Imported: 21 Feb '17 | Published: 01 Mar '05
USPTO - Utility Patents
New genes containing a DNA sequence coding for hydroxynitrile lyase, which genes can be prepared via a primer combination based on the DNA sequence of the 5′-region of the mdl genes from Prunus serotina and from Prunus amygdalus and/or a primer 2 based on the 3′-region of the DNA sequences of one of the hydroxynitrile lyase isoenzymes from Prunus serotina or from Prunus amygdalus, subsequent amplification with a DNA polymerase using a DNA from organisms, containing genes coding for hydroxynitrile lyase, as templates and cloning, and also recombinant proteins which can be prepared in suitable host cells by heterologous expression of the DNA sequence of said HNL genes, and proteins and fusion proteins derived therefrom and use of said proteins for preparing (R)- or (S)-cyanohydrins.
New genes containing a DNA sequence coding for hydroxynitrile lyase, recombinant proteins derived therefrom and having hydroxynitrile lyase activity, and use thereof
1. Field of the Invention
Biocatalytic processes have become very important for the chemical industry. In this connection, carrying out chemical reactions with the aid of biological catalysts is particularly interesting in those fields of application in which it is possible to utilize the frequently found enzyme property of preferably converting or forming in chemical reactions with chiral or prochiral components one of the two enantiomers.
Essential requirements for utilizing said advantageous properties of enzymes are the availability of said enzymes in industrially required amounts and a sufficiently high reactivity and also stability under the real conditions of an industrial process.
2. Description of the Related Art
Cyanohydrins are a particularly interesting class of chiral chemical compounds. Cyanohydrins are important, for example, in the synthesis of α-hydroxy acids, α-hydroxyketones, β-aminoalcohols which are used for producing biologically active substances, for example pharmaceutical active substances, vitamins or pyrethroid compounds.
Said cyanohydrins are prepared by addition of hydrocyanic acid to the carbonyl group of a ketone or aldehyde.
Industrial production of chiral compounds such as, for example, (S)-cyanohydrins was made possible by making use of the enzyme (S)-hydroxynitrile lyase from Hevea brasiliensis and is described, for example, in WO 97/03204, EP 0 951561 and EP 0 927 766.
However, there is a large variety of interesting chemical compounds for which the R enantiomers are important for industrial applications. Up until now, only processes which can be used only on the laboratory scale have been described for preparing a number of products (e.g.: EP 0 276 375, EP 0 326 063, EP 0 547 655). In this connection, mainly enzyme preparations obtained from plants of the Rosaceae family, for example from the kernels of almonds (Prunus amygdalus), were used.
Recently, Prunus species have become more and more important so that attempts were made to investigate said species in greater detail.
The specialist literature, for example Plant Physiology, April 1999, Vol 119, pp. 1535-1546, discloses that Prunus species can contain a plurality of R-HNL isoenzymes. These isoenzymes are expressed at different levels in various tissues of the plant. It was possible to identify in the plant Prunus serotina which is a close relative of Prunus amygdalus 5 different isoenzymes up until now and to sequence their genes. Only one Prunus amygdalus isoenzyme has been described up until now in Planta (1998) 206: 388-393, and this isoenzyme is most strongly expressed in the flower bud. A gene for said R-HNL isoenzyme has already been isolated and the cDNA has been sequenced.
However, no successful (functional) heterologous expression of such a gene has been reported in the specialist literature or patent literature.
Industrial applications on a large scale, too, have not been carried out up until now, the main reason being that enzyme preparations from almond kernels with hydroxynitrile lyase activity have not been available up until now in sufficient quantities and at justifiable costs.
It was therefore an object of the invention to create a basis which can provide an R-hydroxynitrile lyase in amounts required for industrial applications.
This object was achieved by looking for a way of producing an enzyme corresponding to the R-HNL preparation of Prunus amygdalus by genetic engineering strategies with the aid of an appropriate recombinant microorganism strain. Such a recombinant enzyme with R-HNL activity ought to be made technically available in this way in a sufficient amount.
It can be derived from the DNA sequences described in the literature above that particular regions in the genes of the various isoenzymes are highly conserved. According to the invention, this is used as a basis for generating primers for PCR amplification of P. amygdalus R-HNL. With the aid of such primers it is then possible, using DNA isolated from for example almond kernels (P. amygdalus) as template, to amplify by means of PCR DNA pieces which show in the analysis by agarose gel electrophoresis distinct specific bands. According to the invention, bands were found which correspond to the size of mdl genes (Planta (1998) 206: 388-393). Subsequently, appropriate primers for all isoenzymes known in Prunus serotina are generated and corresponding PCR products are obtained. DNA from this region is isolated from appropriate preparative agarose gels and cloned into standard vectors for cloning of PCR-generated fragments in Escherichia coli.
Sequence analysis of a series of selected clones revealed the presence of clones with homologies to the particular, already known R-HNL genes of Prunus species, although the sequences of the clones or genes obtained in this way differ in several sequence positions from the sequences already known or published, whereby important functional differences are established.
As a result, a new variant of HNL genes was unexpectedly found, although primer combinations which made use of the already known sequence of a cDNA obtained from Prunus amygdalus flower material were used.
The new genes were sequenced and the genomic DNA sequence was determined.
