Patent Application
20100184142
This invention relates to bacteria that on the basis of genetic modification display increased secretion of proteins, nucleotide sequences as well as plasmids that contain at least one gene, which codes for a SecA protein with increased secretion for proteins, a SecA with increased protein secretion as well as a method for the production of the desired proteins using the invention-based bacteria.
Gram-positive bacteria (above all, various Bacillus species) can discharge proteins in large quantities into the surrounding culture medium. This ability has been used for a long time for the industrial procurement of a plurality of secreted enzymes (such as, for example, amylases, proteases, lipases). The production strains that are used in industry for these processes are optimized by repeated, non-directed mutagenesis and subsequence screening for an increased exoenzyme production, something that can lead to yields of several grams of enzyme per liter of culture supernatant. Most of the time, the causes of the increased productivities of these strains are not known. Various attempts to use gram-positive bacteria as host organisms for the secretory procurement of heterologous proteins frequently yielded only a disappointing result. The yields, thus obtained in the overwhelming majority of cases, turned out to be definitely less than the quantities that can be attained from the secretion of homologous exoproteins. One of the reasons for this is the inefficient or entirely missing transport of the heterologous protein via the cytoplasm membrane [1-4].
The transport of proteins via the bacterial plasma membrane is catalyzed via so-called Sec-translocase (
An efficient initiation of translocation of a secretory protein via the bacterial cytoplasm membrane requires the development of a functional complex consisting of at least the SecA protein, SecY and the export protein upon the membrane. As a consequence of these functional interactions as well as the exchange of ADP against ATP on the nucleotide bonding point 1 (NBS I) of SecA, a conformation change leads to a membrane insertion of SecA as well as the activation of the SecA ATPase activity and the initiation of translocation. The ATP hydrolysis on SecA is regulated by a double intramolecular mechanism. Investigations on Escherichia coli showed that in the free SecA, which is present cytosolically, the ATPase activity is down-regulated by the interactions of the regulatory elements IRA-1 and IRA-2 (intramolecular regulator of ATP hydrolysis) with NBS 1. It is assumed that the conformation change brought about on the basis of functional interactions between SecA and the export protein of SecA leads to a spatial removal of IRA-1 and IRA-2 from NBS 1, as a result of which, the SecA ATPase is activated [7, 8]. During the secretory procurement of heterologous proteins with gram-positive bacteria, the translocation can represent a restrictive step on the basis of quality control or “proofreading activity” of the translocase. This control mechanism is necessary for the cell in order—with respect to its own proteins—to slot into the secretion path only those that are intended for export out of the cytosol. As for the heterologous proteins that are not adapted to the foreign export apparatus in an optimum fashion, this quality control, however, can represent an essential restrictive step. The ability of the heterologous expert proteins in terms of activating the translocation ATPase activity of the SecA protein is most likely decisive as to whether and with what efficiency a membrane transport of the heterologous export protein takes place or whether and to what extent there is a rejection of the heterologous export protein by the quality control of the Sec-translocase.
It is therefore the object of the invention to provide nucleotide sequences, protein sequences, bacteria as well as a method by means of which one can facilitate a secretory procurement of proteins that will be improved when compared to past known microbiological processes.
Starting with the preamble of Claim 1, the problem is solved according to the invention with the features given in the characterizing part of Claim 1. Furthermore, the problem is solved according to the invention by starting with the preamble of Claim 11 with the features given in the characterizing part of Claim 11. The problem is furthermore solved according to the invention by starting with the preamble of Claim 21 with the features given in the characterizing part of Claim 21. The problem is also solved according to the invention by starting with the preamble of Claim 22 with the features given in the characterizing part of Claim 22. The problem is also solved according to the invention by starting with the preamble of Claim 23 with the features given in the characterizing part of Claim 23. Starting with the preamble of Claim 30, the problem is also solved according to the invention with the features given in the characterizing part of Claim 30. Furthermore, The problem is also solved according to the invention by starting with the preamble of Claim 31 with the features given in the characterizing part of Claim 31.
Using the invention-based nucleic acids as well as polypeptides, it is now possible to take proteins that so far were exported by the microorganisms only in small quantities or not at all and to obtain them in a secretory fashion with microorganisms or to make them with increased efficiency. Compared to the naturally occurring or gene-engineering unchanged nucleic acids, the invention-based nucleic acids code for a translocation ATPase, hereafter referred to as SecA, which causes or displays increased secretion for proteins. The term “increased secretion” is taken to mean a protein secretion that is increased when compared to the wild type of organisms or that is also possible for the first time. Furthermore, it is possible to provide microorganisms and methods by means of which one can facilitate a secretion and production of proteins with yields that are higher when compared to hitherto known microbial methods or that are possible for the first time.
Advantageous developments are given in the subclaims.
