MX2007005219A - Process for constructing strain having compactin hydroxylation ability - Google Patents

Abstract

The present invention is directed to methods and compositions for microbial based production of pravastatin. The compositions of the invention include novel strains of microorganisms that are capable of efficiently hydroxylating compactin (ML-236 B) resulting in production of pravastatin. In particular, the microorganisms of the invention are genetically engineered to express both cytochrome P-450 and thefdxsheorfdxshe-likeprotein. The invention further relates to the use of such microorganisms in processes designed for production of pravastatin for use in treatment of disease such as hypercholesterolemia and hyperlipidemia.

Description

PROCESS FOR BUILDING CEPA THAT HAS COMPACTINE HYDROXYLATION CAPACITY Field of the invention The present invention relates to a novel protein, fdxshe or the fdxshe-like protein, associated with the gene encoding a cytochrome P-450, and necessary for its activity. The present invention relates to methods and compositions for the production of pravastatin based on microbes. Compositions of the invention include vectors containing this gene, the use of those vectors in expression systems, and novel strains of microorganisms that are capable of efficiently hydroxylating compactin (ML-236 B) that results in the production of pravastatin. Specifically, the microorganisms of the invention are genetically produced to express both recently discovered cytochrome P-450 and fdxshe or the fdxshe-like protein. The invention also relates to the use of those microorganisms in processes designed for the production of pravastatin for use in the treatment of diseases such as hypercholesterolemia and hyperlipidemia.

BACKGROUND OF THE INVENTION Pravastatin is an inhibitor of HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase), a key enzyme in cholesterol biosynthesis. This enzyme significantly reduces the levels of cholesterol and lipids in the plasma and is thus of great pharmacological importance in the therapy of hype cholesterolemia and hyperlipidemia. [Serizawa et al, J. Antibiot., 36: 887-891 (1983); Serizawa et al, J. Antibiot., 36: 918-920 (1983); Serizawa et al, J. Antibiot., 36: 604-607 (1983); Tsujita et al, Biochim. Biophys. Acta, 877: 50-60 (1986); Arai et al, Ann. Rep. Sankyo Res. Lab., 40: 1-38 (1983); and Koga et al, Biochim. Biophys. Acta, 1045: 115-120 (1990).] Pravastatin is obtained by microbial sodium hydroxylation ML-236B (compactin), a substance produced by a filamentous fungus, Penicillium citrinum. This hydroxylation can be carried out at different levels by different fungal genes, such as Mucor Rhizopus, Syncephalastrum, Cunninghamella, Mortiterella and bacteria such as Nocardia, Actinomadura and Streptomyces which are described in different patents. [U.S. Patent Nos. 5,179,013; 4. 48,979; 4,346,227; 4,537,859; 6,566,120; 6,750,366; Canadian Patents No. 1,150,170; 1,186,647; Japanese Patent No. 58-10572; and European Patent No. 0605230].

The hydroxylation takes place in position 6 of ML-236B, catalyzed by the Cytochrome P-450sca monooxygenase system, found in Streptomyces carbophilus. [Matsuoka et al, Eur. J. Biochem. , 184: 707-713 (1989); and Serizawa, et al, Biochimica et Biophysica Acta, 1084: 35-40 (1991).] Cytochrome P-450sca was characterized as occurring in three forms: P-450sca_i, P-450sca-2 and P-450sca-3, which, according to US Pat. No. 5,179,013, are suitable for use in hydroxylation processes.

Serizawa et al cloned the DNA encoding P-450sca_2 from Streptomyces carbophilus. [Japanese Patent Kokai No. 6-70780; and Watanabe et al, Gene, 163: 81-85 (1995).] The gene has an open reading frame of 1233 bp, which encodes a protein of 410 amino acids. [Watanabe et al, Gene, 163: 81-85 (1995).] A 2.8 kb DNA insert, together with a 1 kbp part of the 5 'non-coding region of the P-450sca-2 gene, it was cloned into a multi-copy plasmid, pIJ702, and used to transform Streptomyces lividans TK21. The transformed Streptomyces lividans TK21 converted L-236B into pravastatin even faster than S. carbophilus. See Watanabe, I. et al, Gene 163: 81-85 (1995).

Watanabe et al revealed that the expression of P-450sca is subject to the induction of transcription, that is, it was discovered that ML-236B and phenobarbital improved the expression of P-450 as much as 30 times. This was established by Northern blotting analysis, which found no transcription in the absence of L-236B, but found three transcripts when ML-236B was present. Transcript levels increased during a period of six hours at a maximum rate when the substrate was present. The DNA sequence of the 5 'region was published, which encoded a regulatory-like protein and the promoter sequence. [Watanabe et al, Gene 210, 109-116 (1998). ] A length of 1 kbp of the 5 'non-coding region of the gene encoding the cytochrome P-450s ca-2 in Streptomyces carbophilus has transcriptional promoter activity, which is inducible by the substrate. When the 1 kbp region was shortened, the transcription promoter allowed for significant expression of the protein in a suitable expression system without it having to be induced. [U.S. Patent No. 5,830,695] Serizawa and Matsuoka purified an NADH-cytochrome-P-450-reductase from S. carbophilus. They used the purified P-450sca protein to demonstrate in vitro hydroxylation activity in the presence of purified flavoprotein (NADH-cytochrome-P-450-reductase), NADH and 02. They revealed that P-450sca and NADH-cytochrone-P- 50 reductase reconstituted the hydroxylation activity in vitro and did not obtain any evidence of the existence of the iron and sulfur protein in S. carbophilus. Based on these findings, Serizawa and Matsuoka classified the P-450sca cytochrome system as a two-component system similar to those found in eukaryotic microsomal cytochrome systems, which does not require iron and sulfur proteins. [Serizawa and Matsuoka, Biochem. et Biophysica Acta, 1084: 34-40 (1991).] The present invention is based on the discovery that the gene encoding fdxshe or a fdxshe-like protein is located below the cytochrome P-450 gene and is necessary for the function of cytochrome P-450sca. Therefore, the P-450sca cytochrome system is a three-component system, unlike the two-component system proposed by Serizawa and Matsuoka, which is described in Figure 4.

