EP2291534A1 - Microorganisme à sécrétion optimisée - Google Patents

Microorganisme à sécrétion optimisée

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Publication number
EP2291534A1
EP2291534A1 EP09753816A EP09753816A EP2291534A1 EP 2291534 A1 EP2291534 A1 EP 2291534A1 EP 09753816 A EP09753816 A EP 09753816A EP 09753816 A EP09753816 A EP 09753816A EP 2291534 A1 EP2291534 A1 EP 2291534A1
Authority
EP
European Patent Office
Prior art keywords
acid sequence
corynebacterium
nucleic acid
microorganism
cofactor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09753816A
Other languages
German (de)
English (en)
Inventor
Sandra Scheele
Roland Freudl
Johannes Bongaerts
Karl-Heinz Maurer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
Henkel AG and Co KGaA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henkel AG and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of EP2291534A1 publication Critical patent/EP2291534A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)

Definitions

  • the invention is directed to microorganisms characterized in that they contain a nucleic acid sequence which is not naturally present in them and which comprises at least the following sequence segments: a) nucleic acid sequence coding for a protein which contains a cofactor, and b) nucleic acid sequence, which is at least 20% identical to the sequence given in SEQ ID NO.1 or a nucleic acid sequence structurally structurally related to this sequence, wherein the amino acid sequence encoded by the nucleic acid sequence b) interacts functionally with the amino acid sequence encoded by the nucleic acid sequence a) in such a way that at least the amino acid sequence encoded by the nucleic acid sequence a) is secreted by the microorganism, with the proviso that the microorganism belongs to the genus Corynebacterium.
  • microorganisms can be used to improve biotechnological production processes for proteins containing a cofactor. Therefore, the invention is further directed to uses of such microorganisms as well as methods in which such microorganisms are cultured, in particular fermentative uses and methods.
  • the present invention is in the field of biotechnology, in particular the production of recyclables by fermentation of microorganisms which are capable of forming the valuable substances of interest.
  • biotechnology in particular the production of recyclables by fermentation of microorganisms which are capable of forming the valuable substances of interest.
  • These include, for example, the production of low molecular weight compounds, such as food supplements or pharmaceutically relevant compounds, or of proteins, which in turn is due to their diversity, a large technical application.
  • the general aim is to obtain as high a product yield as possible in the fermentation, and secondly that these are discharged from the production organism by secretion from the cell into the production medium. In this way, it is possible to dispense with the complicated digestion of the cells and the further purification or work-up (downstream processing) is considerably simplified, since fewer undesired cell constituents have to be separated off.
  • Most of the technical enzymes that are currently used in detergents and cleaners, especially proteases and amylases, are naturally secreted.
  • the genes of these enzymes contain before the sequence, the for the enzyme (or proenzyme in the case of proteases) coded, a so-called signal sequence, often the so-called Sec signal sequence. This Sec signal sequence encodes an N-terminal signal peptide responsible for the translocation of the unfolded enzyme across the cytoplasmic membrane (sea-dependent secretion).
  • Tat signal peptides The prior art discloses various Tat signal peptides from different species, including E. coli, Bacillus subtilis and representatives of the genera Streptomyces and Corynebacterium.
  • a microorganism which is characterized in that it contains a nucleic acid sequence which is not naturally present in it and which comprises at least the following sequence segments: a) nucleic acid sequence coding for a protein which contains a cofactor, and b) nucleic acid sequence which is at least 20% identical to the sequence given in SEQ ID NO.1 or a nucleic acid sequence structurally homologous to this sequence, wherein the amino acid sequence encoded by nucleic acid sequence b) interacts functionally with the amino acid sequence encoded by nucleic acid sequence a) such that at least the amino acid sequence encoded by the nucleic acid sequence a) is secreted by the microorganism, with the proviso that the microorganism belongs to the genus Corynebacterium.
  • nucleic acid sequences in bacteria of the genus Corynebacterium bring about the secretion of proteins which contain a cofactor, in particular of a protein encoded by a nucleic acid sequence a) which is normally localized in the cytosol of the cell and therefore would not be secreted. Furthermore, they effect this to an extent that such a microorganism is suitable for the biotechnological production of the cofactor-containing protein, in particular in fermentative processes.
  • a microorganism belonging to the genus Corynebacterium is understood as meaning, in addition to bacteria of the genus Corynebacterium itself, also other coryneform bacteria, in particular those belonging to the genera Brevibacterium, Micrococcus, Microbacterium and Mycobacterium.
