Patent Application
20110129894
The invention is directed towards microorganisms containing a nucleic acid sequence that is not naturally present in them, and that includes at least the following sequence segments:
The present invention is in the field of biotechnology, particularly the manufacture of valuable substances by fermentation of microorganisms capable of forming such valuable substances of interest. These include, for example, the manufacture of low molecular weight compounds (e.g., food supplements or pharmaceutically relevant compounds) or proteins, which, due to their diversity, there is a large range of industrial applications.
There exists substantial prior art covering fermentation of microorganisms, particularly on the industrial scale. It ranges from optimization of the strains in question with respect to rates of formation and nutrient utilization, through technical design of the fermentor, to recovery of valuable materials from the cells in question and/or fermentation medium. Both genetic and microbiological as well as process engineering and biochemical approaches are involved.
For economical production of proteins (e.g., enzymes), one generally seeks firstly to obtain the highest possible product yield in the fermentation, and secondly to eject the product from the producing organism by secretion from the cell into the production medium. This avoids costly digestion of the cells and, because less unwanted cell components have to be separated, significantly simplifies further purification and downstream processing. The majority of industrial enzymes are secreted naturally, particularly proteases and amylases which are employed in washing and cleaning agents. The genes of these enzymes have a signal sequence, often called the Sec-signal sequence, before the sequence that codes for the enzyme (or proenzyme in the case of proteases). This Sec-signal sequence codes an N-terminal signal peptide responsible for translocation of the unfolded enzyme over the cytoplasm membrane (see dependent secretion).
Moreover, Tat- (“Twin-arginine translocation”) dependent secretion of proteins is known from the prior art (see inter alia, Schaerlaekens et al., J. Biotechnol., Vol. 112, pp. 279-288 (2004)). This is conveyed over Tat-signal peptides. Various Tat-signal peptides from various species are known from the prior art, including E. coli and Bacillus subtilis, as well as from members of the genera Streptomyces and Corynebacterium.
International Patent Application Publication No. WO 2002/022667 shows that completely folded polypeptide chains are ejected over the Tat-secretion path and this secretion path is also suitable for secretion of proteins comprising a cofactor. It is therefore proposed to use the Tat-secretion path for heterologous expression of proteins. However, this application likewise shows that not every Tat-signal peptide in all microorganisms or in all bacteria also effects a corresponding secretion. For example, the PhoD-signal peptide from Bacillus subtilis is not detected from the Tat-secretion system of E. coli per se (see, Example 4 of WO 2002/022667), but rather only after genetic modification thereof (here by recombinant expression of two components of the B. subtilis Tat-secretion system). The article by Pop et al., J. of Biological Chemistry, Vol. 277(5), pp. 3268-3273 (2002) also comes to the same conclusion.
Accordingly, a heterologous expression system that allows Tat mediated secretion of a cofactor-containing protein, particularly an enzyme, in different microorganisms cannot be concluded from the prior art. In particular, this is not disclosed for bacteria of the genus Corynebacterium. Furthermore, no such system is known for Corynebacterium which enables a satisfactory product yield in fermentation.
Accordingly, the present invention seeks to improve biotechnological production of proteins, particularly those having a cofactor, especially by using bacteria of the genus Corynebacterium. Additionally, the invention seeks to increase, in a fermentation process, the product yield of proteins, particularly those having a cofactor, again by using bacteria of the genus Corynebacterium. In particular, a microorganism should be made available, especially one of the genus Corynebacterium which secretes in an improved manner proteins having a cofactor, and whose use further preferably increases the product yield in a fermentation process.
Accordingly, the present invention provides for a microorganism having a nucleic acid sequence that is not naturally present in it, and that includes at least the following sequence segments:
It was surprisingly found that such nucleic acid sequences in bacteria of the genus Corynebacterium effect secretion of proteins having a cofactor, especially from protein coded from a nucleic acid sequence a) that is normally localized in the cytosol of the cell and was therefore not secreted. Moreover, they affect this in such a degree that a microorganism of this type is suitable for biotechnological production of the cofactor-containing protein, especially in fermentation processes.
A microorganism belonging to the genus Corynebacterium is also understood to mean, in addition to bacteria of the genus Corynebacterium itself, additional coryneform bacteria, particularly those belonging to the genera Brevibacterium, Micrococcus, Microbacterium and Mycobacterium.