Accordingly, the present invention relates to new genes containing a DNA sequence coding for hydroxynitrile lyase, which genes can be prepared via a primer combination of a primer 1 based on the DNA sequence of the 5′-region of the mdl genes from Prunus serotina and from Prunus amygdalus and/or a primer 2 based on the 3′-region of the DNA sequences of one of the hydroxynitrile lyase isoenzymes from Prunus serotina or from Prunus amygdalus, subsequent amplification with a DNA polymerase using a DNA from organisms, containing genes coding for hydroxynitrile lyase, as templates and cloning.
Thus it is possible, for example, to prepare gene-specific PCR primer based on sequence homology of the Prunus amygdalus MDL1 gene and of the Prunus serotina mdl5 gene, and, as a result, a new gene, the HNL5 gene, is obtained after amplification and cloning.
The Prunus amygdalus HNL5 gene produced by PCR amplification, for example, has the nucleotide sequence depicted in FIG. 1, which is likewise a subject of the invention. The invention also relates to HNL5 genes having a nucleotide sequence which is at least 80%, preferably 85%, identical to the sequence depicted in FIG. 1.
The new HNL5 gene differs from the published sequence of the Prunus amygdalus MDL 1 gene in 7 base pairs.
Furthermore it is possible, for example, to prepare gene-specific PCR primers based on sequence homology of the Prunus serotina mdl1 gene, and, as a result, a new gene, the HNL1 gene, is obtained after amplification and cloning.
The HNL1 gene produced by PCR amplification, for example, has the nucleotide sequence depicted in FIG. 8, which is likewise a subject of the invention. The invention also relates to HNL1 genes having a nucleotide sequence which is at least 80%, preferably 85%, identical to the sequence depicted in FIG. 8.
Analogously, it is possible, according to the invention, to prepare further gene-specific PCR primers, for example based on the sequence of the Prunus amygdalus MDL1 gene and/or based on the sequence of the known Prunus serotina mdl1, mdl2, mdl3 and mdl4 genes, and this results in obtaining, after amplification and cloning, further new genes such as, for example, HNL2, 3 or 4 which are all subjects of the present invention.
The genomic clones and genomic DNA thereof form the basis for obtaining enzyme preparations by heterologous expression, for example by inducible or constitutive expression, in various host cells.
Furthermore, sequence analysis of the genomic DNA of the new genes of the invention shows that the proteins encoded by the new genes possess a signal sequence or a signal peptide, and this also makes secretory expression of heterologous proteins in suitable host cells possible.
In this connection, specific expression vectors are used for expressing the protein of one of the cloned new genes as a fusion protein with a signal peptide.
Accordingly, the present invention further relates to recombinant proteins which can be prepared in suitable host cells by heterologous expression of the genomic DNA sequence of the Prunus amygdalus HNL genes (for example HNL1, HNL2, HNL3, HNL4 and HNL5). Examples of suitable host cells in this connection are microorganisms. Preference is given to eukaryotic microorganisms and particular preference is given to fungi. Examples are Saccharomyces cerevisiae or Pichia pastoris.
For example, the amino acid sequence of Prunus amygdalus hydroxynitrile lyase HNL5, derived from the nucleotide sequence of the HNL5 gene, is depicted in FIG. 3 and the amino acid sequence of Prunus amygdalus hydroxynitrile lyase HNL1, derived from the nucleotide sequence of the HNL1 gene, is depicted in FIG. 9.
In addition, the invention relates to the use of a DNA sequence which codes for the signal peptide of a hydroxynitrile lyase, for example of Rosacea species, for secretory expression of heterologous proteins in host cells and to the proteins obtained in this way.
Accordingly, the invention further relates to fusion proteins or heterologous proteins which can be prepared by using a DNA sequence which codes for the signal peptide of a hydroxynitrile lyase, for example of Rosacea species, and by secretory expression of said DNA sequence in suitable host cells.
Examples of suitable host cells in this connection are again microorganisms. Preference is given to bacteria or eukaryotic microorganisms, and particular preference is given to fungi, such as, for example, Saccharomyces cerevisiae or Pichia pastoris.
For example, the nucleic acid sequence of the DNA fragment coding for a secretory hybrid protein (PamHNL5xGOX) with HNL activity, which nucleic acid sequence comprises sequences of the P. amygdalus HNL5 gene and the Aspergillus niger glucose oxidase gene, is depicted in FIG. 4. The amino acid sequence of the PamHNL5xGOX hybrid protein, derived from the nucleotide sequence (FIG. 4), is depicted in FIG. 5.
FIG. 6 shows the comparison of the amino acid sequences of the Prunus amygdalus HNL5 protein and the PamHNL5xGOX hybrid protein.
In order to obtain the recombinant proteins of the invention, the clones, for example, which show homologies with the known genes of P. amygdalus MDL1 and/or with P. serotina mdl1, mdl2, mdl3, mdl4 and mdl5 are treated further.
In order to obtain recombinant proteins with hydroxynitrile lyase activity, the appropriate genes, for example the HNL5 gene, are incorporated, for example, into an expression vector for Pichia pastoris or for Saccharomyces cerevisiae.
The genomic DNA may be spliced beforehand by means of PCR. Preference is given to preparing a base fragment for constructing expression plasmids for heterologous expression of the appropriate gene in bacteria and eukaryotes. For this purpose, a plasmid is constructed from which it is possible to obtain a DNA fragment coding for a gene of the invention for incorporation into various expression vectors by cutting with restriction endonucleases.
Using the genes of the invention, it is thus possible to produce a functional HNL by expression in a heterologous host, using, for example, an inducible promoter system or a constitutively expressed promoter system.