  • Coryneforms are bacterial cells with a characteristic cell morphology thickened at one end.
  • Corynebacterium itself is a genus of aerobic to facultative anaerobically living, Gram-positive bacteria whose representatives are usually between 3 to 5 microns long and whose cells have a mostly characteristic club shape, during growth, the shape can also switch between rod-shaped and coccoid. Often they do not form spores and are immobile.
  • In the cell wall of bacteria of the genus Corynebacterium are typically characteristically meso-2,6-diaminopimelic acids containing sugars galactose and arabinose and mycolic acids.
  • nucleic acid sequence is not a separate sequence of the microorganism, that is, in the wild-type form of the microorganism is not present in this form or can be isolated from this.
  • a natural nucleic acid sequence would therefore be present in the genome of the considered microorganism per se, ie in its wild-type form.
  • microorganisms according to the invention such a sequence has been introduced, preferably introduced selectively, or generated in it, for example and preferably with the aid of genetic engineering methods. Therefore, this sequence was not naturally present in the respective microorganism, so that the microorganism was enriched by this sequence.
  • this sequence is expressed by the microorganism.
  • the Nucleic acid sequence in a microorganism according to the invention thus in addition to the below-described nucleic acid sequences a) and b) further at least one or more sequences, in particular promoter sequences, for expression of the nucleic acid sequences a) and b).
  • the nucleic acid sequence in a microorganism according to the invention thus comprises at least two sequence segments, namely the nucleic acid sequences a) and b), and particularly preferably additionally one or more sequences, in particular promoter sequences, for expression of the nucleic acid sequences a) and b).
  • the nucleic acid sequence a) hereby codes for a protein which contains a cofactor, that is to say that protein which is to be secreted by the microorganism and thus discharged therefrom.
  • the nucleic acid sequence b) encodes an amino acid sequence which interacts with a translocation system used by the microorganism, in the present case by a bacterium of the genus Corynebacterium, in such a way that at least the amino acid sequence encoded by the nucleic acid sequence a) is secreted by the microorganism ,
  • the amino acid sequence encoded by this nucleic acid sequence b) therefore binds directly or indirectly to at least one component of the translocation system of the microorganism according to the invention.
  • direct bonding is meant a direct interaction which may be covalent or non-covalent; Indirect binding is understood to mean that the interaction can be via one or more other components, in particular proteins or other molecules, which act as adapters and accordingly have a bridging function between the amino acid sequence encoded by the nucleic acid sequence b) and a component of the bacterial translocation system, in which case, too, the interactions may be covalent or non-covalent.
  • the translocation system used is a Tat-dependent secretion, ie using at least one component of the Tat secretion system.
  • the nucleic acid sequence b) thus codes for a Tat signal sequence (Tat signal peptide), which is functional in Corynebacterium and allows a secretion of the nucleic acid sequence encoded by the nucleic acid sequence a).
  • Tat signal peptide a cofactor-containing protein (encoded by the nucleic acid sequence a)) is secreted by bacteria of the genus Corynebacterium due to the presence of the amino acid sequence encoded by the nucleic acid sequence b).
  • the amino acid sequences encoded by nucleic acid sequences b) and a) may be part of the same polypeptide chain, but may also be present on non-covalently linked polypeptide chains.
  • the cofactor-containing protein encoded by the nucleic acid sequence a) also differs from the amino acid sequence encoded by the nucleic acid sequence b) Cell is discharged.
  • Functional coupling / functional interaction of the amino acid sequence encoded by the nucleic acid sequence b) and the cofactor-containing protein encoded by the nucleic acid sequence a) is therefore to be understood as described, that the cofactor-containing protein encoded by the nucleic acid sequence a) due to the existence of the nucleic acid sequence encoded by the nucleic acid sequence b) is removed from the cell.
  • nucleic acid sequence b) in the cell would reduce or even eliminate the secretion of the cofactor-containing protein encoded by nucleic acid sequence a).
  • a functional interaction is achieved in that the amino acid sequence encoded by the nucleic acid sequence b) and the amino acid sequence encoded by the nucleic acid sequence a) are constituents of the same polypeptide chain, at least within the cell.
  • amino acid sequences encoded by the respective nucleic acid sequences a) and b) can also be present on separate polypeptide chains as long as the functional interaction of both sequences - ie the advantageousness and / or necessity of the presence of the amino acid sequence encoded by the nucleic acid sequence b) for the secretion of the from the nucleic acid sequence a) encoded cofactor-containing protein - at least within the cell is given, for example by direct or indirect binding of both amino acid sequences to each other, wherein all bonds may be covalent or non-covalent.