The object of the invention is a SecA protein (translocation ATPase) with an amino acid sequence, which, compared to the wild type SecA amino acid sequence, displays at least a change in the amino acid sequence or a modified form of these polypeptide sequences or isoforms, as a result of which, there is formed a SecA with increased secretion for proteins.
It was found quite surprisingly that a change (=exchange) of an amino acid of the amino acid sequence of the SecA protein will already lead to an increased secretion of proteins or to a secretion of proteins that will be possible for the first time. Several exchanged amino acids, for example, 2 to 7, however, can also bring about the increased secretion for proteins.
Changes in the area of the amino acid sequences that are responsible for the development of the regulatory elements IRA-1/IRA-2, or a modified form of these polypeptide sequences or isoforms thereof, turned out to be advantageous for the SecA with increased secretion for proteins, whereby in this case, the term “area” is intended to cover not only changes that are found precisely in the amino acid positions that are responsible for the development of the IRA-1/IRA-2 but also changes that, for example, are by 250 to 300 amino acids in front of or behind the particular amino acid positions for IRA-1 or IRA-2.
The positions of the invention-based changes in the amino acid sequence of SecA can be shifted within different microorganisms. For example, starting with the changes in the amino acid positions of SecA of Staphylococcus carnosus, one can also cover the changes in the amino acid sequence of the SecA of other microorganisms that correspond to these positions.
By means of a single change as well as by several changes in the area of the amino acid sequence that is responsible for the development of IRA-1 or IRA-2 as well as by combinations of changes in these areas, it was possible to obtain a SecA with increased secretion for proteins.
The invention-based polypeptides are distinguished by the following: They come from gram-positive or gram-negative bacteria, preferably from the family of the Bacillaceae, Staphylococcaceae, Enterobacteriaceae or Corynebacteriaceae, particularly preferably the genus Bacillus, Staphylococcus, Escherichia or Corynebacterium, particularly preferably the species of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Staphylococcus carnosus, Escherichia coli or Corynebacterium glutamicum. Examples of bacterial in strain cultures that are obtainable from DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH/German Collection of Microorganisms and Cell Cultures Co., Braunschweig) are, for example, Bacillus subtilis 168 or Staphylococcus carnosus TM 300. The invention at hand is characterized in greater detail by the listing of the previously mentioned bacteria strains, although this is not intended to be a restrictive list.
In the case of Staphylococcus carnosus, the amino acid sequences that form the IRA-1 in SecA extend to positions 721 to 772 and for IRA-2 to positions 448 to 567.
In the case of Bacillus subtilis, the amino acids sequences that form the IRA-1 in SecA extend to positions 716 to 767 and for IRA-2 to positions 442 to 561.
In the Staphylococcus carnosus that is isolated from SecA protein, it turned out to be advantageous to make at least one change in the area of the amino acids from position 198 to 772 or a modified form of these polypeptide sequences or isoforms thereof.
In the SecA protein isolated from Bacillus subtilis, it turned out to be advantageous to make at least one change in the area of the amino acids from position 442 to 767 or a modified form of these polypeptide sequences or isoforms thereof.
The object of the invention is a SecA with increased secretion for proteins or a part thereof containing an amino acid sequence according to SEQ ID No. 2, which displays at least one change in the group of the amino acids in position 198, 470, 474, 493, 537, 665 and/or 734 or a modified form of these polypeptide sequences or isoforms thereof.
The following amino acid exchanges in the SecA of Staphylococcus carnosus provide to be successful by way of example (see
Position 198: Tyrosine into histidine
Position 470: Histidine into glutamine
Position 474: Alanine into valine
Position 493: Alanine into valine
Position 537: Asparaginic acid into alanine
Position 665: Valine into glutamic acid
Position 734: Asparaginic acid into valine.
The invention at hand also relates to a SecA with increased secretion for proteins with an amino acid sequence according to SEQ ID No. 4, which displays at least one change from the group of amino acids in position 464, 468, 487, 531 and/or 729 or a modified form of these polypeptide sequences or isoforms thereof.
The following amino acid exchange in the SecA of Bacillus subtilis proved to be successful by way of example (see
Position 464: Histidine into glutamine
Position 468: Alanine into valine
Position 487: Alanine into valine
Position 531: Asparaginic acid into alanine
Position 729: Asparaginic acid into valine
By isoforms, we mean proteins having the same or comparable action specificity, which, however, display a differing primary structure.
By modified forms according to the invention, we mean proteins where there are changes in the sequence, for example, on the C- or N-terminus of the polypeptide or in the area of conserved amino acids without, however, impairing the function of increased protein secretion. These changes can be made in the form of amino acid exchanges according to known methods.