It is thus possible to find, out of a large number of transformants, recombinant strains of, for example, Pichia pastoris, which overexpress recombinant protein with R-HNL activity. After inducing expression, the majority of the protein with R-HNL activity can be found in said strains in the culture supernatant.
Thus it was possible for the first time to prepare a recombinant protein which has an R-HNL activity comparable to that detectable in almonds (Prunus amygdalus kernels) in amounts usable for industrial applications.
Unexpectedly it was found that the recombinant proteins with R-HNL activity, which are derived, for example, from the HNL1-5 genes of the invention and are denoted Pam-HNL1-5, have, compared with the enzyme preparation isolated from almonds, substantially better properties as biocatalysts in reaction mixtures for the preparation of cyanohydrins and can thus be used particularly advantageously for biocatalytic synthesis of cyanohydrins.
Furthermore, it is also possible to truncate the sequences of the recombinant proteins of the invention at the C-terminal end or to replace the sequences in the N- and C-terminal region by those of a related protein with different functions. Accordingly, the invention also relates to proteins altered in this way.
The recombinant proteins are distinguished in particular also by having a host-specific glycosylation, as a result of which the recombinant proteins are substantially more stable than the native proteins.
A particular advantage of the recombinant proteins of the invention results from the substantially higher stability which causes a substantially lower amount of enzyme compared with the native enzyme to be required, in order to achieve high enantiomeric purity. Thus, comparative experiments show that, when using the recombinant proteins of the invention, merely a tenth of the required amount of native protein is required in order to achieve comparable enantiomeric purities (ee values).
Accordingly, the invention further relates to the use of the recombinant proteins of the invention (HNLs) for preparing (R)- or (S)-cyanohydrins.
The starting materials used for preparing the (R)- or (S)-cyanohydrins are an aldehyde or a ketone as substrate, a cyanide group donor and a recombinant protein of the invention.
Aldehydes mean in this connection aliphatic, aromatic or heteroaromatic aldehydes. Aliphatic aldehydes mean in this connection saturated or unsaturated, aliphatic, straight-chain, branched or cyclic aldehydes. Preferred aliphatic aldehydes are straight-chain aldehydes with in particular 2 to 30 carbon atoms, preferably from 2 to 18 carbon atoms, which are saturated or mono- or polyunsaturated. In this connection, the aldehyde may have both C—C double bonds and C—C triple bonds. The aliphatic, aromatic or heteroaromatic aldehydes may furthermore be unsubstituted or substituted with groups inert under the reaction conditions, for example with unsubstituted or substituted aryl or heteroaryl groups, such as phenyl, phenoxy or indolyl groups, with halogen, hydroxy, hydroxy-C1-C5-alkyl, C1-C5-alkoxy, C1-C5-alkylthio, ether, alcohol, carboxylic ester, nitro or azido groups.
Examples of aromatic or heteroaromatic aldehydes are benzaldehyde or differently substituted benzaldehydes such as, for example, 3,4-difluorobenzaldehyde, 3-phenoxybenzaldehyde, 4-fluoro-3-phenoxybenzaldehyde, hydroxybenzaldehyde, methoxybenzaldehyde, furthermore furfural, methylfurfural, anthracene-9-carbaldehyde, furan-3-carbaldehyde, indole-3-carbaldehyde, naphthalene-1-carbaldehyde, phthaldialdehyde, pyrazole-3-carbaldehyde, pyrrole-2-carbaldehyde, thiophene-2-carbaldehyde, isophthalaldehyde or pyridinaldehydes, thienylaldehydes etc.
Ketones are aliphatic, aromatic or heteroaromatic ketones in which the carbonyl carbon atom is substituted unequally. Aliphatic ketones mean saturated or unsaturated, straight-chain, branched or cyclic ketones. The ketones may be saturated or mono- or polyunsaturated. They may be unsubstituted or substituted with groups inert under reaction conditions, for example with unsubstituted or substituted aryl or heteroaryl groups such as phenyl or indolyl groups, with halogen, ether, alcohol, carboxylic ester, nitro or azido groups.
Examples of aromatic or heteroaromatic ketones are acetophenone, indolylacetone, etc.
Aldehydes and ketones which are suitable according to the invention are known or can be prepared as usual.
The substrates are converted in the presence of the HNLs of the invention using a cyanide group donor.
Suitable cyanide group donors are hydrocyanic acid, alkali metal cyanides or a cyanohydrin of the general formula I
In the formula I, R1 and R2 are independently of one another hydrogen or an unsubstituted hydrocarbon group, or R1 and R2 are together an alkylene group having 4 or 5 carbon atoms, where R1 and R2 are not simultaneously hydrogen. The hydrocarbon groups are aliphatic or aromatic, preferably aliphatic groups. R1 and R2 are preferably alkyl groups having 1-6 carbon atoms, and very preferably the cyanide group donor is acetonecyanohydrin.
The cyanide group donor may be prepared according to known methods. Cyanohydrins, in particular acetonecyanohydrin, may also be obtained commercially. Preference is given to using hydrocyanic acid (HCN), KCN, NaCN or acetonecyanohydrin as cyanide group donor, and particular preference is given to hydrocyanic acid.
In this connection, it is also possible to liberate hydrocyanic acid from one of its salts such as, for example, NaCN or KCN just prior to the reaction and to add it to the reaction mixture undissolved or in soluble form.