Extract of the invention An object of the invention is to provide an effective and efficient method for converting compactin to pravastatin.

Another objective of the invention is to use the coexpression of cytochrome P-450 and ferredoxin or the ferredoxin-like protein to convert compactin to pravastatin.

The invention comprises novel microorganisms capable of hydroxylating compactin (ML-236B) and tproducing pravastatin. The microorganisms of the invention are generally produced to express both the cytochrome P-450 and the ferredoxin or the ferredoxin-like protein. The invention is based on the discovery that the coexpression of cytochrome P-450, together with ferredoxin or ferredoxin-like protein, results in the production of pravastatin.

The invention also encompasses processes for building a genetically modified microorganism having compacty hydroxylation capability comprising transforming a host cell with one or more plasmid constructs comprising a promoter and nucleic acid sequences encoding cytochrome P-450 and ferredoxin or the protein similar to ferredoxin.

Another embodiment of the invention comprises processes for producing pravastatin using fermentation techniques known in the art, which comprise culturing the genetically modified microorganism of the invention in a medium containing ML-236B under conditions in which cytochrome P-450 and ferredoxin or the ferredoxin-like protein are expressed that derive in the catalytic conversion of ML-236B into pravastatin.

In yet another aspect of the invention, the process may comprise recovering pravastatin from the culture medium and its use in the treatment of disorders related to cholesterol biosynthesis such as such as hypercholesterolemia and hyperlipidemia.

The present invention also comprises a nucleic acid molecule encoding fdxshe.

The present invention also comprises the vector containing the nucleic acid molecule.

The present invention also comprises a novel protein encoded by fdxshe and methods for its production using the aforementioned vector.

The foregoing and other objects, features and advantages of the present invention will be better understood from the following specification.

Brief Description of the Drawings > Figure 1 illustrates a nucleic acid and amino acid sequence of fdxshe (SEQ ID No. 1).

Figure 2 illustrates the quantitative determination of pravastatin and compactin: The bars show the soluble remnant form of compactin (compactina-OH) and pravastatin produced after overnight incubation by strains of S. lividans harboring different plasmid constructions by S. helvaticus.

Figure 3 illustrates the production rate of pravastatin from colonies of S. helvaticus with and without the plasmid pWHM-cytP45Oshe-fdxshe.

Figure 4 illustrates the two component model of the hydroxylation of compactin without ferredoxin and the novel three component model of the hydroxylation of compactin of the present invention with ferredoxin.

Detailed description of the invention The present invention describes the discovery that the co-expression of P-450 and ferredoxin or the ferredoxin-like protein in genetically engineered microorganisms, leads to the hydroxylation of ML-236B (compactin) to form pravastatin. The present invention, described in detail below, relates to those microorganisms as well as their use in the production of pravastatin for use in the treatment of disorders related to cholesterol biosynthesis. These disorders include, but are not limited to, hypercholesterolemia and hyperlipidemia.

As used herein, ferredoxin or a protein similar to ferredoxin is the fdxshe gene of Streptomyces helvaticus. It has been found that the nucleic acid molecule corresponds to a sequence of 188 base pairs [See SEQ ID No. 1, Figure 1], located below the open reading frames of cytochrome P-450.

As used herein, ML-236B includes ML-236B and salts thereof. In a preferred embodiment ML-236B is ML-236B sodium. As used herein, pravastatin refers to pravastatin and salts thereof. In a preferred embodiment, pravastatin is pravastatin sodium.

Preferably, ML-236B (compactin) is provided by providing, for example, a microorganism, for example a fungus or bacteria, which produces compactin, or a cell-free extract of a microorganism that produces compactin, or a culture medium free of cells of a precultured culture of a compactin producing microorganism, or a solution comprising compactin, or semi-purified compactin, or substantially purified compactin or salts thereof. The culture is under conditions in which cytochrome P-450 or ferredoxin or a ferredoxin-like protein are expressed, thus allowing the ML-236B to be converted to pravastatin by the catalytic action of cytochrome P450 and then the recovery of the pravastatin from the crop.

The present invention comprises microorganisms transformed with one or more expression vectors capable of encoding cytochrome P-450 and ferredoxin or a ferredoxin-like protein. The choice of expression vectors to be used in the practice of the invention depends on the type of microorganism that is being transformed. For example, it is essential that the sequences of the expression vector necessary for replication, regulation of transcription, translational control, etc. are compatible with the microorganism chosen for the transformation. Bacterial expression vectors include, but are not limited to, those based on bacteriophage DNA or plasmid DNA and include those that are suitable for the transformation of prokaryotes such as actinomycetes, Escherichia and Bacillus subtilis, to name a few. In a preferred embodiment of the invention, expression vectors compatible with actinomycetes are used as the plasmid pWHM-3.

If the expression vectors of the present invention need not have more characteristics than those required for expression in a given host, it will be appreciated that the selection criteria may be useful. These criteria include those by which the plasmid confers on the host properties such as selectivity of expression and transformation, such that the phenotype is modified. Suitable selective markers, ie those that confer a phenotype to the host, include drug resistance marker genes such as those conferring resistance to thiostreptone, ampicillin, tetracycline or chloramphenicol.; however, it will be appreciated that many other selective labels can be used.