The object of this invention includes polypeptides with the function of an SecA with increased secretion for proteins that are so altered in terms of their amino acid sequence that they can export homologous and/or heterologous proteins with increased activity out of the cells.
The object of this invention includes nucleic acids that code for a SecA with increased secretion of proteins containing a gene sequence secA, which in the secA gene, display at least one mutation or an allele, homologue or derivative of these nucleotide sequences or nucleotide sequences hybridizing with them.
It was now found quite surprisingly that already a mutation in a nucleotide of the secA gene leads to an increased secretion for proteins or to a secretion of proteins that would be possible for the first time. Several mutations, for example, 2 to 7, however, can also bring about the increased secretion of proteins.
At least one mutation in nucleotide areas that code for the regulatory elements IRA-1 and/or IRA-2 of the SecA proteins or an allele, homologue or derivative of these nucleotide sequences or nucleotide sequences hybridizing with them turned out to be advantageous for the expression of an SecA with increased secretion for proteins, where the term “area” is intended to cover not only mutations that lie precisely in the areas that code for IRA-1/IRA-2 but also mutations that, for example, are located 750 to 900 nucleotides before or after the areas that in each case code for IRA-1/IRA-2.
Both by means of a single mutation and by means of several mutations in the area of the nucleotide sequences that code for IRA-1 or IRA-2 as well as a combination of mutations in these areas, it was possible to bring about an increased export of proteins.
The invention-based nucleic acids are distinguished as follows: They are isolated from gram-positive or gram-negative bacteria such as, for example, from the family of Bacillaceae, Staphylococcaceae, Enterobacteriaceae or Corynebacteriaceae, preferably the genus of Bacillus, Staphylococcus, Escherichia or Corynebacterium, particularly preferably from Bacillus subtilis, Bacillus licheniformis or Bacillus amyloliquefaciens, Staphylococcus carnosus, Escherichia coli or Corynebacterium glutamicum. The invention at hand is characterized in greater detail by the listing of the abovementioned bacteria strains, which, however, is not intended as a restrictive list.
In the case of Staphylococcus carnosus, the nucleotide area that codes for IRA-1 extends to the nucleotides from position 2161 to 2316 and for IRA-2 from position 1342 to 1701 (see also SEQ ID No. 1).
In the case of Bacillus subtilis, the nucleotide area that codes for IRA-1 extends to the nucleotides from position 2146 to 2301 and for IRA-2 from position 1324 to 1683 (see also SEQ ID No. 3).
In the case of the nucleic acids isolated from Staphylococcus carnosus, it turned out advantageous to have at least one mutation in the area of the nucleotides from position 592 to 2210 or an allele, homologue or derivative of these nucleotide sequences or nucleotide sequences hybridizing with them.
In the case of the nucleic acids isolated from Bacillus subtilis, it turned out advantageous to have at least one mutation in the area of the nucleotides from position 1392 to 2186 or an allele, homologue or derivative of these nucleotide sequences or nucleotide sequences hybridizing with them.
Particularly advantageous was found to be a nucleic acid containing a secA gene according to SEQ ID No. 1, which displayed at least one mutation in the gene from the group of the nucleotides in position 592, 1410, 1421, 1478, 1610, 1994 and/or 2210 or an allele, homologue or derivative of these nucleotide sequences or nucleotide sequences hybridizing with them.
The following mutations turned out to be successful in the secA gene of Staphylococcus carnosus:
Also particularly advantageous was found to be a nucleic acid containing a secA gene according to SEQ ID No. 3, which displayed at least one mutation from the group of the nucleotides in position 1392, 1403, 1404, 1460, 1461, 1592 and/or 2186 or an allele, homologue or derivative of these nucleotide sequences or nucleotide sequences hybridizing with them.
The following mutations turned out to be successful in the secA gene of Bacillus subtilis:
In contrast to the mutations in positions 1392, 1592 and 2186, the two mutations of the nucleotides in position 1403 and 1404 or 1460 and 1461 on the protein level in each case lead to the exchange of only one amino acid.
Using the invention-based nucleic acid, one can efficiently export proteins out of the cells that in the presence of an unchanged SecA are exported only to a minor extent or not at all. The invention-based nucleic acids that code for a SecA with increased secretions for proteins bring about an altered control mechanism of the SecA for the proteins that are slotted out of the cell. In particular, heterologous proteins that are not adapted to the export apparatus of the host cell in an optimum fashion can now be particularly successfully exported with the help of the invention-based secA sequence. But the export of homologous proteins can also be advantageously improved with the help of the altered secA sequence.
By a nucleic acid or a nucleic acid fragment, we mean, according to the invention, a polymer consisting of RNA or DNA that can be single-strand or double-strand and that can contain optionally natural, chemically synthesized, modified or artificial nucleotides. The DNA polymer concept here also includes the genomic DNA, cDNA or mixtures thereof.