Coryneforms are bacterial cells having a characteristic haunch-like, thickened cell morphology at one end. Corynebacterium itself is a genus of aerobic to facultatively anaerobic living, gram-positive bacteria whose representatives are mostly from 3 to 5 μm long and whose cells exhibit a mostly characteristic thickened shape, wherein the shape can also change during growth between rod shaped and coccus shaped. Often they do not form any spores and are non-motile. In general, the cell wall of bacteria of the genus Corynebacterium typically comprise meso-2,6-diamino pimelic acids, the sugars galactose and arabinose, and mycolic acids. In this context, “not naturally present” means that the nucleic acid sequence is not an innate sequence of the microorganism (i.e., is not present in this form in the wild type form of the microorganism or cannot be isolated from it). Consequently, a natural nucleic acid sequence would therefore be present in the genome of the given microorganism per se (i.e., in its wild type form). In contrast, a sequence of this type would be introduced into microorganisms according to the invention, preferably introduced in a targeted manner, or produced in them, for example, preferably with the aid of genetic engineering processes. Therefore this sequence is not naturally present in the particular microorganism, so that the microorganism is enriched by this sequence. This sequence is preferably expressed by the microorganism. Accordingly, the nucleic acid sequence in a microorganism according to the invention preferably further contains, in addition to nucleic acid sequences a) and b) described below, at least one or more sequences, especially promoter sequences for expressing nucleic acid sequences a) and b).
Accordingly, the nucleic acid sequence in a microorganism according to the invention contains at least two sequence segments, namely nucleic acid sequences a) and b), and preferably further contains one or more sequences, particularly promoter sequences, for expressing nucleic acid sequences a) and b). Nucleic acid sequence a) codes here for a protein having a cofactor (i.e., the protein that is secreted from the microorganism and thereby intended to be ejected from it). Nucleic acid sequence b) codes here for an amino acid sequence that interacts with a translocation system used from the microorganism; thus, in the present case from a bacterium of the genus Corynebacterium so that at least the amino acid sequence coded by nucleic acid sequence a) is secreted from the microorganism. Consequently, the amino acid sequence coded from this nucleic acid sequence b) binds directly or indirectly to at least one component of the translocation system of the microorganism according to the invention. Direct binding is understood to mean a direct interaction that can be covalent or non-covalent; indirect binding is understood to mean that the interaction can occur over one or more additional components, especially proteins or other molecules that act as an adapter and accordingly have a bridging function between the amino acid sequence coded by nucleic acid sequence b) and a component of the bacterial translocation system, wherein here as well the interactions can be covalent or non covalent.
The translocation system that is used preferably concerns a Tat-dependent secretion (i.e., uses at least one component of the Tat-secretion system). Nucleic acid sequence b) therefore codes for a Tat-signal sequence (Tat-signal peptide) that is functional in Corynebacterium and enables secretion of the amino acid sequence coded by nucleic acid sequence a). In this way, due to the presence of the amino acid sequence coded by nucleic acid sequence b), a cofactor-containing protein (coded by nucleic acid sequence a)) is secreted from bacteria of the genus Corynebacterium.
Amino acid sequences coded by nucleic acid sequences b) and a) can be components of the same polypeptide chain, but can also be present on polypeptide chains that are not covalently bound with one another. It is possible, for example, that non-covalently bound polypeptide chains nevertheless interact with one another, especially due to non-covalent bonds, in such a way that the cofactor-containing protein coded by nucleic acid sequence a) is also ejected from the cell due to the existence of the amino acid sequence coded by the nucleic acid sequence b). By a functional coupling/functional interaction of the amino acid sequence coded by nucleic acid sequence b) and that of the cofactor-containing protein coded by nucleic acid sequence a) as described, the issue therefore is to understand that the cofactor-containing protein coded by nucleic acid sequence a) is ejected out of the cell due to the existence of the amino acid sequence coded by nucleic acid sequence b). Without the presence of the amino acid sequence coded by nucleic acid sequence b) in the cell, secretion of the cofactor-containing protein coded by nucleic acid sequence a) would therefore be diminished or not at all present. An exemplary and particularly preferred functional interaction of this type is achieved in that the amino acid sequence coded by nucleic acid sequence b) and the amino acid sequence coded by nucleic acid sequence a) are components of the same polypeptide chain, at least inside the cell. In principle, however, the amino acid sequences coded from the relevant nucleic acid sequences a) and b) can also be present on separate polypeptide chains as long as the functional interaction of both sequences—i.e., the advantage and/or necessity of the presence of the amino acid sequence coded by nucleic acid sequence b) for the secretion of the cofactor-containing protein coded by nucleic acid sequence a)—is given, at least inside the cell, for example, by direct or indirect binding of both amino acid sequences to one another, wherein all bonds can be covalent or non-covalent.
In comparative experiments a functional interaction of this type is determined wherein a first microorganism containing a nucleic acid sequence according to the invention having at least one nucleic acid sequence b) and one nucleic acid sequence a) and expresses them, is compared with a second microorganism that differs from the first microorganism only in that it does not contain nucleic acid sequence b). Both microorganisms were cultivated under the same conditions, wherein the conditions were chosen such that at least the first microorganism expresses and secretes the cofactor-containing protein coded by nucleic acid sequence a). The presence of a functional interaction is demonstrated by increased secretion of the cofactor-containing protein coded by nucleic acid sequence a) by the first microorganism when compared with the second microorganism.