Methods which are known to those skilled in the art can be used to construct expression vectors containing cytochrome P-450 coding sequences and / or the coding sequences of ferredoxin or ferredoxin-like protein and the control sequences of transcription and translational. To construct expression vectors, DNA fragments comprising nucleic acids encoding cytochrome P-450 and ferredoxin or ferredoxin-like protein are inserted into an expression vector together with the transcriptional and translational control sequences. These transcription control sequences include, for example, promoter sequences. These translational sequences include, for example, ribosome binding sequences. In one embodiment of the invention the cytochrome P-450 and / or the coding sequences and the coding sequences of ferredoxin or of a ferredoxin-like protein, are inserted into a single vector. Alternatively, the coding sequences can be inserted into separate vectors which are then co-transformed into the host cells. Methods commonly known in the art of recombinant DNA technology that can be used for the cloning of molecules in expression vectors are described in Ausubel et al (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & amp; amp;; Sons, NY: and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

With respect to the sequences encoding cytochrome P-450, those sequences can be obtained from a variety of different microorganisms using a variety of different methods known to those skilled in the art. For example, a genomic DNA library can be screened using a labeled cytochrome P-450 probe. For a guide on hybridization conditions see, for example, Ausubel et al, supra. In addition, cytochrome P-450 nucleic acid sequences can be obtained by performing the PCR (polymerase chain reaction) using two oligonucleotide primers designed on the basis of the known cytochrome P-450 nucleotide sequences that are disclosed in the I presented. In a preferred embodiment of the invention, the cytochrome P-450 coding sequences are isolated from strains that can not be clearly differentiated from the actinomycete, preferably strains that can not be clearly differentiated from Streptomyces, more preferably strains that can not be clearly differentiated from S. carbophílus or S. helvaticus. See Watanabe et al, Gene, 163: 81-85 (1995).

With respect to. the coding sequences of ferredoxin or ferredoxin-like protein, those sequences can be obtained from a variety of different microorganisms using methods described above after isolation of the cytochrome P-450 coding sequences. Various forms of ferredoxin such as 3Fe-4S from S. griseolus, the PimF protein from S. natalensis, and the RimH protein from S. diastaticus are known and can be used in the construction of expression vectors. In addition, the coding sequence of the ferredoxin or the novel ferredoxin-like protein described above can be used in the construction of expression vectors.

The present invention provides a novel ferredoxin gene obtained from S. helvaticus. The novel gene has been identified and cloned under the cytochrome P-450 gene in S. helvaticus and is called fdxshe. The cloned fdxshe gene of the present invention is 188 bp in length and has more than 80% homology with the S. greseolus ferredoxin SauC and more than 70% homology with S. natalensis ferredoxin (PimF) or S. diastaticus (RimH). The nucleotide sequence and deduced amino acid sequence of fdxshe is shown in Figure 1. The fdxshe nucleotide sequences of the invention include: (a) the DNA sequences shown in Figure 1; (b) a nucleotide sequence encoding the amino acid sequence shown in Figure 1; (c) all nucleotide sequences which (i) hybridize to the nucleotide sequence indicated in (a) or (b) under stringent conditions, eg, hybridization to DNA bound to the filter in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C, and washed in 0.1 X sSC / 0.1% SDS at 68 ° C (Ausubel FM et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, on page 2.10.3) and (ii) encodes a functionally equivalent gene product.

The invention also includes nucleic acid molecules that can encode or act as fdxshe antisense molecules, useful, for example, in the regulation of the fdxshe gene (for and / or as antisense primers in amplification reactions of the nucleic acid sequences of the fdxshe gene). The invention also comprises nucleotide sequences encoding fdxshe mutant, fdxshe peptide fragments, truncated fdxshe, and fdxshe fusion proteins.

The invention also comprises (a) DNA vectors containing any of the preceding fdxshe sequences and / or their complements (eg, antisense RNAi); (b) DNA expression vectors containing any of the preceding fdxshe sequences operatively associated with a regulatory element that directs the expression of the coding sequences of fdxshe; and (c) technically created host cells containing any of the preceding fdxshe sequences operatively associated with a regulatory element that directs the expression of the fdxshe coding sequences in the host cell. As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, operators, and other elements known to those skilled in the art that excite and regulate gene expression.

Figure 1 shows the deduced amino acid sequence of the fdxshe protein. The amino acid sequences of fdxshe of the invention includes the amino acid sequence shown in Figure 1. The invention also comprises proteins that are functionally equivalent to the fdxshe encoded by the nucleotide sequences described in Figure 1, as judged by either of numerous criteria that include, but are not limited to, the ability to catalyze, in conjunction with cytochrome P-450, the conversion of ML-236B to pravastatin. Those functionally equivalent fdxshe proteins include, but are not limited to, proteins that have aggregates or substitutions of amino acid residues within the amino acid sequence encoded by the fdxshe nucleotide sequences described above, but which result in a silent change, thus producing a functionally equivalent gene product.

The expression of the sequences encoding cytochrome P-450 and ferredoxin or the ferredoxin-like protein can be modulated with any regulatory promoter known in the art to function in the particular microorganism chosen. These promoters can be inducible or constitutive. It is generally preferred to use a promoter with significantly higher activity, rather than a lower activity, such that maximum transcription can be achieved. These promoters include, but not limited to, the promoters of lac, tac, cat, xyl, ptipA, tsr, or pkg.