The conversion may be carried out in an organic, aqueous or 2-phase system or in emulsion. The aqueous system used is an aqueous solution containing the inventive HNL or a buffer solution. The examples thereof are Na citrate buffer, phosphate buffer, etc.
Organic diluents which may be used are aliphatic or aromatic hydrocarbons which are not or negligibly water-miscible and which are unhalogenated or halogenated, alcohols, ethers or esters or mixtures thereof. Preference is given to using methyl tert-butyl ether (MTBE), diisopropyl ether, dibutyl ether and ethyl acetate or a mixture thereof.
In this connection, the HNLs of the invention may be present either as such or immobilized in the organic diluent, but the conversion may also be carried out in a two-phase system or in emulsion using nonimmobilized HNL.
The present invention will now be described in more detail by the following Examples. However, the following Examples are merely illustrative in nature. Other Examples within the scope of the claims are also possible. Thus, the following should not be construed to limit the spirit and scope of the claims.
Isolation of Genomic DNA from Almonds (Prunus amygdalus Kernels)
Dried almonds (Farmgold, batch number L4532, 1999 harvest) were finely chopped using a knife and frozen in a mortar with liquid nitrogen and ground using a pestle under liquid nitrogen to give a fine powder. 0.1 gram of frozen almond powder was directly admixed with 65° C. warm “breaking puffer” (100 mM NaAc; 50 mM EDTA; 500 mM NaCl, adjusted to pH 5.5; 1.4% SDS and 20 μg/ml RNAse A). After stirring for 15 minutes, the insoluble cellular residues were removed by centrifugation (10 min at 7000 g) and the supernatant was admixed with the same volume of 10 M ammonium acetate and then incubated on ice for 10 min. After centrifugation at 10,000 g for 15 minutes, the supernatant was extracted 2× with phenol/chloroform (1/1, phenol equilibriated with 50 mM Tris, pH 8.0). After another extraction with twice the volume of chloroform/isoamyl alcohol (24/1), the DNA was precipitated from the supernatant with the same volume of isopropanol, removed by centrifugation, and the DNA pellet was washed with 70% ethanol and dried in air. The DNA was then dissolved in 200 μl of water at 68° C. for 20 min and purified by ethanol precipitation (Ausubel et al., 1999). After the centrifugation, the DNA pellet was dried in air and dissolved in 50 μl of water.
Amplification and Cloning of a Genomic DNA Section of Almond (Prunus amygdalus) DNA Homologous to known Rosaceae mdl Genes
Since it was known that a plurality of hydroxynitrile lyase isoenzymes whose sequences are highly homologous to one another can appear in Prunus species (Hu and Poulton, 1999), gene-specific PCR primers based on sequence homology of the Prunus serotina mdl5 gene and the Prunus amygdalus MDL1 gene (Suelves et al., 1998) were prepared:
The amplification was carried out in a 50 μl mixture with 1.2 U of “Hotstar” Taq DNA polymerase (Qiagen, Hilden, Germany), with 50 ng of genomic almond DNA as template, in each case 200 ng of primers 1 and 2, 5 μl of a dNTP (2 mM each) mix, all of these in 1×PCR buffer according to the “Hotstar Kit” manual (Qiagen, Hilden, Germany), starting with a denaturation step of 15 minutes at 95° C., followed by 30 cycles (1 min 95° C., 30 sec 64° C., 1 min 72° C.) for amplification and a final incubation at 72° C. for 5 min for preparation of complete products.
Said PCR produced a DNA fragment of approx. 2.16 kb in size (determined by analysis by means of agarose gel electrophoresis). This PCR product was purified by means of the “Qiaquick Kit” (Qiagen, Hilden, Germany) according to the enclosed manual and sequenced using the “Dye Deoxy Terminator Cycle Sequencing” kit (Applied Biosystems Inc., Forster City, Calif., USA) according to the primer walking strategy starting from the two primers used for the PCR. The obtained DNA sequence of the PCR fragment of 2162 base pairs total length is depicted in FIG. 1.
Approx. 0.5 μg of the purified PCR product was cut with restriction endonuclease EcoRI and cloned into plasmid vector pBSSK(−) (Stratagene Cloning Systems, La Jolla, Calif., USA) via the EcoRI cleavage site. The insert of a resultant recombinant molecule (the corresponding plasmid was denoted pBSPamHNL5g) was sequenced according to the method described above, and the sequence of the cloned fragment was 100% identical to the sequence of the above-described PCR product obtained with the two primers (1 & 2).
Sequence Analysis of the Genomic Prunus amygdalus DNA Fragment obtained by PCR Amplification with Primers 1 and 2
In the region of the PCR-amplified and sequenced DNA section, an open reading frame which is interrupted by 3 introns was found. Said three introns were identified with the aid of the “GT . . . AG” intron consensus sequence. The reading frame starts with an ATG codon at position +13 and ends with a stop codon at position +2151.
For the coding region, the fragments of positions 13 to 115 (exon I), positions 258 to 917 (exon II), 1121 to 1961 (exon III) and 2078 to 2150 (exon IV) were joined together. The assembled DNA sequence codes for a protein with 559 amino acids and a calculated molecular weight of 61 kDa or 57.9 kDa for an N-terminally processed form. The peptide masses were calculated with the aid of the GCG program package (Genetics Computer Group, Wisconsin, USA). Said protein was denoted PamHnI5.