  • a functional interaction is determined by a first microorganism containing a nucleic acid sequence according to the invention, comprising at least one nucleic acid sequence b) and a nucleic acid sequence a), and expressing them, with a second microorganism, the possible only of the first microorganism distinguishes that it does not include the nucleic acid sequence b) is compared.
  • Both microorganisms are cultured under the same conditions, the conditions being such that at least the first microorganism expresses and secretes the cofactor-containing protein encoded by the nucleic acid sequence a).
  • the presence of a functional interaction results from the increased secretion of the cofactor-containing protein encoded by the nucleic acid sequence a) in the first microorganism in comparison with the second microorganism.
  • the nucleic acid sequence b) is in this respect at least 20% identical to the nucleic acid sequence given in SEQ ID NO.1 or at least 20% identical to the amino acid sequence encoded by it (stated in SEQ ID NO.2), in each case based on the total length of the stated sequences.
  • the nucleic acid sequence b) is more preferably at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%.
  • sequences allow efficient Tat-dependent secretion of a cofactor-containing protein in bacteria of the genus Corynebacterium.
  • sequences homologous to these sequences are meant a sequence encoding an amino acid sequence whose amino acid sequence causes such spatial folding of that sequence to interact with the translocation system used by Corynebacterium such that the cofactor-containing protein is secreted from the translocation system of the Corynebacterium cell becomes.
  • the amino acid sequence encoded by this nucleic acid sequence therefore binds directly or indirectly to at least one component of the translocation system of the microorganism according to the invention.
  • direct binding is meant a direct interaction
  • indirect binding means that the interaction can be via one or more further components, in particular proteins or other molecules, which act as adapters and accordingly have a bridging function between the nucleic acid sequence encoded by the structural homologous nucleic acid sequence Amino acid sequence and a component of the bacterial translocation system.
  • a preferred structural homologous nucleic acid sequence of the invention encodes a Tat signal peptide comprising three motifs: a positively charged N-terminal motif, a hydrophobic region, and a C-terminal region containing a short consensus motif (AxA), and preferably with this motif ends, which specifies the cleavage site by a signal peptidase.
  • an Tat signal peptide encoded by a structural homologous nucleic acid sequence of the invention comprises a consensus sequence [ST] -RRXFLK.
  • the amino acids are given in the one-letter code for amino acids in protein sequences which is familiar to the person skilled in the art, where x stands for any amino acid in the protein sequence and ST means that it can be serine or threonine.
  • amino acid sequence encoded by the structural homologous nucleic acid sequence is not any of the prior art Tat signal peptides, but an amino acid sequence that is recognized by, or interacts with, the translocation system used by Corynebacterium occurs and therefore causes a secretion of cofactor-containing proteins in bacteria of the genus Corynebacterium.
  • a microorganism of the genus Corynebacterium which allows Tat-mediated secretion of a cofactor-containing protein, in particular an enzyme, and which in particular enables a satisfactory product yield in a fermentation.
  • Act-mediated secretion is understood to mean that at least one component of the Tat secretion system of the subject microorganism is involved in the outflow of the cofactor-containing protein.
  • the microorganism is characterized in that the folding of the nucleic acid sequence encoded by the nucleic acid sequence a) takes place in the cytoplasm of the microorganism.
  • This is essential because many proteins that contain a cofactor are already partially or fully folded in the cytoplasm, and therefore, in particular, with it they are capable of receiving the cofactor normally present in the cytoplasm of the cell.
  • the tertiary structure of the protein In order to be able to take up a cofactor, therefore, the tertiary structure of the protein must be at least partially or completely formed.
  • the secretion of such a protein which has already at least partially assumed its tertiary structure, is usually much more complicated compared to the discharge of an amino acid sequence in its primary structure or at most secondary structure.
  • the microorganism is therefore characterized in that it secretes at least the amino acid sequence encoded by the nucleic acid sequence a) together with at least one cofactor.
  • Coenzymes are usually not proteins, but organic molecules that often carry chemical groups or serve to transfer chemical groups between different proteins or subunits of a protein complex. As a rule, they are not covalently linked to the protein carrying them, in particular enzyme.