In one embodiment of the invention, the promoter located immediately adjacent to and associated with a cytochrome P-450 ORF of an actinomycete, preferably a Streptomyces, more preferably S. carbophilus or S. helvaticus can be used. The 5 'region of approximately 1 kilobase and adjacent to the P-450 ORF does not necessarily contain the entire promoter region for this gene and according to US Patent No. 5,830,695 ("the' 695" patent) the DNA of the Promoter region can be considerably shorter than 1 Kb preferably more than 160 bp, more preferably more than 300 bp. The promoter of the present invention can vary as much as desired from the 5 'promoter, provided that the resulting promoter still exhibits the required promoter activity. The patent ? 695 is hereby incorporated by reference to disclose the different sizes of promoters that can be used to express cytochrome P-450 and / or ferredoxin or the ferredoxin-like protein.

In addition, the following methods can be used to identify DNA sequences that have promoter activity for insertion into the expression vectors of the invention. To identify, or assaying the DNA sequences for the activity of the transcription promoter (i) a recombinant expression vector is constructed, wherein the DNA encoding a suitable protein is operatively linked to the putative promoter DNA and inserted into a suitable vector, for example, the plasmid of actinomycete plJ702 or pWHM-3 [Katz E. , et al, (1983), J. Gen. Microbial. , 129, 2703-2714], and then (ii) a suitable host cell that allows the vector to replicate in stable form, for example, Streptomyces Ixvidans when the vectors pIJ 02 or pWH -3 are used are transformed with the vector.

The transformation can be carried out according to Howood et al, [c.f. Hopwood, D.A. et al (1985), "Genetic Manipulation of Streptomyces: A Laboratory Manual", The John Innes Foundation, Norwick, United Kingdom] when the transformant is, for example, a streptomycete. It will be appreciated that the gene used to assay the activity of the promoter should not be generally present or expressed in the host, prior to transformation.

Transcription levels can be easily established by Northern blotting or PCR (Polymerase Chain Reaction) analysis of RNA [Polymerase Chain Reaction, c.f. Innis, MA, et al (1990), PCR PROTOCOLS ", Academic Press, New York.] For example, the expression levels of the product can be determined by determining the physiological activity of the protein produced. it can prepare a recombinant DNA vector, wherein the DNA encoding a protein with a given activity, such as an enzyme, is operatively connected, for example by ligation, to the 3 'end of the putative promoter. connected to the putative promoter can then be assayed in a manner appropriate for the expression product.

It will be appreciated that the methods for testing the products of expression can be tailored specifically to the relevant products. For example, the putative promoter can be operably linked to a drug resistance gene, such as a chloramphenicol acetyl transferase gene [c.f. Gorman, C.M., et al (1982), Mol. Cell. Biol., 2, 1044-1051], or with a luciferase gene [c.f. de Wet, J.R., et al, (1987), Mol. Cell. Biol, 7, 725-7371], which can be detected by methods known in the art. Other methods may also be employed to test expression by the activity of the expression product.

Another method for measuring expression, for example, is through recognition of the product using an appropriate antibody. Measurement techniques include radioimmunoassay [c.f. Berson, R.S., et al (1973), "Methods in Investigative and Diagnostic Endocrinology", Vol. 2A, 2B, North-Holland Publishing Co. , Amsterdam], enzyme immunoassay [c.f. Engwall, E., (1980), Methods in Enzymology, 70 (A), 419-439], Western blotting analysis [c.f. Harlow, E., et al (1988), "Antibodies-A Laboratory Manual," p. 471, Cold Spring Harbor Laboratory, New York] and immunoprecipitation [c.f. Kessler, W.W., 81981), "Methods in Enzymology", 73 (B), 442-459], which depends on how one wants to measure the interaction, and whether the antibody or antigen is labeled in any way. In any case, it will be appreciated that the present description of these techniques is not exhaustive, and that other methods will be readily apparent to those skilled in the art.

In cases where that expression vector encodes cytochrome P-450 and ferredoxin or the ferredoxin-like protein, and the plasmid is compatible with S. lividans, then the plasmid can be introduced into a strain of Streptomyces lividans that does not produce the cytochrome P-450 or ferredoxin, and the transformant can then be cultured in the presence of L-236B. The amount of pravastatin produced then indicates the level of expression of cytochrome P-450 and / or ferredoxin and the ferredoxin-like protons promoted by the putative promoter.

Once it has been established that the desired DNA to be used as a promoter of the present invention has the necessary activity as a promoter, then it can be used to construct an expression vector for the expression of cytochrome P-450 and / or ferredoxin or a protein similar to ferredoxin. As stated above, cytochrome P-450sca-2 is the preferred expression product, a suitable vector is pSCA1013-DELTA. (1013/428), said vector can be isolated from Streptomyces lividans TK-21.

Transformations may generally occur in the promoter sequences, in the sequences that encode ferredoxin or ferredoxin-like proteins, ie fdxshe, or the sequences that encode cytochrome P-450 throughout the transformation process, or can be done for convenience, for example to introduce a restriction site, for example. Therefore, the present invention contemplates promoters, ferredoxins, or cytochrome P-450, which can vary through eliminations, inversions, insertions and substitutions. Preferably, the promoters, ferredoxin and cytochrome P-450 of the invention share a very substantial sequence homology with the relevant parts of the molecules to which they correlate.

Other differences and alterations in the sequence and the means to effect them will be readily apparent to those skilled in the art and the present invention contemplates all of them. The promoters, ferredoxins, and cytochrome P-450 of the present invention which vary in a different from the natural variation, are also referred to herein as mutants in such a way that both the mutants and the variants are provided insofar as they work together to derive in the production of a microorganism capable of hydroxylating ML-236B '(compactin) to form pravastatin.

If desired, the mononucleotide sequence of any cloned DNA can be determined, for example, by the chemical modification method of Maxam-Gilbert [Maxam, AM and Gilbert, W. (1980), Methods in Enzymology, 65, 499- 599] or the dideoxy chain termination method using the M13 phage [Messing, J. and Vieira, J. (1982), Gene, 19, 269-276].