The protein sequence derived for the open reading frame (without introns) is shown in FIG. 3. It was possible to determine distinctive homologies to known Rosaceae hydroxynitrile lyases (Blast program, GCG package, version 10.1, Genetics Computer Group, Wisconsin, USA), the highest homologies being to the published sequences of Prunus amygdalus Mdl1 (99 percent identical, Suelves et al. 1998) and of Prunus serotina Mdl5 (94 percent identical, Hu and Poulton, 1999). With the aid of this homology, a cleavable signal sequence with cleavage between S27 and L28 was identified. It was possible to detect a cleavage site of this type in the two Prunus serotina Mdl1 and Mdl4 isoenzymes by N-terminal sequencing of the native proteins purified from plant material (Zihua Hu and Jonathan E. Poulton, Plant Physiology, 119, 1535-1546, 1999). The sequences of various HNL isoenzymes present in Rosacea species are known only for Prunus serotina. Due to the highest homologies to the Prunus serotina Mdl5 sequence, the new HNL gene from Prunus amygdalus was established as HNL5. Searching for sequence motifs in the PROSITE sequence motif database (GCG package, version 10.1, Genetics Computer Group, Wisconsin, USA) revealed the presence of 13 potential N-glycosylation sites (drawn into FIG. 3).
Obtaining an intron-free Prunus amygdalus HNL5 gene by PCR splicing. By means of a specific PCR strategy using overlapping primers, the coding regions were linked to one another (according to FIG. 2) by 4 successive PCR reactions.
In the first round of PCR (PCR1-1 and PCR1-2) exons II and III were amplified using the primer pairs PamHNL5b/PamHNL5c (PCR1-1) and PamHNL5d/PamHNL5e (PCR1-2), respectively. The 50 μl PCR mixtures in 1×PCR buffer (Qiagen) contained: in each case 100 pmol of the appropriate primers, 2.5 U of “Hotstar” Taq DNA polymerase (Qiagen), 5 μl of a dNTP (2 mM each) mix, 10 ng of plasmid pBSPamHNL5g as template. The following program was run: 15 min 95° C., 30 cycles 1 min 95° C., 30 sec 68° C., 1 min 72° C., then finally 5 min 7° C. for preparation of complete products). After electrophoretic separation in an agarose gel, the products from PCR1-1 and PCR1-2 were eluted from the gel by means of the Qiaexll kit.
Amplification of approx. 50 ng of the product from PCR1-1 with in each case 100 pmol of primers PamHNL5a2 and PamHNL5c led to an extension of said first PCR product in the second round of PCR (PCR2). The following program was run: 15 min 95° C., 30 cycles 1 min 95° C., 30 sec 68° C., 1 min 72° C., then finally 5 min 72° C. for preparation of complete products). The other conditions were the same as for PCR1. After electrophoretic separation, this PCR product was likewise purified via an agarose gel and eluted.
In the third round of PCR (PCR3), the products from PCR1-2 and PCR2 were linked to one another by primer-less PCR with the aid of the overlapping ends (5 cycles of 1 min at 94° C., 30 sec at 68° C. and 1.5 min at 72° C., in each case approx. 100 ng of the two products from PCR1-1 and PCR2 in 50 μl mixtures in 1×PCR buffer (Qiagen), 5 μl of the dNTP (2 mM each) mix and 2.5 U of “Hotstar” Taq DNA polymerase (Qiagen).
The full length of the coding Prunus amygdalus hn15 gene was completed and the complete product amplified in a fourth round of PCR (PCR4) using in each case 100 pmol of primers PamHNL5al and PamHNL5f. Said primers were directly added to the PCR 3 reaction mixture. The following program was run: 20 cycles of 1 min 95° C., 30 sec 63° C., 1.5 min 72° C. and, finally, 5 min 72° C.). The other conditions were the same as for PCR1.
The product obtained in the final round of PCR was fractionated via a preparative agarose gel and DNA of 1.6-1.8 kb in size was eluted from the gel by means of the “Quiaexll” kit (Qiagen) and cloned into plasmid pBSSK(−) via the EcoRI cleavage site. A clone having the correct restriction pattern was selected and sequenced.
The sequence in the coding region was 100% identical to the exons of the genomic DNA sequence. This clone was denoted pBSPamHNL5orf.
The preparation of a base fragment for constructing expression plasmids for heterologous expression of the Prunus amygdalus HNL5 gene in bacteria and eukaryotes.
The aim of this experiment was to construct a plasmid from which a DNA fragment coding for Prunus amygdalus HNL5 for incorporation into various expression vectors can be obtained by restriction endonuclease cleavage. In this connection, PCR amplification added suitable sequences to the ends of the Prunus amygdalus HNL5 gene contained in pBSPamHNL5orf via appropriate primers.
The insert of plasmid pBSPamHNL5orf was amplified by means of PCR using the primers PCRHNL5-a and PCRHNL5-e (10 ng of DNA of plasmid pBSPamHNL5orf as template, 400 ng of primer PCRHNL5-a, 200 ng of primer PCRHNL5-e). The PCR reaction was carried out in 50 μl mixtures in 1×PCR buffer (Qiagen), containing 5 μl of the dNTP (2 mM each) mix and 1.2 U of “Hotstar” Taq DNA polymerase (Qiagen). The following program was run: 15 min 95° C., 30 cycles 1 min 95° C., 30 sec 68° C., 1.5 min 72° C., then finally 5 min 72° C. for preparation of complete products).