  • coenzymes according to the invention as cofactors are selected from the group consisting of nicotinamide dinucleotide (NAD + ), nicotinamide dinucleotide phosphate (NADP + ), coenzyme A, tetrahydrofolic acid, quinones, especially menaquinone, ubiquinone, plastoquinone, vitamin K, ascorbic acid (vitamin C) ), Coenzyme F420, riboflavin (vitamin B2), adenosine triphosphate S-adenosylmethionine, 3'-phosphoadenosine 5'-phosphosulfate, coenzyme Q, tetrahydrobiopterin, cytidine triphosphate, nucleotide sugar, glutathione, coenzyme M, coenzyme B, methanofuran, tetrahydromethanopterin , Methoxatin.
  • the invention is not limited to the group consist
  • Prosthetic groups form a permanent part of the protein structure and are usually covalently bound to the protein, especially the enzyme.
  • the prosthetic group is particularly preferably selected as cofactor from the group consisting of flavin mononucleotide, flavin adenine dinucleotide (FAD), pyrroloquinoline quinone, pyridoxal phosphate, biotin, methylcobalamin, thiamine pyrophosphate, heme, molybdopterin and disulphites or thiols, especially lipoic acid ,
  • the invention is not limited to the said prosthetic groups as Cofactors limited, but also represent all other prosthetic groups cofactors in the context of the invention.
  • the microorganism is thus characterized in that the cofactor of the protein encoded by the nucleic acid sequence a) is a coenzyme or a prosthetic group.
  • the cofactor may be a coenzyme or a prosthetic group.
  • the cofactor comprises several coenzymes or several prosthetic groups, in particular two, three, four, five, six, seven or eight coenzymes or two, three, four, five, six, seven or eight prosthetic groups or combinations thereof .
  • cofactors are often important in electron transfer processes and are often part of enzymes that catalyze redox reactions, they can exist in different oxidation states. For example, NAD + , NADP + or FAD are the oxidized compounds, while NADH, NADPH and FADH 2 are the reduced counterparts.
  • cofactors may be protonated or deprotonated as acid or as base or, in general, provided that they change between several forms, are present in all possible forms, for example with or without the chemical group transferred by the respective cofactor, such as, for example, a methyl group or a phosphate group Quinone or hydroquinone or as disulfide or dithiol.
  • the amino acid sequence encoded by the nucleic acid sequence a) contains a cofactor which can not be assigned to any of the two groups of cofactors described above. It is essential that the amino acid sequence coded by the nucleic acid sequence a) contains at least one cofactor, it being generally necessary for the presence of the cofactor that the amino acid sequence has a tertiary structure, ie has reached a higher degree of folding in comparison with the amino acid sequence in their primary or secondary structure, where the primary structure is the linear sequence of the individual amino acids and the secondary structure is the presence of the basic structural elements alpha-helix and ⁇ -sheet in the otherwise largely linear amino acid sequence.
  • cofactors may also be, for example, metal ions (trace elements).
  • cofactors are preferably divalent or trivalent metal cations, for example Cu 2+ , Fe 3+ , Co 2+ or Zn 2+ .
  • Metal ions for example, can favor the attachment of the substrate or the coenzyme or, on the other hand, participate directly in the catalytic process as part of the active center or the prosthetic group. Furthermore, these metal ions cause the stabilization of the three-dimensional structure of proteins, in particular enzymes, and thus protect them from denaturation.
  • the microorganism is characterized in that the amino acid sequence encoded by the nucleic acid sequence b) is a signal sequence for the Tat secretion pathway.
  • Tat-dependent secretion allows the outflow of fully folded polypeptide chains. Therefore, this secretion pathway is particularly suitable for the secretion of proteins containing a cofactor.
  • gene expression is its translation into the gene product (s) encoded by said gene (s), ie into one protein or into several proteins.
  • gene expression comprises transcription, ie the synthesis of a ribonucleic acid (mRNA) based on the DNA (deoxyribonucleic acid) sequence of the gene and its translation into the corresponding polypeptide chain.
  • mRNA ribonucleic acid
  • the expression of a gene leads to the formation of the corresponding gene product which has and / or effects a physiological activity and / or contributes to an overall physiological activity in which several different gene products are involved.
  • the gene product, ie the corresponding protein is supplemented by a cofactor.
  • the microorganism is characterized in that the amino acid sequence encoded by the nucleic acid sequence b) and the amino acid sequence encoded by the nucleic acid sequence a) are constituents of the same polypeptide chain.
  • Tat-mediated secretion of a cofactor-containing protein is effected by interacting the Tat signal sequence portion of the polypeptide chain with the Tat-dependent translocation system used by Corynebacterium such that the cofactor-containing protein is removed from the translocation system of Corynebacterium Cell is discharged.