Nucleic acid sequence b) in this regard is at least 20% identical to the nucleic acid sequence listed in SEQ ID NO. 1 or at least 20% identical to the amino acid sequence coded by it (listed in SEQ ID NO. 2), each based on total length of the listed sequences. Nucleic acid sequence b) is increasingly preferably identical to at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% identical to the nucleic acid sequence listed in SEQ ID No. 1 or to at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% identical to the amino acid sequence coded by it (listed in SEQ ID NO. 2). Unexpectedly, these sequences enable an efficient Tat-dependent secretion of a cofactor-containing protein in bacteria of the genus Corynebacterium.
Instead of the cited sequences that enable a secretion of a cofactor-containing protein, their structurally homologous sequences can also be used. A structurally homologous nucleic acid sequence is understood to mean a sequence that codes an amino acid sequence whose order of amino acids causes such a spatial folding of this sequence that it interacts in such a way with the employed translocation system of Corynebacterium that the cofactor-containing protein of the translocation system is ejected from the Corynebacterium cell. Consequently, the amino acid sequence coded by this nucleic acid sequence binds directly or indirectly to at least one component of the translocation system of the microorganism according to the invention. A direct binding is understood to mean a direct interaction; an indirect binding is understood to mean that the interaction can occur over one or more additional components, especially proteins or other molecules that act as an adapter and accordingly have a bridging function between the amino acid sequence coded by the structurally homologous nucleic acid sequence and a component of the bacterial translocation system
A preferred structurally homologous nucleic acid sequence according to the invention codes for a Tat signal peptide containing three motifs: a positively charged N-terminal motif, a hydrophobic region and a C-terminal region that comprises a short consensus motif (A-x-A) and preferably ends with this motif that specifies the cleavage site by a signal peptidase. A Tat signal peptide coded by a structurally homologous nucleic acid sequence according to the invention likewise preferably includes a consensus sequence [ST]-R-R-x-F-L-K. The amino acids are listed using the one letter code commonly used by experts for amino acids in protein sequences, wherein x is any amino acid in the protein sequence and ST means serine or threonine. It is important that the amino acid sequence coded by the structurally homologous nucleic acid sequence is not just any Tat signal peptide of the prior art, but is rather an amino acid sequence recognized by the translocation system of the used Corynebacterium, or as described, interacts with this and therefore effects secretion of cofactor-containing proteins in bacteria of the genus Corynebacterium.
In this way a microorganism of the genus Corynebacterium is inventively provided which enables a Tat-mediated secretion of a cofactor-containing protein, especially an enzyme, and which in particular enables a satisfactory product yield in a fermentation process. Tat-mediated secretion is understood to mean that at least one component of the Tat secretion system of the considered microorganism is involved in ejection of the cofactor-containing protein.
In a separate embodiment, the microorganism is characterized in that the folding of the amino acid sequence coded by the nucleic acid sequence a) occurs in the cytoplasm of the microorganism. This is of considerable importance, as many proteins having a cofactor are already partially or completely folded in the cytoplasm, especially as they are then capable of taking up the cofactor generally present in the cytoplasm of the cell. In order to be able to take up a cofactor, the tertiary structure of the protein must therefore be at least partially or completely formed. Secretion of such a protein that has already at least partially assumed its tertiary structure is generally disproportionately more complicated in comparison to ejection of an amino acid sequence in its primary structure or, at best, secondary structure. In the first named case it is necessary, at least as far as possible, to retain the tertiary structure (i.e., the spatial form)—for example, also so as not to lose again a non-covalently bound cofactor—whereas in the second case, a not yet folded protein is secreted which first assumes its later tertiary structure after the secretion step. Ejection of such cofactor-containing proteins whose tertiary structure has already formed in the cytoplasm, especially those having been heterologously expressed in the bacterium, therefore represents a particular challenge that is made possible with the present invention, principally in regard to biotechnological fermentation processes for the recombinant production of such cofactor-containing proteins. Consequently, in a preferred embodiment of the invention the microorganism is characterized in that it secretes at least the amino acid sequence coded by nucleic acid sequence a) together with at least one cofactor.
Cofactors are classified into different groups. Two large groups are the coenzymes and the prosthetic groups. Coenzymes typically are not proteins but rather are organic molecules that often carry chemical groups or serve to transfer chemical groups between different proteins or subunits of a protein complex. They are generally non-covalently bonded with the protein, particularly the enzyme that carries them. As cofactors, inventively particularly preferred coenzymes are chosen from nicotinamide dinucleotide (NAD+), nicotinamide dinucleotide phosphate (NADP+), coenzyme A, tetrahydrofolic acid, quinones, especially menaquinone, ubiquinone, plastoquinones, vitamin K, ascorbic acid (vitamin C), coenzyme F420, riboflavin (vitamin B2), adenosine triphosphate S-adenosyl methionine, 3′-phosphoadenosine-5′-phosphosulfate, coenzyme Q, tetrahydrobiopterin, cytidine triphosphate, nucleotide sugar, glutathione, coenzyme M, coenzyme B, methanofuran, tetrahydromethanopterin, methoxatin. However, the invention is not limited to these coenzymes as cofactors; rather, all further coenzymes represent cofactors in the context of the invention.