By alleles according to the invention, we mean equivalent nucleotide sequences that code for SecA proteins with increased secretion for proteins. Equivalent sequences are those sequences which, in spite of a deviating nucleotide sequence, for example, caused by the degeneration of the genetic code, will still code for the SecA protein with the desired increased secretion for proteins. Equivalent nucleotide sequences thus comprise naturally occurring variants of the sequences described herein as well as artificial nucleotide sequences that are obtained, for example, by chemical synthesis and that are possibly adapted to the codon custom of the host organism.
By equivalent nucleotide sequences, we also mean sequences with mutations, in particular, natural or artificial mutations of an originally isolated sequence that continue to code for a SecA protein with increased secretion for proteins.
Mutations of the equivalents comprise substitutions, additions, deletions, exchanges or insertions of one or several nucleotide residues. This also includes so-called sense mutations that on the protein level can, for example, result in the exchange of conserved amino acids, which, however, do not lead to any basic change of the increased secretions of proteins of the invention-based SecA protein and thus are neutral in terms of function. That includes also changes of the nucleotide sequence that on the protein level relate to the C-terminus or N-terminus of a protein without, however, essentially impairing the function of the protein.
The invention at hand also covers those nucleotide sequences that one obtains by modification of the nucleotide sequence, resulting in corresponding derivatives. The objective of such a modification, for example, can be the further delimitation of the coding sequence contained therein or, for example, also the insertion of additional restriction enzyme interfaces.
The object of the invention at hand also includes artificial DNA sequences so long as they have the desired properties as described above. Such artificial DNA sequences can, for example, be determined by the re-translation of amino acid sequences by means of computer assisted programs. Particularly suitable are coding DNA sequences that were obtained by re-translation of a polypeptide sequence according to the codon use that is specific for the host organism. The specific codon utilization can easily be determined by an expert who is familiar with molecular genetic methods by computer analysis of other already known genes of the organism that is to be transformed.
By homologous sequences according to the invention, we mean those that are complementary for the invention-based nucleotide sequences and/or that hybridize with them. The “hybridizing sequences” concept according to the invention includes substantially similar nucleotide sequences from the group of DNA or RNA that, under known stringent conditions, enter into a specific interrelationship (bonding) with the previously mentioned nucleotide sequences. That includes also short nucleotide sequences with a length of, for example, 10 to 30, preferably 12 to 15 nucleotides. According to the invention, this, among other things, also covers so-called primers or probes.
According to the invention, that also includes the sequence areas that precede (structural genes) (5′- or upstream) and/or follow (3′- or downstream) the coding areas. This especially includes sequence areas with regulatory function. They can influence the transcription, the RNA stability or the RNA processing as well as the translation. Examples of regulatory sequences, among other things, are promoters, enhancers, operators, terminators, translation amplifiers or ribosome bonding points.
Basically, genes can be amplified and subsequently isolated by known methods such as, for example, the polymerase chain reaction (PCR) with the help of short synthetic nucleotide sequences (primers). The primers used are generally produced on the basis of known gene sequences resting on existing homologies in conserved areas of the genes and/or considering the GC content of the DNA of the microorganism that is to be investigated.
Another procedure for the isolation of coding nucleotide sequences is the complementation of so-called defect mutants that, at least in phenotypical terms, display a function loss in the activity of the gene to be investigated or of the corresponding protein. By “complementation,” we mean the elimination of the gene defect of the mutant and extensive restoration of the original phenotype prior to mutagenesis, which is attained by the introduction of functional genes or gene fragments.
A conventional mutagenesis method for the production of defect mutants, for example, is the treatment of the bacterial cells with chemicals such as, for example, N-methyl-N-nitro-N-nitrosoguanidine or UV radiation. Such methods for triggering mutation are generally known and, among other things, can be followed up in Miller (A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992)) or in the handbook “Manual of Methods for General Bacteriology” by the American Society for Bacteriology (Washington D.C., USA, 1981).
The object of the invention furthermore includes a gene structure containing at least one of the previously described nucleotide sequences that code for a SecA with increased secretion for proteins as well regulatory sequences that are tied in with them in operational terms, which control the expression of the coding sequences in the host cell. Corresponding gene structures, for example, can be chromosomes, plasmids, vectors, phages or other nucleotide sequences that are not closed in a circular manner.
The invention at hand relates to a vector containing a nucleotide sequence of the kind described previously and coding for a SecA with increased secretion for proteins, regulative nucleotide sequences that are operationally tied in with it as well as additional nucleotide sequences for the selection of transformed host cells, for replication within the host cell or for integration into the corresponding host cell genome.