  • the Tat signal sequence portion of the polypeptide chain directs the entire polypeptide chain to a component of the Tat-dependent translocation system by binding directly or indirectly to that component, which binding is likely to be noncovalent.
  • nucleic acids encoding such polypeptide chains can be generated by per se known methods of altering nucleic acids. Such are illustrated, for example, in pertinent handbooks such as those of Fritsch, Sambrook, and Maniatis, "Molecular cloning: a laboratory manual,” CoId Spring Harbor Laboratory Press, New York, 1989.
  • the principle is to produce a nucleic acid containing the nucleic acid sequences a) - the coding sequence for the cofactor-containing protein - and b) - the sequence coding for the Tat signal sequence - in the same reading frame, wherein preferably the nucleic acid sequence b) upstream, ie at the 5 ' end of the nucleic acid sequence a) Therefore, in the resulting polypeptide, the Tat signal sequence is preferentially located at the N-terminus of the polypeptide, optionally between the nucleic acid sequences b) and a), ie between Tat signal sequence (Tat signal peptide) and the secreting cofactor-containing protein, a spacer.
  • the spacer may be 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in length.
  • the microorganism is characterized in that it is selected from the group of Corynebacterium ammoniagenes (Brevibacterium ammoniagenes), Corynebacterium glutamicum, Brevibacterium taipei, Micrococcus glutamicus, Brevibacterium roseum, Brevibacterium flavum, Corynebacterium herculis, Brevibacterium lactofermentum, Corynebacterium acetoacidophilum, Brevibacterium divaricatum, ammoniaphilum Brevibacterium saccharolyticum, Brevibacterium immariophilium, Microbacterium, Corynebacterium lilium, Corynebacterium callunae, Brevibacterium thiogenitalis, Corynebacterium afermentans, Corynebacterium amycolatum, Corynebacterium auris, Corynebacterium atypicum, Corynebacterium bovis, Coryne
  • the microorganism is selected from the group consisting of Corynebacterium ammoniagenes ATCC6872, Corynebacterium glutamicum ATCC13032, Brevibacterium Taipei ATCC13744, Micrococcus glutamicus ATCC 13761, Brevibacterium roseum ATCC13825, Brevibacterium flavum ATCC13826, Corynebacterium herculis ATCC13868, Brevibacterium lactofermentum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Brevibacterium divaricatum ATCC14020 , Brevibacterium saccharolyticum ATCC14066, Brevibacterium immariophilium ATCC14068, Microbacterium ammoniaphilum ATCC15354, Corynebacterium lilium ATCC15990, Corynebacterium callunae ATCC15991, Brevibacterium thiogenitalis ATCC19240, and most preferably the microorganism Corynebacterium glut
  • Gram-positive bacteria of the genus Corynebacterium have the fundamental difference from gram-negative bacteria to readily release secreted proteins into the medium surrounding the bacteria, usually the nutrient medium, from which, if desired, the expressed proteins are directly recovered or purified to let. They can be isolated directly from the medium or further processed. Preference is therefore given to secretion into the surrounding medium.
  • Gram-positive bacteria are related or identical to most of the organisms of origin for technically important enzymes and usually form even comparable enzymes, so they have a similar codon Usage and their protein synthesizer is naturally aligned accordingly.
  • Codon usage is understood to mean the translation of the genetic code into amino acids, i. which nucleotide sequence (triplet or base triplet) for which amino acid or for which function, for example the beginning and end of the region to be translated, binding sites for various proteins, etc., encoded.
  • nucleotide sequence triplet or base triplet
  • codon usage code for the same amino acids and can be better translated depending on the respective host. This possibly necessary rewriting thus depends on the choice of the expression system.
  • the present invention is applicable in principle to all microorganisms of the genus Corynebacterium, in particular to all fermentable microorganisms of this genus, and leads to the fact that can be realized by the use of such microorganisms as production organisms an increased product yield in a fermentation.
  • proteins containing a cofactor in particular enzymes, especially enzymes catalyzing redox reactions, are considered. Examples which may be mentioned are oxidases, peroxidases, hydrogenases, dehydrogenases, reductases, biotin-dependent redox enzymes, CO 2 -fixing enzymes, inter alia
  • microorganism ie by living cells
  • transformation a microorganism according to the invention
  • the preferred microorganisms are characterized by good microbiological and biotechnological handling. This concerns, for example, easy culturing, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins. Frequently, from the abundance of different according to the state of the Technique available systems, the optimal expression systems are determined experimentally for the individual case.