After cutting with restriction endonuclease EcoRI, the DNA fragment obtained was cloned into vector PBSSK(−) (Stratagene, USA) and verified by sequencing. The resultant plasmid was called pBSPamHNL5ex.
Construction of Expression Constructs for Heterologous Expression of the HNL5 Gene in Pichia pastoris
DNA of plasmid pBSPamHNL5ex was cut with EcoRI, and the HNL fragment was separated from the vector part by means of preparative gel electrophoresis. After eluting the HNL fragment DNA by means of Qiaex II kit (Qiagen), said fragment was cloned into plasmids pHILD2 and pGAPZ (Invitrogen, San Diego, Calif., USA) via the EcoRI cleavage sites. The correct orientation of the insert toward the promoters was checked with the aid of control cuts. In each case, one clone having a correctly orientated insert was selected and preserved. The correct transitions from vector part to incorporated HNL fragment were verified by sequencing. Said two plasmids for expression in Pichia pastoris formed were denoted pHILDPamHNL5a (for inducible expression) and pGAPPamHNL5a (for constitutive expression).
Inducible Expression of the Prunus amygdalus HNL5 Gene in Pichia pastoris
DNA of plasmid pHILDPamHNL5a was cut with restriction endonuclease Not1 and transformed into Pichia pastoris GS115 (Invitrogen, San Diego, Calif., USA). Transformation was carried out according to the protocols of the Pichia expression kit (Invitrogen, San Diego, Calif., USA). 100 histidine-prototrophic clones were cultivated in liquid medium (500 ml shaker cultures) according to the protocols of the Pichia expression kit and induced with methanol for 48 hours. The cells were removed by centrifugation and suspended with disruption buffer (50 mM Tris-HCl pH 7.6) to an optical density (OD600) of 50.0 and disrupted in a “Merckenschlager” homogenizer (Braun, Melsungen, FRG) according to the “glass bead method” (Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-Interscience, New York, 1999). Both the culture supernatants and cell lysates were assayed for HNL enzyme activity using a standard activity assay (analogous to WO 97/03204) and using racemic mandelonitrile as substrate. Two clones which showed, in these shaker flask experiments, the best activities in the culture supernatant (Pichia pastoris PamHNL5-a37 and Pichia pastoris PamHNL5-a4) were selected and preserved. Analysis of the methanol utilization type resulted in the phenotype Mut□ (good methanol utilization) for Pichia pastoris PamHNL5-a37 and the phenotype Muts (slow methanol utilization) for Pichia pastoris PamHNL5-a4.
Constitutive Expression of the Prunus amygdalus HNL5 Gene in Pichia pastoris
Plasmid pGAPPamHNL5a was transformed into P. pastoris GS115. Transformation was carried out according to the protocols of the Pichia expression kit from Invitrogen Corp. (San Diego, Calif., USA). Transformants were selected from YPD complete medium plates with 100 mg/l Zeocin. 100 Zeocin-resistant clones were cultivated in in each case 500 ml of YPD complete medium and incubated with shaking at 30° C. for 96 hours. The cells were removed by centrifugation and HNL activity was determined in the culture supernatant. A clone which showed the best activity in the culture supernatant was preserved and denoted Pichia pastoris PamHNL5-a2.
HNL Production in a Laboratory Bioreactor using a Pichia pastoris Strain Transformed with the Prunus amygdalus HNL5 Gene (Methanol-inducible Expression System)
Pichia pastoris PamHNL5-a37 was grown in a standard laboratory bioreactor (total volume 42 1) in a three-phase process. Said process consisted of a first exponential and a second linear growth phase for biomass formation and a subsequent expression phase for formation of the recombinant Prunus amygdalus HNL enzyme, in principle following the method described for the production of recombinant Hevea brasiliensis HNL (Hasslacher, M., Schall, M., Hayn, M., Bona, R., Rumbold, K., Lückl, J., Griengl, H., Kohlwein, S. D., Schwab, H.: High level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts. Protein Expression and Purification 11, 61-71, 1997).
In detail, the following conditions were kept to: Chemicals 1.-8., measured for 20 liters and dissolved in 15 liters of water, were initially introduced into the bioreactor and sterilized together with the reactor at 121° C. for 1 hour. After cooling to 29° C., the pH of the medium was adjusted to pH 5.0 with ammonia (chemical 9, initially introduced in a sterile feed bottle). Subsequently, approx. 200 ml of a sterile-filtered trace element solution (chemicals 10-18, appropriate amounts for 20 l) were introduced into the bioreactor via a feed bottle.
The bioreactor prepared in this way was inoculated with 2 liters of preculture which had been cultivated in shaker flasks at 30° C. according to the conditions stated in the manual of the Pichia expression kit (Invitrogen Corp., San Diego, Calif., USA). Culturing was carried out at a constant temperature of 29° C. Controlling aeration (between 15 and max. 40 liters of air/min) and stirrer revolutions (between 250 to 500 rpm) maintained the O2 partial pressure at a value above 30% of the saturation concentration. After 21 hours, the biomass had grown to a value in the region of 22 g/l of dry cell mass (DCM). From this time onward, sterile glycerol was metered in in constant small portions at 15 min intervals, and 130 g of glycerol were added per hour. In this, second linear growth phase, it was possible to reach a biomass concentration in the range of 70 g/l DCM over a period of 42 hours.