Suitable as vectors are those that are replicated in bacteria such as, for example, pWH1520 [17], pCU3 or pXR100 (see also
Using the invention-based nucleic acid sequences, one can synthesize corresponding probes or also primers, and one can use them for amplifying and isolating preferably gram-positive bacterial, for example, with the help of genes from other microorganisms that are analogous to the PCR technique.
The object of the invention at hand thus also includes a probe for the identification and/or isolation of genes coding for proteins that participate in the export of proteins, whereby this probe is made starting with the invention-based nucleic acid sequences of the kind described earlier and contains a marking suitable for detection. The probe can be a segment out of the invention-based sequence, for example, out of a conserved area, which, for example, has a length of 10 to 30 or preferably 12 to 15 nucleotides and which can hybridize under stringent conditions specifically with homologous nucleotide sequences. Numerous suitable markings are known from the literature on the subject. Instructions for this purpose can be found by the expert, among other things, for example, in the handbook by Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and by Newton and Graham: PCR (Spektrum Akademischer Verlag/Publishers, Heidelberg, Germany, 1994) or, for example, in the handbook: “The DIG System Users Guide for Filter Hybridization” by Firma Roche Diagnostics/the Roche Diagnostics Company (Mannheim, Germany) and by Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260).
The object of the invention at hand furthermore includes the transfer of at least one of the invention-based nucleic acid sequences or a part thereof coding for a SecA with increased secretion for proteins, an allele, homologue or derivative thereof into a host system. That also includes the transfer of an invention-based gene structure into a host system. This transfer of DNA into a host cell is accomplished according to gene engineering methods. As a preferred method, one might mention here the transformation and, especially in a preferred manner, the transfer of DNA by electroporation. Gram-positive host organisms proved to be particularly suitable. A transformed microorganism, resulting from a successfully performed nucleic acid transfer, will differ from the correspondingly non-transformed microorganism in that it contains nucleic acids of the kind according to the invention and can bring them to expression accordingly. As representative of a suitable host system, we might mention organisms of the genus of Bacillus or Staphylococcus and preferably the species of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens or Staphylococcus carnosus. As culture medium, depending on the requirements, we find usable a complex medium such as, for example, LB Medium (T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Clonin [sic]: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989)) or also a mineral salt medium such as, for example, CGXII medium (Keilhauer, C. et al., 1993, J. Bacteriol., 175: 5593-5603). After corresponding cultivation, the bacteria suspension can be harvested and can be used for further examination, for example, for transformation or isolation of nucleic acids according to currently customary methods. This procedure can analogously be applied also to other gram-positive or gram-negative bacteria strains. Bacteria of the family of Bacillus or Staphylococcus are preferred here as host systems. In a particularly preferred manner, one might mention here the species Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens or Staphylococcus carnosus.
Moreover, the invention at hand also includes bacteria strains as host systems that are distinguished as mutated or wild type strains suitable for protein production because their metabolism flow runs increasingly in the direction of a biosynthesis of proteins. Suitable according to the invention are also those microorganisms that are known to the expert from microbial production methods such as, for example, Enterobacteriaceae or Corynebacteriaceae.
As host organisms, one can also use microorganisms where one or several gene(s), coding for proteins, components or factors that are responsible for the transport of proteins through the bacterial plasma membrane, are so altered that, in addition to the invention-based SecA protein, they contribute to an increased transport of proteins through the plasma membrane. That, for example, could be the other known constituents (secY, secE, secG, secD, secF and yajC) of Sec-translocase. By way of example, we might mention here the host organisms of the family of Bacillus or Staphylococcus.
The invention at hand will be explained in greater detail by means of the selected examples referring to microorganisms, although it is in no way restricted by them.
The invention at hand furthermore relates to a genetically altered microorganism, containing in a replicable form an invention-based nucleic acid of the kind previously described.
The invention also comprises a genetically altered microorganism, containing in replicable form a gene structure or a vector of the kind previously described.
The object of the invention furthermore also includes a genetically altered microorganism, containing an invention-based polypeptide with the function of an increased secretion for proteins when compared to the microorganism that is accordingly not altered in genetic terms. An invention-based genetically altered microorganism is furthermore distinguished by the following: It is a gram-positive or gram-negative bacterium such as, for example, an organism from the family of the Bacillaceae, Staphylococcaceae, Enterobacteriaceae or Corynebacteriaceae, particularly preferably the genus of Bacillus or Staphylococcus. Particularly preferred, for example, are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens or Staphylococcus carnosus.
The invention at hand furthermore relates to a method for the microbial production of proteins where at least one of the invention-based nucleic acids isolated from a gram-positive or gram-negative bacterium is transferred into a host organism or is generated there with the means known to the expert and is expressed there, where this genetically altered microorganism is employed for the microbial production of proteins and where the correspondingly formed protein is isolated out of the culture medium.