In addition, it will be appreciated that the terms "expression product", "protein", and "polypeptide" are generally interchangeable, and are used in that sense herein. In certain circumstances, the polypeptide translated from the original DNA is not the final product, but in an intermediate form of the final product, post-translational modifications are required to obtain the required product. In the case of P-450sca-2, it is necessary to incorporate iron in a heme ring in the protein to generate the final expression product. Therefore, while the terms "expression product", "protein" and "polypeptide" are used synonymously herein, the differences between the terms will be recognized by those skilled in the art in the relevant context.

After the construction of the appropriate expression vector (s) designed to express the cytochrome P-450 and ferredoxin or the ferredoxin-like protein, the vector (s) is transformed into a suitable host. Preferred hosts include actinomycete and ascomycote, although other prokaryotes and eukaryotes may also be used, such as, for example, Escherichia coli and Bacillus subtilis. In preferred embodiments of the invention, Streptomyces lividans TK 21 and Penicillium citrinum are transformed.

Suitable transformation methods for use with an actinomycete comprise forming the actinomycete culture into spheroplasts using lysozyme. Then a buffer solution containing recombinant DNA vectors and polyethylene glycol is added to introduce the expression vector into the host cells, using the Thompson or Hopwood methods [c.f. Thompson, C.J., et al, (1982), J. Bacteriol., 151, 668-677 or Hopwood, D.A. et al (1985), "Genetic Manipulation of Streptomyces: A Laboratory Manual", The John Innes Foundation, Norwich], for example. In a non-exhaustive embodiment of the invention, a thiostrepton resistance gene is used as a selective marker in the expression vector plasmid [c.f. Hopwood, D.A., et al (1987), "Methods of Enzymology" 153, 116, Academic Press, New York].

If transformation of E. coli is desired to express a product under the control of a promoter of the present invention, then an appropriate general method is one in which the relevant recombinant DNA vector is added to the competent cells. Competent cells are generally prepared in the presence of salts such as calcium chloride, magnesium chloride and rubidium chloride [c.f. Hanaban, D. (1983), J. Mol. Biol. 166, 557-580]. An alternative method comprises electroporation, which comprises the use of high voltage pulses applied to a suspension comprising the host E. col! and the expression vector, thus producing the incorporation of the vector in the cells [Electroporation: Dover W.J. et al (1988), Nucleic Acid Res., 16, 6127, and Calvin, N.M. et al (1988), J. Bacteriol. , 170, 2796].

In the case that B. subtilis is desired as the host cell, then a suitable method wherein the host cells are made into protoplasts using lysozyme. Then a buffer solution containing recombinant DNA vectors and polyethylene glycol is added to the protoplasts, and then the vector is incorporated into the host cells by electroporation (supra) [Cheng, S., et al (1979), Mol. Gen. Genet. , 168, 111]. In a preferred embodiment, a marker of drug resistance, such as that for resistance to chloramphenicol, is used as a selective marker for the transformed cell line, but it will be appreciated that many other selective labels can be used.

Regardless of host, the desired transformant can be cultured using methods known in the art, with the desired polypeptide (s) that produce the culture in intracellular or extracellular form, or both. The media that is used in the culture can be suitably selected from different types of media commonly used for the relevant host cells. In general, those culture conditions, which are accepted as normal for the particular host may also be used for the expression of the desired polypeptide, subject to any modifications required by the properties of the polypeptide, for example. In addition, if the expression vector contains an inducible promoter, it may be necessary to add compounds capable of inducing the expression of the cloned genes.

The particular culture technique employed is not critical to the invention and any technique commonly used for cultivation can also be employed for the present invention. In general, the techniques used that refer to industrial efficiency are chosen. The actinomycete nutrients, for use as a source of assimilable carbon include glucose, sucrose, starch, glycerol, starch syrup, molasses and soybean oil for use as the carbon source. Examples of sources of assimilable nitrogen include soybean meal, wheat germ, meat extract, peptone, corn infusion liquid, dry yeast and ammonium sulfate. In addition to the above, inorganic salts such as sodium chloride, potassium chloride, calcium carbonate or phosphate, and additives may also be suitably used to assist the growth of the microorganism or to promote the production of the desired polypeptide, in combination if necessary .

Again, culture techniques generally appropriate for the host in question are also applicable to the transformed microorganisms, which include methods such as liquid culture and deep culture, suitable for production on an industrial scale. The culture conditions, unless otherwise generally contraindicated, or as specified herein, comprise temperatures between 20 ° C and 37 ° C, preferably between 26 ° C and 28 ° C. The expression product, ie, cytochrome P-450 and ferredoxin or a ferredoxin-like protein, under the control of a promoter of the present invention is generally produced intracellularly or extracellularly, and occasionally both.

In yet another embodiment of the invention, cytochrome P-450 and ferredoxin can be isolated, purified and recovered by different methods, which are known to those skilled in the art, particularly those based on the physical or chemical properties of the polypeptide. In the case where the polypeptides are expressed externally, the polypeptides can be isolated, purified and recovered from the resulting supernatant by centrifuging the culture medium, for example, to remove cells.

To isolate and purify the cytochrome P-450 and the ferredoxin proteins that have accumulated within the cells, the cells are first suspended in a solution containing a protease inhibitor and then homogenized using a medium, such as one commonly known. for those skilled in the art, such as, for example, an ultrasonic homogenizer.