  • Preferred embodiments are those microorganisms which are regulatable in their activity due to genetic regulatory elements which are provided, for example, on the expression vector, but may also be present in these cells from the outset. For example, by controlled addition of chemical compounds that serve as activators, by changing the culture conditions or when reaching a specific cell density, these can be excited for expression. This allows a very economical production of the products of interest.
  • microorganisms may also be altered in their requirements of culture conditions, have different or additional selection markers, or express other or additional proteins.
  • it may be those microorganisms which express a plurality of products, in particular a plurality of cofactor-containing proteins, in particular enzymes, and secrete them into the medium surrounding the microorganisms.
  • the microorganisms according to the invention are cultured and fermented in a manner known per se, for example in discontinuous or continuous systems.
  • a suitable nutrient medium is inoculated with the microorganisms (host cells) and the product is harvested from the medium after an experimentally determined period of time.
  • Continuous fermentations are characterized by achieving a flow equilibrium in which over a relatively long period of time cells partly die off but also regrow and at the same time product can be removed from the medium.
  • the present invention is therefore suitable for the production of recombinant proteins, in particular enzymes. According to the invention, these are to be understood as meaning all genetic engineering or microbiological processes which are based on the genes for the products of interest being introduced into a microorganism according to the invention.
  • a gene according to the present invention comprises the nucleic acid sequences b) and a) explained in detail above, in order to effect a secretion of the cofactor-containing protein encoded by the nucleic acid sequence a), as a rule together with the gene encoded by the nucleic acid sequence b) Signal sequence (Tat signal peptide), and it particularly preferably additionally comprises one or more sequences, in particular promoter sequences, for expression of the nucleic acid sequences a) and b).
  • vectors in particular expression vectors, but also those that cause the gene of interest in the host cell in an existing genetic element such as the chromosome or other vectors can be inserted.
  • the functional unit of gene and promoter and any other genetic elements is referred to as expression cassette according to the invention.
  • expression cassette it does not necessarily have to exist as a physical entity.
  • vectors are understood to be elements consisting of nucleic acids which contain a gene for the purposes of the present invention. They can establish this in a species or cell line over several generations or cell divisions as a stable genetic element.
  • Vectors, especially when used in bacteria, are special plasmids, ie circular genetic elements.
  • vectors which serve the storage and thus to a certain extent also the genetic engineering, the so-called cloning vectors, and on the other hand those which fulfill the function of realizing the gene of interest in the host cell, that is, the expression of the protein.
  • cloning vectors those which fulfill the function of realizing the gene of interest in the host cell, that is, the expression of the protein.
  • expression vectors are referred to as expression vectors.
  • the nucleic acid (the gene) is suitably cloned into a vector.
  • Another object according to the invention is thus a vector which contains a gene in the sense of the present invention.
  • a vector which contains a gene in the sense of the present invention.
  • vectors may include those vectors derived from bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins.
  • vectors are able to establish themselves as stable units in the relevant host cells over several generations. It is irrelevant in the context of the invention whether they establish themselves as extrachromosomal units or integrate them into a chromosome or into chromosomal DNA. Which of the numerous systems known from the prior art is chosen depends on the individual case. Decisive factors may be, for example, the achievable copy number, the selection systems available, in particular antibiotic resistances, or the cultivability of the host cells capable of accepting the vectors.
  • Expression vectors comprise partial sequences which enable them to replicate in the microorganisms of the invention optimized for the production of proteins and to express the contained gene there.
  • Preferred embodiments are expression vectors which themselves carry the genetic elements necessary for expression.
  • expression is influenced by promoters that regulate transcription of the gene.
  • the expression may be carried out by the natural, originally located in front of a gene promoter, but also after genetic engineering, both by a promoter provided on the expression vector of the host cell and by a modified or a completely different promoter of another organism or another host cell.
  • Expression vectors may be regulatable via changes in culture conditions or addition of certain compounds, such as cell density or specific factors.
  • Expression vectors allow the associated protein to be produced heterologously, that is in a cell or host cell other than that from which it can naturally be obtained.
  • the cells may well belong to different organisms or come from different organisms.
  • homologous protein recovery from a gene cell naturally expressing the gene via a suitable vector is within the scope of the present invention, as long as the host cell is a microorganism designed according to the invention. This may have the advantage that natural translational-related modification reactions on the resulting protein are performed exactly as they would naturally occur.
  • An insertable expression system may further include additional genes, such as those provided on other vectors, which affect the production of the protein of the invention which contains a cofactor and is encoded by the nucleic acid sequence a). These may be modifying gene products or those which are to be purified together with the protein secreted according to the invention, for example in order to influence its enzymatic function. These may be, for example, other proteins or enzymes, inhibitors or elements which influence the interaction with various substrates.
