according to the invention or in a microorganism according to the invention (Bacillus licheniformis
• AaprE auxiliary protease / target protease, cf. also FIG. 3) by means of native polyacrylamide gel electrophoresis.
• the increased protease activity is based essentially on the activity of the target protease, since their expression and secretion was significantly increased (t: culture time, AP103: auxiliary protease (two bands), AP217: target protease, Mpr: further size marker).

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Tropical Medicine & Parasitology (AREA)
• Virology (AREA)
• Enzymes And Modification Thereof (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (12)

Applications Claiming Priority (2)

Publications (1)

Family

ID=45952535

Family Applications (1)

Country Status (14)

Cited By (3)

Families Citing this family (2)

Citations (24)

Family Cites Families (5)

• 2011
  • 2011-04-13 DE DE102011007313A patent/DE102011007313A1/de not_active Withdrawn
• 2012
  • 2012-04-05 JP JP2014504258A patent/JP6099627B2/ja not_active Expired - Fee Related
  • 2012-04-05 WO PCT/EP2012/056262 patent/WO2012139964A1/de active Application Filing
  • 2012-04-05 EP EP12713714.9A patent/EP2697376B1/de not_active Not-in-force
  • 2012-04-05 BR BR112013025951A patent/BR112013025951A2/pt not_active Application Discontinuation
  • 2012-04-05 CN CN201280028836.1A patent/CN103608457A/zh active Pending
  • 2012-04-05 US US14/110,724 patent/US9663798B2/en not_active Expired - Fee Related
  • 2012-04-05 ES ES12713714.9T patent/ES2639114T3/es active Active
  • 2012-04-05 MX MX2013011816A patent/MX348766B/es active IP Right Grant
  • 2012-04-05 KR KR1020137029683A patent/KR20140019436A/ko not_active Application Discontinuation
  • 2012-04-05 RU RU2013150276A patent/RU2642324C2/ru not_active IP Right Cessation
  • 2012-04-05 PL PL12713714T patent/PL2697376T3/pl unknown
  • 2012-04-05 CA CA2831473A patent/CA2831473A1/en not_active Abandoned
  • 2012-04-05 DK DK12713714.9T patent/DK2697376T3/en active
• 2016
  • 2016-12-15 JP JP2016243563A patent/JP2017079768A/ja not_active Withdrawn

Patent Citations (24)

Non-Patent Citations (11)

Also Published As

Similar Documents

Legal Events