Although not generally necessary for the elucidation of the present invention, it will be appreciated that examples of specific methods for the isolation, purification and collection of the desired polypeptides include techniques such as protein precipitation, ultrafiltration, molecular sieve chromatography (filtration). of gel), adsorption chromatography, ion exchange chromatography, affinity chromatography, the different appropriate types of liquid chromatography, including high performance liquid chromatography (HPLC), dialysis and combinations thereof.

In any case, it will be appreciated that the desired polypeptides can be easily produced on an industrial scale, both with high yield and with high purity, using the present invention. It will also be appreciated that it is possible to use the cytochrome P-450 and ferredoxin or ferredoxin-like protein produced by transformed host cells of the present invention using a doped or partially purified sample of preparation. Cytochrome P-450 and ferredoxin or ferredoxin-like protein can be obtained from recombinant Streptomyces lividans in a manner described above and can be used directly in the production of pravastatin, for example.

Pravastatin produced by any of the processes of the present invention can be recovered by the method of Serizawa et al, [c.f. Serizawa, N. et al (1983), J. Antibiotics, 36, 608]. In a specific embodiment, Streptomyces lividans TK-21 can be used in that manner.

This invention also provides a pharmaceutical composition comprising pravastatin or a fragment thereof, prepared using the methods and compositions of the present invention, and a pharmaceutically acceptable carrier.

The carrier, as used herein, includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to the cell or mammal that is exposed to them at the dosages and concentrations employed. Generally the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other acids; antioxidants that include ascorbic acid; low molecular weight polypeptide (less than 10 residues) / proteins, such as serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; against ions that form salts such as sodium; and / or non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

The active ingredients may also be entrapped in microcapsules prepared, for example, by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, microspheres of albumin, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. The formulations that should be used for in vivo administration must be sterile. This is achieved quickly by filtration through sterile filtration membranes. Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), L-glutamic acid copolymers and. gamma. ethyl-L-glutamate, ethylene and non-degradable vinyl acetate, copolymers of lactic acid and degradable glycolic acid such as LÜPRON DEPOT® (injectable microspheres composed of the copolymer of lactic acid and glycolic acid and leuprolide acetate), and polyhydric acid D- (-) -3-hydroxybutyric. While polymers such as ethylene and vinyl acetate and lactic acid and glycolic acid allow the release of the molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time.

Treatment means all treatments of a disease in an animal and includes (1) preventing the occurrence of the disease in a mammal that may be predisposed to the disease but does not experience or exhibit symptoms of the disease, for example, prevention of onset of clinical symptoms; (2) inhibit the disease, for example stop its development; or (3) alleviating the disease, for example, causing the regression of the symptoms of the disease.

In a specific embodiment of the invention, pravastatin prepared using the methods and compositions of the present invention are used to treat disorders related to cholesterol biosynthesis. These disorders include, but are not limited to, hypercholesterolemia and hyperlipidemia.

The effective amount for the treatment of a disease means that the amount that, when administered to a mammal in need thereof, is sufficient for the treatment of the effect, as defined above, for that disease.

In any case, it will be appreciated that the desired pravastatin can be easily produced on an industrial scale, both in high yield and high purity, using the present invention.

Having described the invention with reference to certain preferred embodiments, other embodiments will become apparent to one skilled in the art upon consideration of the specification. All methods, preparations, solutions and the like, which are not specifically defined, can be found in "Molecular Cloning-A Laboratory Handbook" (supra) which is incorporated herein by reference in its entirety as a reference. The invention is also defined as reference to the following examples which describe in detail the preparation of the compound of the present invention. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the invention.

Examples Example 1: Comparative Example This example exemplifies that, by transforming the cytochrome P-450 gene into S. lividans TK-21 without the fdxshe gene or fdxshe-like, it is not sufficient to induce the hydroxylation of compactin to pravastatin.

Construction of plasmid pWHM-CytP450she Total DNA was extracted from S. helvaticus using the modified PUREGENE DNA Isolation Case for Yeast and Gram-positive Bacteria. The cytP450she gene was amplified by PCR using the synthetic oligonucleotide primers 5 'TAT AAG CTT TGC GGT AGA CCG CCG TCC 3' and 5 'TTT CTA GAC CAG GTG ACC GGG AGT TCG TTG 3' based on Genbank sequences: E06907 and E13579. The PCR fragment was purified from the PCR mixture (V-Gene PCR Purification Kit) and digested with restriction enzymes HindIII and EcoRI at 37 ° C for 2 hours using buffers and enzymes recommended by the producer (MBI Fermentas) .

The product of the digestion reaction was separated by agarose gel electrophoresis on a 1% w / v agarose gel in an underwater-type electrophoresis tank containing TAE solution and a cycle at 100 V for 3 hours. The agarose slice containing the relevant 1.2 kb fragment was excised from the gel and the DNA was extracted from the gel using the Gene V DNA Gel Extraction Kit. The purified and digested PCR fragment was ligated into the vector of PWHM-3 (Vara et al, 1989) (cut with HindIII and EcoRI and purified in a manner similar to the purification of the PCR fragment).

The ligation mixture was electroporated into blue E. coli XLl cells using the BIO-RAD MicroPulser Electroporation Apparatus and plated onto ampicillin containing the LB medium. The ampicillin-resistant colonies were isolated and grown overnight in the LB medium containing 100 g / ml ampicillin. From the overnight cultures, the plasmid DNA was extracted using the Rapid Plasmid DNA Mini-Preparation Kit of Gene V. The extracted plasmid DNA was digested with EcoRI and HindIII and passed over 1% gel of agarose in TAE buffer. The construction of the plasmid containing the insert of the desired size was selected and sequenced and used in subsequent works (pWHM-CytP450she).