Prosthetic groups form a permanent part of the protein structure and in general are covalently bound to the protein, especially the enzyme. As the cofactor, the prosthetic group is particularly preferably chosen from flavin mononucleotide, flavin adenine dinucleotide (FAD), pyrroloquinoline quinone, pyridoxal phosphate, biotin, methylcobalamin, thiamine pyrophosphate, heme, molybdopterin and disulfides or thiols, especially lipoic acid. However, the invention is not limited to these prosthetic groups as cofactors; rather all further prosthetic groups represent cofactors in the context of the invention.
In a further preferred embodiment of the invention, the microorganism is characterized in that the cofactor of the protein for which nucleic acid sequence a) codes is a coenzyme or a prosthetic group. In particular, coenzymes or prosthetic groups of this type can be present in various oxidation states. Moreover, the cofactor can concern a coenzyme or a prosthetic group. However it is also possible that the cofactor includes a plurality of coenzymes or a plurality of prosthetic groups, especially two, three, four, five, six, seven or eight coenzymes or two, three, four, five, six, seven or eight prosthetic groups or combinations thereof. As cofactors are frequently important in electron transfer processes and, for example, are often components of enzymes that catalyze redox reactions, they can be present in different oxidation states. Thus NAD+, NADP+ or FAD can be the oxidized compounds, whereas NADH, NADPH as well as FADH2 can be the reduced counterparts. Analogously, cofactors can be present in their protonated or deprotonated form as the acid or base respectively, or generally—in so far as they alternate between a plurality of forms—can be present in all possible forms, for example, with or without the chemical group transferred from the cofactor under consideration, such as a methyl group or a phosphate group, as a quinone or hydroquinone or as a disulfide or dithiol.
Furthermore it is possible that the amino acid sequence coded by nucleic acid sequence a) contains a cofactor assigned to neither of the two previously mentioned groups of cofactors. It is important that the amino acid sequence coded by nucleic acid sequence a) have above all a cofactor, wherein in general it is required for the presence of the cofactor that the amino acid sequence has a tertiary structure (i.e., has attained a higher degree of folding when compared with the amino acid sequence in its primary or secondary structure, wherein primary structure refers to the linear sequence of the individual amino acids and secondary structure to the existence of the basic structural elements α-helix and β-pleated sheet in the otherwise essentially linear amino acid sequence). Formation of a spatial configuration of secondary structural elements towards one another is part of the formation of the tertiary structure in the context of the present application. Additional cofactors can also be metal ions (trace elements), for example. Preferably, such cofactors concern divalent or trivalent metal cations such as Cu2+, Fe3+, Co2+ or Zn2+. Metal ions, for example, can facilitate the addition of the substrate or coenzyme, or can participate directly as a component of the active center or of the prosthetic group in the catalytic process. In addition, these metal ions can effect stabilization of the three-dimensional structure of proteins, especially enzymes, and protect them from being denatured.
In a particularly preferred embodiment of the invention, the microorganism is characterized in that the amino acid sequence coded by nucleic acid sequence b) is a signal sequence for the Tat secretion path. As previously mentioned, Tat-dependent secretion enables ejection of completely folded polypeptide chains. Consequently, this secretion path is particularly suited for secretion of proteins having a cofactor. Accordingly, in bacteria of the genus Corynebacterium, it is inventively preferred to use the Tat secretion path for secretion of heterologously expressed proteins having a cofactor.
Expression of a gene is its translation into the gene product(s) coded from this gene (i.e., into a protein or into a plurality of proteins). In general, the gene expression includes the transcription, that is, synthesis of a ribonucleic acid (mRNA) on the basis of the DNA (deoxyribonucleic acid) sequence of the gene and its translation into the corresponding polypeptide chain. Expression of a gene leads to formation of the corresponding gene product that exhibits a physiological activity and/or effects and/or contributes to a higher-level physiological activity, to which a plurality of different gene products are involved. In the context of the present invention, the gene product (i.e., the corresponding protein) is further complemented by a cofactor.