The genetically altered microorganism, made according to the invention, can be cultivated continuously or discontinuously by way of the batch method (set cultivation) or by the fed batch (feed method) or repeated fed batch methods (repetitive feed method) for the purpose of producing proteins. A summary of known cultivation methods can be found in the textbook by Chmiel (Bioprozesstechnik 1. Einfuehrung in die Bioverfahrenstechnik—Bioprocess technique 1. Introduction into Bioprocess Technique (Gustav Fischer Verlag/Publishers, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere Einrichtungen—Bioreactors and Peripheral Devices (Vieweg Verlag/Publishers, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used for this purpose must suitably meet the requirements of the particular strains. Description of culture media of various microorganisms can be found in the handbook “Manual of Methods for General Bacteriology” of the American Society of Bacteriology (Washington D.C., USA, 1981). As a source of carbon, one might use sugars and carbohydrates such as, for example, glucose, saccharose, lactose, fructose, maltose, molases, starch and cellulose, oils and fats such as, for example, soybean oil, sunflower oil, peanut oil and coconut fat, fatty acids such as, for example, palmitinic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerin and ethanol, and organic acids such as, for example, acetic acid. These substances can be used individually or as a mixture. As nitrogen source, one can employ organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn spring water, soybean flower and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually or as a mixture. As a phosphorus source, one can employ phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium furthermore must contain metal salts such as, for example, magnesium sulfate or iron sulfate that are necessary for growth. Finally, one can employ essential growth substances such as amino acids and vitamins in addition to the abovementioned substances. Suitable preliminary stages can, moreover, be added to the culture medium. The substances to be used and listed can be added to the culture in the form of a one-time preparation or they can be fed-in suitably throughout cultivation.
To check the pH control of the culture, one can suitably employ basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid. To check the foam development, one can employ anti-foaming agents such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids, one can add suitable selectively acting substances to the medium, for example, antibiotics. To maintain aerobic conditions, one introduces oxygen or oxygen-containing gas mixtures into the culture such as, for example, air. The temperature of the culture normally is between 30° C. and 38° C., and preferably it is 37° C. The culture is continued until a maximum of the desired protein has been formed. This objective is normally attained within 8 to 72 hours.
The proteins can be analyzed by activity determination, sequencing or electrophoresis.
The microorganisms that are the object of the invention at hand can, for example, make proteins from glucose, saccharose, lactose, mannose, fructose, maltose, molasses, starch, cellulose or from glycerin and ethanol. This may involve the already previously more thoroughly described representatives of the gram-positive or gram-negative bacteria. The microorganisms that are genetically altered according to the invention here are distinguished by increased protein secretion when compared to the correspondingly unchanged microorganisms (wild type) or the microorganisms that merely contain the vector without the gene insert. In a particular embodiment of the invention at hand, it is shown that the expression of the invention-based secA gene in the homologous Bacillus subtilis system (that is to say, all components of the Sec transport apparatus come from Bacillus subtilis) will result in an at least triple increase in protein accumulation in the medium when compared to the control strains. By overexpression of additional genes that have a positive effect upon the general metabolism of protein biosynthesis, one can expect an additional increase in protein production.
Using the invention-based method, one can make pharmaproteins, hormones, enzymes, growth factors or, for example, cytokines. In that way, for example, one can make proteases, amylases, carbohydrases, lipases, epimerases, tautomerases, mutases, transferases, kinases or phosphatases in a microbial manner.
By way of example, the figures show plasmids, a schematic overview of the Sec protein secretion apparatus, a representation of the SecA proteins with the invention-based mutations as well as an experimental evidence of the increased protein secretion using the invention-based nucleic acids.
The following are shown:
First of all, a Bacillus subtilis strain was constructed with an artificially increased quality control of the SecA protein (hereafter called RMA=replacement mutant SecA).
The strain was generated in that the Bacillus subtilis secA gene, lying on the chromosome, was exchanged against the secA gene of Staphylococcus carnosus. This exchange resulted in an artificially increased quality control of Sec-translocase. The heterologous protein OmpA from E. coli was used as a model protein. The artificially increased quality control of the SecA protein meant that the heterologous protein OmpA of E. coli was specifically excluded from export. Moreover, the RMA displayed a cold-sensitive growth (that is to say, no growth at 25° C.) as well as defects in the formation of spores and the development of natural competence.