Transformation of the construction of p H -CytP450she in S. llvldans TK-21: The construction of the plasmid was transformed into protoplasts of S. lividans TK-21 as described in Hopwood (1985) and selected on a medium R2YE containing thiostrepton. The thiostreptone-resistant colonies were transformed to separate R2YE Petri dishes containing thiostrepton and allowed to sporulate at 28 ° C. The spores were washed separately from each Petri dish in 10% glycerol and stored at -20 ° C until use. From each, 0.5 mL of spore suspension was used to inoculate 100 mL of YEME medium (Hopwood et al, 1985) containing 25 g / g thiostrepton and allowed to culture for 3 days.

The plasmid DNA was purified and digested with EcoRI and HindIII and separated on agarose gels. All constructs of the plasmid tested showed the expected restriction pattern. One of the suspensions of spores tested was selected and used in the subsequent work.

Test of the hydroxylation of compactina of S. lividans TK-21 that transports the construction of pWHM-cytP450she: The previously selected spore suspension was used to inoculate 50 mL of PSI medium (2% glucose, 0.5% soy food, 0.5% soy peptone, 0.01% KH2P04 and 0.1% of CaCOs) and incubated for 2 days with continuous orbital shaking at 300 rpm at 28 ° C. The preculture was used to inoculate 50 mL of the PSF-2 medium (1.8% glucose, 5% soy food, 0.4% CSL and 0.3% CaC03 pH = 7.2) using a 10 % inoculum The culture was cultured for 30-40 hours and 1 mL 40 mg / ml compactina sodium solution was added to the fermentation. The fermentation was allowed to take place for another 24-48 hours. The samples were then collected and analyzed by high performance liquid chromatography. The production of pravastatin was Example 2: This example illustrates that transforming the cytochrome P450 gene in S. lividans TK-21 with the fdxshe gene or fdxshe-like is sufficient to induce the hydroxylation of compactin to pravastatin.

Isolation of the fdxshe gene by inverted PCR: Total DNA was extracted from S. helvaticus using the modified PÜREGENE DNA Isolation Case for Yeast and Gram-positive Bacteria. The genomic DNA was digested with BamHI, diluted tenfold and self-ligated. Using the ligation mixture, the PCR was done using the primers: 5 'CGA ACT CCC GGT CAC CTG GTG AC G3' and 5 'GTC ATG CGC CGA CGC GTC CCG TGC T3'. The PCR products were separated on 0.7% agarose gel and a single PCR product was observed. The PCR product was sequenced: See Figure 1 (SEQ ID No. 1).

An ORF was identified under the cytP450she gene, which showed 70% identity with the S. griseolus ferredoxin-1 gene (suaB), and was named fdxshe.

Construction of the construction of pWHM-cytP450she-fdxshe: Based on the sequence information, a new primer was designed and positioned below the stop codon of fdxshe. Using the 5 'TAT AAG CTT TGC GGT AGA CCG CCG TTC 3' and the primers 5 'AAA GAA TTC GTG ACC GAT CCG CTG TGA CGC C 3', the cytP450she and the 'fdxshe were amplified together from the genomic DNA of S helvaticus The desired sized PCR fragment was cloned into a p HM-3 vector as in Example 1.

Transformation of the construction of pWHM-CytP450she-fdxshe in S. lividans TK-21: The construction of the plasmid was transformed into S. lividans TK-21 in the same manner as described in Example 1. the spores were prepared and the inserts desired dimensions were selected in the same manner as in Example 1.

Hydroxylation assay of compactin of S. lividans TK-21 carrying the construction of pWHM-cytP450she-fdxshe: The previously selected spore suspension was used to inoculate the PSI and PSF-2 medium. The fermentation was done as described in Example 1. The production of pravastatin from the colonies of S. lividans carrying the plasmid of pWHM-cytP450she-fdxshe had 478-502 Example 3 This example exemplifies that transforming the cytochrome P-450 gene in S. lividans TK-21 with the fdxshe gene or fdxshe-like is sufficient to induce the hydroxylation of compactin to pravastatin. i Transformation of the construction of pWH -CytP 50she-fdxshe in S. helvatic s: The plasmid construction of pWHM-CytP450she-fdxshe (described in Example 2) was transformed into protoplasts of S. helvaticus by electroporation. The protoplasts were prepared as described by Bibb et al, 1978, except that the cells were cultured in the ISP-2 medium in place of YEME for 48 hours at 28 ° C. The protoplasts were washed in 10% sucrose solution before electroporation. Electroporation was performed in a 2 mm separation electroporation chamber (BIO-RAD) with the following parameter settings: 1, 5 kV, 600 O and 10 μG in a Bio-Rad icroPulser ElectroPoration Apparatus. The transformed protoplasts were regenerated in an ISP-2 + 10% sucrose medium for 24 hours at 28 ° C and then superimposed with 5 ml of the SNA medium containing 15 μ? of 50 mg / ml of thiostrepton solution. After solidification of the agar overlap the incubation was continued at 28 ° C. Colonies of regenerated transformant appeared after 5-7 days. Cultured colonies were transferred to thiostrepton IPS-2 + 25 medium and cultured for another 5-7 days. The colonies that were still being cultivated were used in later steps. The selected colonies were cultured in the ISP-2 + 25 μg / ml thiostrepton medium and the plasmid DNA was purified from the mycelium and assayed by restriction digestion. The isolated plasmid DNAs presented a similar restriction pattern with the construction of pWHM-CytP450she-fdxshe.

Hydroxylation assay of compactin of S. helvaticns transporting the construction of pWHM-cytP450she-fdxshe Two previously selected colonies were used to inoculate the PSI and PSF-2 medium. The fermentation was done as described in Example 1. The production rate of pravastatin from the colonies of S. helvaticus carrying the plasmid of pWHM-cytP450she-fdxshe was 212-263 μg / g / 2 hours compared to the strains originals that did not transport construction cytP450sca-fdxshe, where the pravastatin production only at 114-116 hours.