In a further preferred embodiment of the invention, the microorganism is characterized in that the amino acid sequence coded by nucleic acid sequence b) and the amino acid sequence coded by nucleic acid sequence a) are components of the same polypeptide chain. In this way, Tat mediated secretion of a cofactor-containing protein is effected, especially of an enzyme, in that the Tat signal sequence fraction of the polypeptide chain interacts with the Tat-dependent translocation system of the Corynebacterium in such a way that the cofactor-containing protein of the translocation system is ejected out of the Corynebacterium cell. The Tat signal sequence fraction of the polypeptide chain therefore directs the whole polypeptide chain to a component of the Tat-dependent translocation system, in that it directly or indirectly binds to this component, wherein the bond is probably non-covalent.
Nucleic acids that code for such polypeptides can be produced by known processes for modification of nucleic acids. Some are illustrated, for example, in pertinent handbooks such as that from Fritsch, Sambrook and Maniatis “Molecular cloning: a laboratory manual”, Cold Spring Harbour Laboratory Press, New York, 1989. The principle is producing a nucleic acid that includes in the same reading frame nucleic acid sequences a)—the coding sequence for the cofactor-containing protein—and b)—the coding sequence for the Tat signal sequence, wherein nucleic acid sequence b) is preferably located up stream (i.e., at the 5′-end of nucleic acid sequence a)). Consequently, the Tat signal sequence is preferably located in the resulting polypeptide at the N-terminus of the polypeptide. A spacer can be optionally located between nucleic acid sequences b) and a) (i.e., between Tat signal sequence (Tat signal peptide) and the cofactor-containing protein to be secreted). The spacer can 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 long. On the nucleic acid level, this means that a spacer sequence is located between nucleic acid sequences b) and a), and based on the genetic code, the spacer is three times as many nucleotides long as amino acids comprised in the spacer.
In a further preferred embodiment of the invention the microorganism is characterized in that it is chosen from Corynebacterium ammoniagenes (Brevibacterium ammoniagenes), Corynebacterium glutamicum, Brevibacterium taipei, Micrococcus glutamicus, Brevibacterium roseum, Brevibacterium flavum, Corynebacterium herculis, Brevibacterium lactofermentum, Corynebacterium acetoacidophilum, Brevibacterium divaricatum, Brevibacterium saccharolyticum, Brevibacterium immariophilium, Microbacterium ammoniaphilum, Corynebacterium lilium, Corynebacterium callunae, Brevibacterium thiogenitalis, Corynebacterium afermentans, Corynebacterium amycolatum, Corynebacterium auris, Corynebacterium atypicum, Corynebacterium bovis, Corynebacterium callunae, Corynebacterium casei, Corynebacterium confusum, Corynebacterium diphtheriae, Corynebacterium equi, Corynebacterium halotolerans, Corynebacterium hanseni, Corynebacterium glucuronolyticum, Corynebacterium jeikeium, Corynebacterium minutissimum, Corynebacterium mycetoides, Corynebacterium nigricans, Corynebacterium pseudodiptheriticum, Corynebacterium pseudotuberculosis, Corynebacterium resisters, Corynebacterium striatum, Corynebacterium tuscaniae, Corynebacterium tuscaniense, Corynebacterium ulcerans, Corynebacterium urealyticum, Corynebacterium xerosis.
The microorganism is preferably further chosen from 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, and Brevibacterium thiogenitalis ATCC19240, wherein the microorganism Corynebacterium glutamicum is particularly preferred.
Such bacteria are characterized by short generation times and low demands on cultivation conditions. In this manner, cost effective processes can be established. Moreover, there exists an extensive wealth of experience with bacteria in fermentation technology. For a wide variety of reasons that have to be experimentally determined for each individual case, such as nutrient sources, product formation rate, time required etc., various bacterial strains can be suitable for a specific production.
Gram-positive bacteria of the genus Corynebacterium are basically different from gram-negative bacteria in that they immediately release secreted proteins into the medium surrounding the bacteria, in general the culture medium from which, when desired, the expressed proteins can be directly recovered or purified. They can be isolated directly from the medium or be further processed. Therefore a secretion preferably occurs into the surrounding medium. In addition, gram-positive bacteria are related or identical to most of the organisms of origin of industrially important enzymes and themselves mostly produce comparable enzymes, so that they have similar codon usage and their protein synthesis apparatus is naturally appropriately configured.
Codon usage refers to the translation of the genetic code in amino acids (i.e., which nucleotide order (triplet or base triplet) codes for which amino acid or for which function, for example, beginning and end of the area to be translated, binding sites for different proteins, etc.). Thus each organism, especially each production strain, possesses a defined codon usage. Bottlenecks can occur in the protein biosynthesis if the codons laying on the transgenetic nucleic acid in the host cell face a comparatively low number of charged tRNAs. In contrast, synonym codons code for the same amino acid and can be better translated depending on the relevant host. This optionally necessary transcription therefore depends on the choice of expression system. Especially for nucleic acid sequences expressed from unknown, possibly non-cultivatable organisms, an appropriate matching of codon usage can be necessary on the microorganism that is to express them.