In the following, suppressor mutants of the RMA were selected, which again can grow at 25° C. and/or which again have the ability for the formation of spores. The characterization of these suppressor mutants meant that in these mutants, changes had occurred in the foreign secA gene. The corresponding amino acid changes related above all to the two areas IRA-1 and IRA-2 that are involved in the regulation of ATP hydrolysis on the NBS-1 of SecA. The further characterization of the suppressor mutants, moreover, showed that the heterologous OmpA protein, which is almost completely excluded from export in the RMA, can be exported in a definitely better manner in the suppressor mutants. On the basis of comparisons with Escherichia coli SecA mutants from bibliography data [10], which partly were identical to the invention-based mutations in the secA gene or the invention-based changes in the amino acid sequence of the SecA protein on the corresponding positions, one may assume that the mutations, identified in the IRA elements of Staphylococcus carnosus SecA, weakened the repression of ATP hydrolysis on the NBS-1 in SecA, as a result of which, the basal ATPase activity was increased [9, 10]. The altered Staphylococcus carnosus SecA variants thus represent SecA proteins that permit a better export of normally inefficient or not at all exported heterologous proteins.
All of the RMA and the isolated suppressor mutants still have the foreign secA gene of Staphylococcus carnosus and thus still represent an artificial situation with a mixed Sec-translocase; therefore, in the following, the discovered changes (individually and also in combination) were transferred to the homologous Bacillus subtilis SecA. The co-expression of the mutated Bacillus subtilis SecA proteins with various inefficiently transported export proteins in Bacillus subtilis showed that also in the now homologous system (that is to say, all components of the Sec transport apparatus come from B. subtilis), one could observe an improved export of the examined proteins (shown in
1. Construction of the Bacillus subtilis SecA Exchange Mutant (RMA)
The pDEL6 plasmid, which was used for the construction of the RMA, was constructed as follows:
A pOrf3 plasmid was obtained by ligation of a 0.5 kb Asp718/Pst1 fragment of plasmid pMKL4 [11] into the plasmid pGEM3Z (Promega). A 2.4 kb Nsil/Pst1 fragment of plasmid pBO1 [12] was ligated into the pOrf3 plasmid, which was digested with Pst1. The secA gene of the resultant plasmid pDEL1 was exchanged by digestion with Spe1 and Sal1 against a 1.4 kb EcoR1/Sal1 fragment of pDG268 plasmid [13], which carries the gene for chloramphenicol acetyl transferase (cat) from which resulted the plasmid pDEL3. Subsequently, various secA gene were ligated into the plasmid pDEL3, which was digested with Sal1 and Pst1. The pDEL5 plasmid carries the B. subtilis secA gene, which was obtained by Sal1/Pst1 digestion from pMKL4 plasmid [11] as 2.5 kb fragment. The pDEL6 plasmid carries the S. carnosus secA gene, which was obtained by Sac1/Pst1 digestion from pMA2 plasmid [14] as 2.6 kb fragment. The pDEL6 fragment, which was used for the construction of the RMA, thus contains areas from five different plasmids:
The bacillus subtilis SecA exchange mutant (RMA) was obtained in that the wild type strain B. subtitles DB104 (his, nprR2, nprE18, ΔaprA3) [15] was transformed with the linearized plasmid pDEL6 and that clones were selected, which were able to grow in the presence of 10 μg/ml of chloramphenicol. The secA gene exchange was verified by Southern Blot analyses, whereby the B. subtilis or S. carnosus secA gene was used as probe.
2. Isolation of Suppressor Mutants of B. subtilis RMA
The B. subtilis RMA was used as parental strain in order to isolate suppressor mutants that again can grow at 25° C. or that can sporulate better than the RMA.
To isolate the cold-resistant suppressor mutants of the RMA, Lurai-Bertani (LB) medium was inoculated after addition of 10 μg/ml of chloramphenicol with 106 cells of RMA and was incubated for 48 hours at 25° C. Between 100 and 1,000 cells were plated out on LB agar plates and were incubated for another 24 hours at 25° C. As an alternative, 106 cells from a culture of RMA that was incubated overnight at 37° C. were plated out directly on LB agar plates and were incubated for 24 hours at 25° C. The stability of the mutation of the resultant suppressor mutants was examined in that the mutants, after inoculation and incubation at 37° C., also after renewed inoculation and incubation at 25° C., displayed additional growth.
To isolate suppressor mutants, which again can sporulate better than RMA, 5 ml of sporulation medium [16] 1:100 were inoculated with a washed overnight solution of RMA and were incubated for at least 24 hours at 37° C. 100 μl of the preparation was heated for 30 minutes at 80° C. after the addition of the same quantity of chloroform. The spores were plated out on LB agar plates and were incubated for 2 days at 25° C. Mutants that were able to grow under these selection conditions were isolated.
3. Cloning and Sequencing of the secA Gene of the Suppressor Mutants
B. subtilis DB 104 was used in order to differentiate whether the suppressing mutations of the suppressor mutants are located in the secA gene of S. carnosus or another gene of B. subtilis. For this purpose, B. subtilis DB 104 was transformed with chromosomal DNA of the suppressor mutants and the chloramphenicol-resistant clone was selected. The resultant transformants were tested to determine whether they can also grow at 25° C., in other words, they also had integrated into the chromosome, in addition to the gene for chloramphenicol acetyl transferase, the neighboring, downstream-located S. carnosus secA gene with the suppressing mutation instead of the B. subtilis secA gene. It was found that the suppressing mutations of the suppressor mutants are located in the secA gene of S. carnosus. Therefore, a cloning of the secA gene was subsequently performed.