  • a further subject of the invention is a process for the preparation of a protein containing a cofactor by a microorganism belonging to the genus Corynebacterium, comprising the following process steps: a) introduction of a nucleic acid sequence which is not naturally present in it and which contains at least comprises the following sequence sections: i. Nucleic acid sequence encoding a protein containing a cofactor, and ii.
  • nucleic acid sequence which is at least 20% identical to the sequence given in SEQ ID NO.1 or a nucleic acid sequence structhomologous to said sequence, into a microorganism, wherein the sequence sections i) and ii) are functionally coupled, b) expressing the nucleic acid sequence according to a ) in the microorganism
  • the method is therefore characterized in that at least the amino acid sequence encoded by the nucleic acid sequence a) is secreted by the microorganism together with at least one cofactor.
  • the method is further characterized in that the cofactor of the protein encoded by the nucleic acid sequence a) is a coenzyme or a prosthetic group.
  • a further subject of the invention is therefore processes for the preparation of a protein containing a cofactor, characterized in that these processes comprise, as a process step, the cultivation of a microorganism according to the invention as described above, which encodes the protein in its surrounding Medium secreted.
  • Cofactor-containing proteins in particular enzymes produced by such methods, are used in a variety of ways. These include, in particular, oxidases, peroxidases, hydrogenases, dehydrogenases, reductases, biotin-dependent enzymes, in particular CO 2 -fixing enzymes, or redox enzymes in general. Redox enzymes are used, for example, for enzymatic bleaching in detergents and cleaners. Also in the textile and leather industries they serve the processing of natural raw materials. Furthermore, all enzymes which can be prepared according to the process according to the invention can in turn be used in the sense of biotransformation as catalysts for chemical reactions.
  • the process is accordingly characterized in that the protein is an enzyme, in particular one which is selected from the group consisting of redox enzyme, oxidase, peroxidase, hydrogenase, dehydrogenase, reductase, biotin-dependent enzyme, CO 2 -fixing enzyme, protease, amylase, cellulase, lipase, hemicellulase, pectinase, mannanase or combinations thereof.
  • redox enzyme oxidase, peroxidase, hydrogenase, dehydrogenase, reductase, biotin-dependent enzyme, CO 2 -fixing enzyme, protease, amylase, cellulase, lipase, hemicellulase, pectinase, mannanase or combinations thereof.
  • Proteins, and in particular enzymes are optimized for their intended use and, in particular, genetically modified to give them improved properties for their intended use.
  • the enzymes produced in the process according to the invention can therefore be the respective wild-type enzymes or further developed variants. Under wild-type enzyme is to be understood that the enzyme is present in a naturally occurring organism or in a natural habitat can be isolated from this.
  • An enzyme variant is understood as meaning enzymes which have been generated from a precursor enzyme, for example a wild-type enzyme, by altering the amino acid sequence.
  • the alteration of the amino acid sequence is preferably carried out by mutations, wherein amino acid substitutions, deletions, insertions or combinations thereof may be made.
  • the incorporation of such mutations into proteins is well known in the art and to those skilled in the art of enzyme technology.
  • 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 the product produced, for example the recombinant protein. All fermentation processes which are suitable for the production of the recombinant proteins therefore represent preferred embodiments of this subject matter of the invention. Such a process is considered suitable if a corresponding product is formed.
  • proteins that contain a cofactor in particular enzymes, in particular enzymes that catalyze redox reactions, are considered.
  • redox enzymes are oxidases, peroxidases, hydrogenases, dehydrogenases, reductases, biotin-dependent redox enzymes, CO 2 -fixing enzymes, among others
  • the optimum conditions for the production processes used, for the microorganisms and / or the products to be prepared on the basis of the previously optimized culture conditions of the strains concerned according to the knowledge of the skilled person, for example in terms of fermentation volume, media composition, oxygen supply or stirrer speed, must be determined experimentally.
  • Fermentation processes which are characterized in that the fermentation is carried out via a feed strategy, are also contemplated.
  • the media components consumed by the ongoing cultivation are fed;
  • considerable increases in both the cell density and in the dry biomass and / or above all the activity of the product of interest can be achieved.
  • the fermentation can also be designed so that unwanted metabolites are filtered out or neutralized by the addition of buffer or matching counterions.
  • the product produced can be harvested subsequently from the fermentation medium. It was preferably secreted into the medium according to the invention. This fermentation process is correspondingly preferred over the preparation of the product from the dry mass, but requires the provision of suitable secretion markers and transport systems.