Example 4: From the initial codon (ATG) to the stop codon (TGA) the CytP450she ORF is amplified by RCR and cloned between the pkg promoter and the trpC terminator in the MCS of the vector pBC-Hygro (Silar, FGN 42: 73). From the initial codon (ATG) to the stop codon (TGA) the fdxshe ORF is amplified by PCR and cloned between the pgk promoter and the trpC terminator in the MCS of the pBC-Phleo vector (Silar, FGN 42:73). The constructs are tested by restriction enzyme digestion and suitable clones showing the sizes of the expected fragments are also tested by sequencing. Clones with the expected DNA sequence are selected and used in subsequent steps.

Both constructions are transformed in sequence into the protoplasts of Penicillium citrinum, selecting 200 hygromycin and 500 ^ g / ml fleomycin respectively. The isolated products that exhibit both hygromycin and phleomycin resistance are selected and tested for the conversion of compactin to pravastatin after a sodium feeding dose of compactin. Also isolated products that can convert compactin to pravastatin are tested.

Claims (30)

1. A nucleic acid molecule comprising a nucleotide sequence of SEQ ID. No. 1
2. The nucleic acid molecule according to claim 1, having at least 85% sequence identity with SEQ ID No. 1.
3. The nucleic acid molecule according to claim 1, having at least 90% sequence identity with SEQ ID No. 1.
4. The nucleic acid molecule according to claim 1, having at least 95% sequence identity with SEQ ID No. 1.
5. The nucleic acid molecule that encodes or acts as an antisense to the nucleic acid molecule according to any of claims 1 to 4.
6. The nucleic acid molecule that hybridizes to the nucleotide sequence according to any of claims 1 to 4.
7. A vector comprising a polynucleotide according to any of claims 1-4.
8. A host cell comprising a vector according to claim 7.
9. A method for producing a polypeptide, comprising culturing a host cell according to claim 8 under in vitro conditions in which the protein encoded by the nucleic acid molecule is expressed; and recover the protein from the crop.
10. . A polypeptide molecule having at least 85% sequence identity with the amino acid sequence of SEQ ID No. 1.
11. . A polypeptide molecule having at least 90% sequence identity with the amino acid sequence of SEQ ID No. 1.
12. . A polypeptide molecule having at least 95% sequence identity with the amino acid sequence of SEQ ID No. 1.
13. 13. A process for constructing a genetically modified strain having compacty hydroxylation capability by transforming a host with a plasmid construct containing a promoter DNA sequence, a cytochrome P-450 coding gene, and a fdxshe coding gene.
14. 14. The process according to claim 13, wherein the host is selected from the group consisting of prokaryotic and eukaryotic hosts.
15. 15. The process according to claim 14, wherein the prokaryote is an actinomycete.
16. 16. The process according to claim 15, wherein the actinomycete is a Streptomyces.
17. 17. The process according to claim 16, wherein the Streptomyces is selected from the group consisting of S. lividans, S. carbophilus, and S. helvaticus.
18. 18. The process according to claim 14, wherein the prokaryotic host is selected from the group consisting of Escherichla 'coli and Bacillus subtilis.
19. 19. The process according to claim 14, wherein the eukaryotic host is a fungus.
20. 20. The process according to claim 19, wherein the fungus is a Penicillium citrinum.
21. 21. The process according to claim 20, wherein the host Penicillium citrinum is a strain that produces ML-236B (compactin).
22. 22. The process according to claim 13, wherein the plasmid is selected from the group consisting of pWHM-3 and pSCA.
23. 23. The process according to claim 13, wherein the plasmid is a multi-copy plasmid.
24. 24. The process according to claim 23, wherein the multi-copy plasmid is PIJ702.
25. 25. The process according to claim 13, wherein the gene coding for cytochrome P-450 is obtained from actinomycete.
26. 26. The process according to claim 13, wherein the gene coding for cytochrome P-450 is obtained from Streptomyces.
27. 27. The process according to claim 26, wherein the cytochrome P-450 coding gene is selected from the group consisting of S. lividans, S. carbophilus and S. helvaticus.
28. The process according to claim 27, wherein the cytochrome P-450 obtained from S. carbophilus is selected from the group consisting of cytP-450sca-l, cytP450sca-2 and cytP-450sca-
29. 29. The process according to claim 13, wherein the promoter DNA sequence is operably linked to the cytochrome P-450 coding gene.
30. 30. The process according to claim 13, wherein the fdxshe gene has the nucleotide sequence in SEQ ID . The process according to claim 13, wherein the fdxshe gene has 80% sequence identity with the nucleotide sequence SEQ ID No. 1. . The process according to claim 13, wherein the fdxshe gene has 85% sequence identity with the nucleotide sequence SEQ ID No. 1. . The process according to claim 13, wherein the fdxshe gene has 90% sequence identity with the nucleotide sequence SEQ ID No. 1. . The process according to claim 13, wherein the fdxshe gene has 95% sequence identity with the nucleotide sequence SEQ ID No. 1. . The process according to claim 13, wherein the fdxshe gene encodes the amino acid sequence of SEQ ID No. 1. . A process for producing pravastatin comprising: cultivating the genetically modified strain by the processes according to claims 13-35 under conditions in which ML-236B is hydroxylated to form pravastatin; and recover pravastatin. . The process according to claim 36, wherein the ML-236B is provided in the medium. . The process according to claim 36, wherein the ML-236B is produced with Penicillium citrinum, which is co-cultivated with the host. . The process according to claim 36, wherein the genetically modified strain is Penicillium citrinum which produces ML-236B.