Fundamentally, the present invention is applicable to all microorganisms of the genus Corynebacterium, particularly to all fermentable microorganisms of this genus, and leads to an increased production yield that can be achieved in fermentation by adding such microorganisms as the production organisms. The products formed during fermentation are proteins having a cofactor, especially enzymes, among which are enzymes that catalyze redox reactions. Examples include oxidases, peroxidases, hydrogenases, dehydrogenases, reductases, biotin-dependent redox enzymes, and CO2-fixing enzymes.
In vivo synthesis of such a product (i.e., by living cells) requires transfer of the associated gene into a microorganism according to the invention, that is, its transformation. Those microorganisms are preferred which can be genetically handled with ease, for example, in relation to transformation with the expression factor and its stable establishment. In addition, preferred microorganisms are characterized by good microbiological and biotechnological handleability. For example, this relates to ease of cultivation, high growth rates, low demands on fermentation media and good production rates and secretion rates for foreign proteins. Frequently, the optimum expression system for the individual case must be experimentally determined from the abundance of different systems available from the prior art. Those microorganisms, which can be regulated in their activity due to genetic regulation elements that are, for example, made available to the expression vector, but which can also be already present in these cells, represent preferred embodiments. For example, they can be stimulated to expression by controlled addition of chemical compounds that serve as activators, by changing cultivation conditions, or by attaining a specific cell density. This allows for very economical production of the products of interest.
The microorganisms can be further modified in regard to their demands on the conditions of culture, exhibit other or additional selection markers or express other or additional proteins. In particular, the microorganisms can concern those that express a plurality of products, especially a plurality of cofactor-comprising proteins, especially enzymes, and secrete them into the medium surrounding the microorganisms.
Microorganisms according to the invention are cultivated and fermented in conventional manner, for example, in discontinuous or continuous systems. In the first case, a suitable nutrient medium is inoculated with the microorganisms (host cells) and the product is harvested from the medium after an experimentally determined time. Continuous fermentations are characterized by the attainment of a flow equilibrium, in which, for a comparatively long time, cells partially die off but also grow again, with product removed from the medium.
The present invention is therefore suitable for producing recombinant proteins, especially enzymes. According to the invention this is understood to include all genetic engineering or microbiological processes that are based on incorporating genes for the products of interest into an inventive microorganism. In the context of the present invention, a gene of this type includes the nucleic acid sequences b) and a) that were previously mentioned in detail and which effect a secretion of the cofactor-containing protein coded by the nucleic acid sequence a), generally together with the Tat signal sequence (Tat signal peptide) coded by the nucleic acid sequence b), and it particularly preferably further includes one or more sequences, especially promoter sequences, for the expression of the nucleic acid sequences a) and b). In this regard the gene in question is inserted by means of vectors, especially expression vectors, but also by those that cause the gene of interest in the host organism to be incorporated into an already present genetic element such as the chromosome or other vectors. The functional unit of gene and promoter and possibly additional genetic elements is inventively designated as the expression cassette. However, for this it must not also necessarily be present as a physical unit.
In the context of the present invention, vectors refer to elements that consist of nucleic acids, which comprise a gene in the context of the present invention. They are able to establish the gene as a stable genetic element in a species or a cell line over several generations or cell divisions. Vectors, particularly when used in bacteria, especially plasmids, are therefore circular genetic elements. In gene technology, a differentiation is made between those vectors that serve the storage and thereby to a certain extent also the technical genetic work, the so called cloning vectors, and those that fulfill the function of realizing the gene of interest in the host cells (i.e., to enable the expression of the protein in question). These vectors are called expression vectors.
In the context of the present invention, the nucleic acid (the gene) is suitably cloned into a vector. Accordingly, a further inventive subject matter is a vector that in the context of the present invention comprises a gene. For example, this includes those vectors that derive from bacterial plasmids, from viruses or from bacteriophages, or essentially synthetic vectors or plasmids with elements from the most different origin. Vectors with each of the additional available genetic elements are able to establish themselves in the relevant host cells for several generations to as far as stable units. Accordingly, in the context of the invention, it is irrelevant whether they establish themselves extrachromosomally as their own units or are integrated into a chromosome or in chromosomal DNA. Whichever of the numerous systems known from the prior art is selected, depends on the individual case. The achievable number of copies, the available selection systems, principally among them resistance to antibiotics, or the ability to cultivate host cells that can take up the vectors, for example, can be decisive.