  • Microorganisms according to the invention are therefore advantageously used in the described method according to the invention and are used in these methods to produce a product, in particular a protein which contains a cofactor. Consequently, a further subject of the invention is accordingly the use of a microorganism described above for the production of a protein which contains a cofactor.
  • the use is characterized in that the protein is an enzyme.
  • the enzyme is selected from the group consisting of redox enzyme, oxidase, peroxidase, hydrogenase, dehydrogenase, reductase, biotin-dependent enzyme, CO 2 -fixing enzyme, protease, amylase, cellulase, lipase, hemicellulase, pectinase, mannanase or combinations hereof.
  • SoXy is a normally cytosolic cofactor-containing protein
  • a Tat-specific signal peptide was added to allow the export of the protein along with its cofactor via the Tat pathway of Corynebacterium glutamicum , It is the heterologous signal peptide TorA, which mediates a strictly Tat-dependent membrane transport in E. coli.
  • the gene of the SoXy was amplified by polymerase chain reaction (PCR), wherein an EcoRI site for ligation in the Corynebacterium glutamicum expression vector pEKEx2 (Eikmanns et al. (1991) Gene 102: 93-98) was inserted at the 3 'end (see Figure 1).
  • the DNA fragment of the TorA signal peptide attached to the first hundred base pairs of the SoXy gene was synthesized and cloned into the expression vector pEKEx2 using the NotI site located in the initial region of the SoXy (see Figure 1).
  • Corynebacterium glutamicum ATCC13032 (Abe et al., (1967) J Gen Appl Microbiol, 13: 279-301) was transformed with the SoXy expression vector.
  • the activity of the SoXy was examined by means of the qualitative activity assay for hydrogen peroxide-forming enzymes in agar plate agar plates using 4-chloronaphthol (Delagrave, S., et al., (2001) Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase , Prot. Eng., 14: 261-267).
  • the more hydrogen peroxide is formed the more likely a blue coloration of the medium occurs.
  • an incipient blue staining in the presence of the SoXy expression vector could be detected within 4 h after the addition of 30 ⁇ l of the culture supernatant (see FIG. The control with empty vector, however, showed no blue color.
  • microorganisms according to the invention are capable of efficiently secreting functional cofactor-containing proteins, above all those which are normally localized in the cytosol.
  • FIG. 1 Cloning scheme for the sorbitol xylitol oxidase. Shown is the expression vector pEKEx2 into which the DNA sequence of the E. coli TorA signal peptide and attached to the 5 'end of the SoXy gene was introduced via the PstI and the NotI interface. In a second cloning step, the 3 'end of the SoXy gene was then inserted via the NotI and EcoRI sites.
  • Figure 2 Coomassie stained polyacrylamide gel for the localization of the sorbitol xylitol oxidase SoXy in samples of the supernatant. Comparison of empty vector (c) in Corynebacterium glutamicum with the three SoXy transformants S1, S2 and S3. Cultivation took place in CGXII medium, the induction of SoXy was carried out with 100 ⁇ M IPTG over a period of 18 hours.
  • Figure 3 Qualitative activity test for hydrogen peroxide-forming enzymes in colonies on agar plate using 4-chloronaphthol. Comparison of empty vector (K) in Corynebacterium glutamicum with two transformants (1 and 2) containing the SoXy expression vector.

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Abstract

L’invention concerne des protéines qui comprennent un cofacteur et qui peuvent être sécrétées de manière améliorée dans un microorganisme qui appartient au genre Corynebacterium, à condition que le microorganisme contienne une séquence d’acides nucléiques qui n’est pas présente naturellement dans celui-ci et qui comprend au moins les sections de séquence suivantes : a) une séquence d’acides nucléiques qui code pour une protéine qui contient un cofacteur et b) une séquence d’acides nucléiques qui est identique à au moins 20 % à la séquence donnée dans SEQ ID NO.1 ou une séquence d’acides nucléiques de structure homologue à cette séquence. Selon l’invention, la séquence d’acides aminés codée par la séquence d’acides nucléiques b) coopère fonctionnellement avec la séquence d’acides aminés codée par la séquence d’acides nucléiques a) de manière à ce qu’au moins la séquence d’acides aminés codée par la séquence d’acides nucléiques a) soit sécrétée par le microorganisme.
EP09753816A 2008-05-29 2009-05-20 Microorganisme à sécrétion optimisée Withdrawn EP2291534A1 (fr)

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