Subsequently, the third phase was initiated by inducing expression via metering in methanol. In this connection, the methanol content of the culture broth was adjusted to a value of 0.8-1% by weight. At the start of and after two days of induction, in each case another portion of sterile trace element solution (chemicals 10-18, appropriate amounts for 20 l, dissolved in approx. 200 ml of water) was added. After an induction phase of 110 hours, it was possible to obtain an amount of enzyme of 110 U/ml of culture broth. After removing the cells, for example by centrifugation, it is possible to obtain a crude enzyme preparation which can be used directly for biocatalytic conversions.
The following chemicals were used for preparing the culture medium (amount per liter):
Trace elements and vitamin H (all chemicals in analytical grade):
Construction of a clone for expressing the Prunus amygdalus HNL5 gene as fusion protein with N-terminal and C-terminal parts of Aspergillus niger glucose oxidase.
This construct was designed such that the fusion protein formed is directed into the secretory pathway via the heterologous signal sequence.
In a PCR in a 50 μl mixture in 1×PCR buffer (Qiagen), with in each case 100 pmol of primers Glucox2 and Glucoxct, 10 ng of plasmid pPamHNL5orf as template, 5 μl of dNTP (2 mM each) mix, and 1.2 U of “Hotstar” Taq polymerase (Qiagen), the C-terminal and N-terminal ends of the HNL5 gene were replaced by a sequence derived from glucose oxidase and truncated, respectively (program: 15 min 95° C., 30×: 1 min 95° C., 1 min 68° C., 2 min 72° C., and finally 10 min 72° C). Finally, in a 2nd PCR in a 50 μl mixture in 1×PCR buffer (Qiagen) with 0.1 μl of product from the first PCR as template, in each case 100 pmol of primers Glucoxl and Glucoxct, 2 μl of dNTP (2 mM each) mix, and 2.5 U of Pwo polymerase (Roche Diagnostics, Mannheim, Germany), (program: 5 min 95° C., 30 times: 1 min 95° C., 0.5 min 68° C., 3 min 72° C. and finally 3 min 72° C.) the 5′ region of the gene was completed.
The PCR product was incorporated into plasmid pHILD2 (Invitrogen, San Diego, Calif., USA) after cutting with EcoRI, via EcoRI cleavage sites simultaneously introduced at the ends of the DNA fragment. A clone having the correct orientation of the insert toward the aox promoter of plasmid pHILD2 was verified by sequencing the transition regions from vector to insert and preserved. The plasmid constructed in this way was denoted pHILDPamHNL5gox.
Not1-linearized DNA of plasmid pHILDPamHNL5gox was transformed into the strain Pichia pastoris GS115 and also into the protease-deficient strain Pichia pastoris SMD1168. From each mixture, several histidine-prototrophic clones were cultured in shaker flasks and HNL activity was determined in the cultured supernatant (standard assay). These experiments were carried out analogously, as described in example 7. It was possible to find in the culture supernatant of some clones HNL activity and thus to state that the signal sequence of the Aspergillus niger gox gene is capable of directing the heterologous HNL5 protein, when expressed in Pichia pastoris, into the secretory pathway.
Oligonucleotide primers used
The nucleic acid sequence of the DNA fragment for a secretory hybrid protein (PamHNL5xGOX) with HNL activity is depicted in FIG. 4, and the amino acid sequence derived therefrom is represented in FIG. 5. A comparison of the amino acid sequences of Prunus amygdalus HNL5 and the hybrid protein PamHNL5xGOX can be found in FIG. 6.
Recombinant protein with HNL activity, which had been produced using the recombinant strain Pichia pastoris PamHNL5-a37, as described in example 9, was subjected to a glycosylation analysis by endoglycosidase digest. For this purpose, 100 ml of the culture supernatant were 10 times concentrated by ultrafiltration (Biomax 30,000 NMWL, Millipore, Bedford, Mass., USA). Samples 1-2 were treated with N-glycosidase F (N-glycosidase F kit, Roche Diagnostics, Mannheim, D).
For samples 4-5, an HNL preparation from almonds from Roche was used, and for samples 6 and 7 the concentrated culture supernatant was treated with endoglycosidase H (Roche Diagnostics, Mannheim, D). All mixtures were carried out in a total volume of 10 μl.
After treatment with the glycosidases, the samples were separated on a 12 percent strength SDS polyacrylamide gel and stained with Coomassie Blue.
These results (see FIG. 7) show that a large part of the oligosaccharides bound to PamHNL5 can be removed by endoglycosidase H even without denaturation of the PamHNL5 protein.
Cleaving off the oligosaccharides leads from a protein smear visible around sizes of from 70 to over 100 kDa to a sharp band at about 60 kDa, corresponding to the calculated molecular weight of a nonglycosylated PamHNL5 protein.
A comparable protein band is not present in the Roche preparation or present only to a negligible extent. In addition, it is impossible to see a significant difference between an untreated protein preparation and a preparation treated with endoglycosidase F. From this finding, it can definitely be stated that the recombinant PamHNL5 enzyme is completely different from the enzyme material obtained from almonds.
Cloning of a genomic DNA fragment having the Coding Region of the Prunus amygdalus HNL1 Gene
A genomic DNA fragment having the coding region of the Prunus amygdalus HNL1 gene was amplified from genomic almond DNA (preparation, see example 1) with the aid of a PCR using primers mandlp2f (5′-ACTACGAATTCGACCATGGAGAAATCAAC-3′) (SEQ ID No. 15) and ecpamHNL1e (5′-CAGAATTCGCCCTTGTGCATGCATCGATTAAAGAACCAAGGATGCTGCTGAC-3′) (SEQ ID No. 16).