Expression vectors include partial sequences that enable them to replicate inventive microorganisms optimized for production of proteins and bring the comprised gene to expression there. Preferred embodiments are expression vectors that themselves carry the genetic elements required for expression. The expression is influenced, for example, by promoters that regulate the transcription of the gene. Thus, the expression can occur by means of the natural, original, localized promoter with this gene, but also after gene technical fusion, both by a prepared promoter of the host cell on the expression vector and also by a modified or a completely other promoter of another organism or of another host cell. Expression vectors can be regulated by changing the conditions of culture or by adding certain compounds such as the cell density or specific factors. Expression vectors permit the associated protein to be produced heterologously (i.e., in a different cell or host cell as that from which it can be obtained naturally). In this regard, the cells can belong to quite different organisms or derive from different organisms. A homologous protein production from a host cell that naturally expresses the gene over an appropriate vector also lies within the field of protection of the present invention, in so far as the host cell is an inventively designed microorganism. This can have the advantage that natural, modification reactions in a context of the translation on the resulting protein can be carried out in exactly the same way as they would normally be in nature.
Moreover, additional genes can be included for a useful expression system, for example, those that are made available on other vectors and which influence inventive production of the protein having a cofactor and coded by nucleic acid sequence a). They can be modified gene products or those intended to be purified together with the inventively secreted protein, for example, to influence its enzymatic function. They can, for example, be other proteins or enzymes, inhibitors or such elements that influence the interaction with various substrates.
A further subject matter of the invention is represented by a process for preparation of a protein having a cofactor by means of a microorganism that belongs to the genus Corynebacterium, said process comprising the following process steps:
With this type of process it is therefore possible to produce cofactor-containing proteins with bacteria of the genus Corynebacterium, especially in a biotechnological fermentation. Due to Tat-mediated secretion of a cofactor-containing protein, especially an enzyme, its purification or further processing in such a process is significantly easier. Furthermore, a process of this type particularly enables a satisfactory product yield in fermentation. All the previously mentioned aspects for the microorganisms and vectors according to the invention also apply to the process according to the invention, so that they will not be repeated again here, but reference is made to the previous embodiments.
Consequently, in a preferred embodiment, the process is characterized in that at least the amino acid sequence coded by nucleic acid sequence a) is secreted together with at least one cofactor from the microorganism.
In a further preferred embodiment the process is therefore further characterized in that the cofactor of the protein for which nucleic acid sequence a) codes is a coenzyme or a prosthetic group.
A microorganism according to the invention is particularly preferably employed in the process according to the invention. Therefore, a further subject matter of the invention is represented by processes for the preparation of a protein that comprises a cofactor, wherein said processes include as a process step the cultivation of a microorganism according to the invention, as has been previously described that secretes the protein into the medium that surrounds said microorganism.
Cofactor-containing proteins, especially enzymes, which are manufactured in this type of process find a wide variety of uses. Among these in particular should be cited oxidases, peroxidases, hydrogenases, dehydrogenases, reductases, biotin-dependent enzymes, especially CO2-fixing enzymes, or redox enzymes in general. For example, redox enzymes are employed for the enzymatic bleach in washing and cleaning agents. They are particularly used in the textile and leather industry for downstream processing of natural raw materials. Moreover, all enzymes manufactured according to the process of the invention can be employed as catalysts for chemical reactions, once again in the context of biotransformation.
Consequently, in a further embodiment of the invention the process is characterized in that the protein is an enzyme, especially one chosen from redox-enzyme, oxidase, peroxidase, hydrogenase, dehydrogenase, reductase, biotin-dependent enzyme, CO2-fixing enzyme, protease, amylase, cellulase, lipase, hemicellulase, pectinase, mannanase or combinations thereof.
Proteins and especially enzymes are optimized and especially genetically modified for their proposed field of application so as to provide them with improved properties for their respective purpose. Enzymes produced in processes according to the invention can therefore be the respective wild type enzymes or further developed variants. Wild type enzymes refer to enzymes present in a naturally occurring organism or in a natural habitat which can be isolated from this. An enzyme variant is understood to mean enzymes that were produced from a precursor enzyme, for example, a wild type enzyme, by modification of the amino acid sequence. The amino acid sequence is preferably modified by mutation, wherein amino acid substitutions, deletions, insertions or combinations thereof can be undertaken. The incorporation of such mutations into proteins is known from the prior art and has long been known to the person skilled in the art of enzyme technology.
Fermentation processes per se are well known from the prior art and represent the actual industrial production step, in general followed by a suitable purification method for the produced product, for example, the recombinant protein. All fermentation processes suitable for producing recombinant proteins therefore represent preferred embodiments of this inventive subject matter. A process of this kind is considered to be suitable if an appropriate product is formed. Products formed during fermentation are considered proteins having a cofactor, especially including enzymes, among which are especially enzymes that catalyze redox reactions. Exemplary redox enzymes are inter alia oxidases, peroxidases, hydrogenases, dehydrogenases, reductases, biotin-dependent redox enzymes, CO2-fixing enzymes.
Optimal conditions for the production processes employed, for the microorganisms and/or the products being produced have to be experimentally determined by the person skilled in the art with the help of the previously optimized culture conditions of the strains in question, for example, in regard to fermentation volumes, medium composition, oxygen demand or stirring rate.