In order to clone the S. carnosus secA gene of the suppressor mutants under the control of the xylose-inducible promoter xylA, an amplification was performed of secA by means of PCR using chromosomal DNA of the suppressor mutants as matrices. The following primers were used for this purpose:
(underlined bases indicate the BamH1 or Sph1 interfaces). After amplification, a 2.56 kb fragment was obtained, which was digested with BamH1 and Sph1 and which was ligated into the likewise BamH1/Sph1 digested vector pWH1520 [17], as a result of which, the plasmid pWEsecAS.c.Supp. were obtained. The sequencing of the secA genes was performed with IRD800 marked primers:
(MWG) using the Thermo Sequenase Fluorescent Labeled Primer Cycling Sequencing Kit (Amersham Pharmacia Biotech) as specified by the manufacturer.
4. Transfer of Suppressing Mutations into the SecA of B. subtilis
The transfer of suppressing mutations in the SecA of S. carnosus upon the corresponding amino acid residues in the B. subtilis was performed by means of specifically targeted mutagenesis of plasmid-coded B. subtilis SecA using QuickChange Site-Directed Mutagenesis Kit by Stratagene according to data provided by the manufacturers. As matrix for the PCR reaction, we used the plasmid pMKL40 and the following primer pairs for insertion of the desired mutations (the altered bases are underlined):
A 1.86 kb SnaB1/Sph1 secA fragment from pMKL40 in which the desired mutation was present was subsequently ligated with the 8.67 kb fragment of the mWMKL1 vector that was digested with SnaB1 and Sph1. From this resulted the plasmid pWAX (X here stands for one of the constructs 464, 468, 487, 531 or 729), which contained the secA genes with the desired mutations in a B. subtilis expression vector under the control of the xylose-inducible promoter pxylA. The sequencing of the altered B. subtilis secA genes was performed with the following primers:
The expression of the SecA proteins was checked out in that LB medium was inoculated at an OD600 of 0.4 after addition of 0.2% (w/v) xylose with the strains B. subtilis DB104, which contained the plasmid pWAx and were then incubated for 4 hours at 37° C. Cells from 2 ml of culture were centrifuged off and were boiled up after solution with 50 μl of lysis buffer (10 mM Tris/HCl, pH 8.0, 25 mM MgCl2, 200 mM NaCl, 5 mg/ml lysozyme) in the same volume of 2× Laemmli sample buffer. The proteins were separated in a 10% SDS polyacrylamide gel and the SecA was documented by means of Western Blot analysis using B. subtilis SecA-specific antibodies. The functional mode of the altered B. subtilis SecA proteins was checked in that the temperature-sensitive B. subtilis secA mutant NIG1152 (met, his, div341ts) [18] was transformed with the pWAX plasmids and the growth defect of the mutant was complemented at the nonpermissive temperature of 42° C. by induction of the SecA expression with 0.2% (w/v) xylose.
A study was made to determine as to whether protein export in B. subtilis wild type is improved by expression of the altered B. subtilis SecA proteins. As export substrate, we used a variant of the alkaline phosphatase PhoB, which, on the basis of an amino acid exchange of leucine into glutamine on position 15 (PhoBL15Q) in the signal sequence, is exported only very inefficiently in the B. subtilis wild type. B. subtilis DB104 was transformed with the plasmids pCU3phoB or pCU3phoBL15Q, which contained the genes for the wild type PhoB or the variant PhoBL15Q as well as the plasmids pWAx that contained the altered secA B. subtilis genes. Using the strains thus obtained, LB medium was mixed with 0.5 mM IGTG and 0.2% xylose, it was inoculated at an OD600 of 0.4, and the cultures were incubated for 4 hours at 37° C. Then, 2 ml of cells were separated from the supernatant by centrifugation, they were dissolved with 50 μl of lysis buffer and were boiled up in the same volume of 2× Laemmli sample buffer.
Proteins from the supernatants were precipitated overnight with 13% trichloracetic acid at 4° C., washed twice with 80% acetone and boiled up in 50 μl of Laemmli sample buffer. A volume corresponding to 0.2 OD cells of the cell extracts and a volume corresponding to 1.0 OD cells of the supernatants were applied upon a 12.5% SDS polyacrylamide gel and the PhoB was documented by means of PhoB-specific antibodies in Western Blot. The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10325026.3 | Jun 2003 | DE | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10554755 | US | |
| Child | 12615209 | US |