The amplification was carried out in 50 μl reactions with 1.2 units of “Hotstar” DNA polymerase (Qiagen GmbH, Hilden, Germany), in each case 10 pmol of the two primers, 2 μl of a dNTP mix (5 mM each) and 100 ng of genomic almond DNA in standard PCR buffer (Qiagen GmbH, Hilden, Germany). The following PCR program was used: 15 min 95° C., then 10 cycles of 1 min at 94° C., 1 min 45° C. and 1 min 20 sec at 72° C., followed by 30 cycles of 1 min at 94° C., 1 min at 64° C. and 1 min 20 sec at 72° C. and a final extension step at 72° C. for 5 min.
Analysis of the DNA obtained showed that this PCR produced a plurality of DNA fragments of different sizes. Amplified DNA was separated in a preparative agarose gel. DNA from a band of the size to be expected for the HNL1 gene of approx. 2.1 kb was isolated from said agarose gel (Qiaquick Gel Extraction Kit, Qiagen GmbH, Hilden, Germany). The DNA obtained, after digest with restriction endonuclease EcoRI, was cloned into cloning vector pBSSK(−) (Stratagene Cloning Systems, La Jolla, Calif., USA) via the EcoRI cleavage site. 5 clones with appropriate inserts were isolated and the inserts were sequenced by means of the primer walking strategy. A clone corresponding to the consensus sequence obtained in this way was selected and the contained plasmid was denoted pSKpamHNL1—5—3.
The DNA sequence of the Prunus amygdalus HNL1 gene was verified and finally determined by amplifying another genomic DNA fragment using primers mandlp3f (5′-ACTACGAATTCGACCATGGAGAAATCAACAATG-3′) (SEQ ID No. 17) and pamHNL1end (5′-ATGCTGCTGACTTGAGGGAATC-3′) (SEQ ID No. 18). The amplification was carried out in 50 μl reactions with 2.5 units of “Hotstar” DNA polymerase (Qiagen GmbH, Hilden, Germany), in each case 10 pmol of the two primers, 2 μl of a dNTP mix (5 mM each) and 50 ng of genomic almond DNA in standard PCR buffer (Qiagen GmbH, Hilden, Germany). The following PCR program was used:
After fractionation of the PCR product in an agarose gel, a single DNA band was detected.
The PCR product was purified by means of the Qiaquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany) and directly sequenced by means of the primer walking strategy. The DNA sequence is depicted in FIG. 8. Possible introns were identified by their general 5′ and 3′ splice sites and their homology to the Prunus serotina HNL1 gene. The nucleotides in the regions of the three introns are shown in lower case letters in order to recognize the intron regions. The cloned insert in plasmid pSKpamHNL1—5—3 has the same sequence. The protein sequence of the Prunus amygdalus HNL1 protein was derived from the coding region of the DNA sequence and is depicted in FIG. 9.
0.5 g (3.9 mmol) of octanal was dissolved in 6 ml of tert-butyl methyl ether and 7.5 ml of an aqueous enzyme solution with recombinant R-HNL from example 9 (pH 3.8) were added. After addition of 0.33 ml (8.4 mmol) of hydrocyanic acid, the mixture was vigorously stirred on a magnetic stirrer at room temperature in order to form an emulsion, and the reaction was followed by means of GC on a cyclodextrin column. After a reaction time of 3 hours, cyanohydrin was formed with 81.1%ee and 48% conversion.
80 ml (34 units/mmol aldehyde) of an aqueous enzyme solution with recombinant R-HNL (34 units/mmol aldehyde) were diluted with 10 ml of 200 mM potassium phosphate/sodium citrate buffer pH 3.8 and added to a solution of 42.2 g (300 mmol) of 2-chlorobenzaldehyde and 42 ml of tert-butyl methyl ether, precooled to 10° C. Subsequently, 19.6 ml (501 mmol) of hydrocyanic acid were metered into the reaction mixture with stirring at 950 rpm within 40 min. After derivatization of cyanohydrin with acetyl chloride, the course of the reaction was followed by means of GC on a cyclodextrin column.
0.25 to 1 ml of R-oxynitrilase solution (E.C.18.104.22.168; 2187 units/ml) was diluted to 4 ml with 50 mM citrate/phosphate buffer (pH 4.0), and the pH of the enzyme solution was adjusted to pH 4.0, where appropriate, with a few drops of citric acid solution. A solution of 3 ml of tert-butyl methyl ether and 0.8 g (5.69 mmol) of 2-chlorobenzaldehyde was added to said solution and, subsequently, 445 μl (11.38 mmol) of hydrocyanic acid were added. The reaction mixture was stirred at 900 rpm by means of a magnetic stirrer at room temperature.
Conversion and enantiomeric purity of the (R)-cyanohydrin formed were analyzed by means of GC. For this purpose, a sample of the reaction solution was centrifuged and 50 μl of the organic phase were diluted with dichloromethane. After derivatization with acetyl chloride, a gas chromatographic analysis on a cyclodextrin column was carried out.
The comparative experiment showed that when using the recombinant proteins of the invention in analogy to example 13 only a tenth of the required amount of native protein is required in order to achieve comparable enantiomeric purities (ee values).