Fermentation processes, wherein the fermentation is carried out with a supply strategy, can also be considered. For this the ingredients of the medium that are used up by the ongoing cultivation are fed in; this is also known as a feed strategy. Considerable increases in both the cell density and in the dry biomass and/or above all in the activity for the product of interest can be achieved in this way.
In analogy with this, the fermentation can also be designed in such a way that unwanted metabolic products can be filtered off or be neutralized by the addition of buffer or matching counter ions.
The manufactured product can be subsequently harvested from the fermentation medium. It was preferably inventively secreted into the medium. This fermentation process is correspondingly preferred over the product purification from the dry mass, but requires the availability of suitable secretion markers and transport systems.
Numerous combination possibilities for the process steps are conceivable for each product that is to be produced or is produced with microorganisms or processes according to the invention. The optimum process has to be determined experimentally for each particular case.
Microorganisms according to the invention are therefore advantageously employed in the described processes according to the invention and are used in these processes to produce a product, especially a protein that comprises a cofactor. Consequently, a further subject matter of the invention is therefore the use of an above-described microorganism for production of a protein having a cofactor.
In a preferred embodiment, the use is characterized in that the protein is an enzyme. The enzyme is advantageously chosen from redox-enzyme, oxidase, peroxidase, hydrogenase, dehydrogenase, reductase, biotin-dependent enzyme, CO2-fixing enzyme, protease, amylase, cellulase, lipase, hemicellulase, pectinase, mannanase or combinations thereof.
The following example further exemplifies the present invention without limiting it in any way.
All molecular-biological procedures were carried out by standard methods, as can be found, for example, in the manual by Fritsch, Sambrook and Maniatis “Molecular cloning: a laboratory manual”, Cold Spring Harbour Laboratory Press, New York, 1989, or in comparable specialized works. Enzymes, construction kits and equipment were employed in accordance with the respective manufacturer's instructions.
a) Construction of the Sorbitol-Xylitol-Oxidase (SoXy)-Expression Vector
As the sorbitol-xylitol-oxidase SoXy concerns a cofactor-containing protein that normally occurs in the cytosol, a Tat-specific signal peptide was introduced in order to enable the export of the protein together with its cofactor over the TAT path of Corynebacterium glutamicum. Here this concerns the heterologous signal peptide TorA that mediates a strictly Tat-dependent membrane transport in E. coli. The gene from the SoXy was amplified using polymerase chain reactions (PCR), wherein an EcoRI segment was introduced on the 3′-end for the ligation into the Corynebacterium glutamicum expression vector pEKEx2 (Eikmanns et al. (1991) Gene 102: 93-98) (see
The DNA fragment of the TorA signal peptide was prepared synthetically and the first hundred base pairs of the SoXy gene attached to it and by using the Notl segment located in the starting region of the SoXy cloned into the expression vector pEKEx2 (see
b) Expression and Secretion of the Sorbitol-Xylitol-Oxidase
Corynebacterium glutamicum ATCC13032 (Abe et al., J. Gen. Appl. Microbiol., Vol. 13, pp. 279-301 (1967)) was transformed with the SoXy-expression vector in order to analyze the expression and secretion of the SoXy.
The cultivation was carried out in CGXII medium (Keilhauer et al., J. Bacteriol., Vol. 175, pp. 5595-5603 (1993)) and the induction of the expression by adding 100 μM IPTG. The proteins were than worked up from the cell faction and the supernatant and separated over polyacrylamide gel. The sorbitol-xylitol-oxidase having a size of 44 kDa in the cell fraction was not visible in a gel dyed with Coomassie. After the induction with IPTG, a protein band with a size of 44 kDa could be seen in the samples of the supernatant for each of the SoXy transformants and did not appear in the supernatant of the negative control (see
c) Determination of the Activity
Activity of the SoXy was investigated with the help of the qualitative activity test for hydrogen peroxide-forming enzymes in colonies on agar plates by means of 4-chloronaphthol, (S. Delgrave et al., “Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase”, Protein Engineering, Vol. 14, pp. 261-267 (2001)). With this method, the more hydrogen peroxide that is formed, the sooner a blue coloration of the medium appears. Using this activity test a commencement of the blue coloration could be detected in the presence of the SoXy expression vector within 4 h after adding 30 μl of the culture supernatant (see
This clearly demonstrated that microorganisms according to the invention are capable of efficiently secreting functional cofactor-containing proteins, above all also those that are normally localized in the cytosol.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2008 025 926.8 | May 2008 | DE | national |
The present application is a continuation of International Patent Application No. PCT/EP2009/056142 filed 20 May 2009, which claims priority to German Patent Application No. 10 2008 025 926.8 filed 29 May 2008, both of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2009/056142 | May 2009 | US |
| Child | 12955219 | US |
