# Patent

## Title
Rhodococcus nitrile hydratase

## Abstract

The invention relates to a Rhodococcus polynucleotide cluster which contains nucleotide sequences which encode polypeptides having the activity of a nitrile hydratase, of an auxiliary protein P15K which activates this enzyme and of a cobalt transporter, to transformed microorganisms in which the nucleotide sequences encoding these proteins are present in increased quantity, and to the use of the transformed microorganisms for preparing amides from nitriles.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a division of U.S. application Ser. No. 11/083,327 , filed Mar. 18, 2005 now U.S. Pat. No. 7,288,402 . U.S. Pat. No. 11/083,327 claims priority to German application Ser. No. 10 2004 013 824.9 , filed on Mar. 20, 2004 . These previous applications are incorporated by reference herein in their entirety.


The invention relates to a Rhodococcus polynucleotide cluster which contains nucleotide sequences which encode polypeptides having the activity of a nitrile hydratase, of an auxiliary protein P15K which activates this enzyme, and of a cobalt transporter, to microorganisms which are transformed with this cluster and in which the nucleotide sequences encoding these proteins are present in increased quantity, and to the use of the transformed microorganisms for preparing amides from nitriles.
A large number of nitrile hydratases have already been described in the literature (Synthetic applications of nitrile-converting enzymes; Martinkova, Ludmila; Mylerova, Veronika; Current Organic Chemistry (2003), 7(13), 1279-1295). Nitrile hydratases have been used since 1983 for producing acrylamide on a scale of several thousand tons per year. This biocatalytic process has proved to be able to compete with the chemical processes (Enzymic synthesis of acrylamide: a success story not yet over; Kobayashi, Michihiko; Nagasawa, Toru; Yamada, Trends in Biotechnology (1992), 10(11), 402-8).
In addition to the nitrile hydratases which can be used for converting acrylonitrile, nitrile hydratases which are are particularly suitable for converting methacrylonitrile (A nitrile hydratase of Pseudonocardia thermophila and the genes encoding and manufacture of the enzyme for conversion of nitriles to amides (EP 790310), 3-cyanopyridine (Process for producing amides with Rhodococcus nitrile hydratase (WO 2002055670) or 2-hydroxynitriles such as 2-hydroxy-4-methylthiobutyro-nitrile (A nitrile hydratase of Rhodococcus and its use in the manufacture of amides (WO 2002070717) and Enzymic conversion of α-hydroxynitriles to the corresponding α-hydroxyamides, acids or acid salts, (WO 9832872) have, for example, also been described. By contrast, no nitrile hydratases which can be used to efficiently convert 2-aminonitriles are thus far known. While the Rhodococcus sp. Cr4 nitrile hydratase converts 2-hydroxynitriles, for example, with a high degree of activity, it does not convert a simple 2-aminonitrile such as aminoacetonitrile at all (WO 2002070717).
The enzymic conversion of aminonitriles into the corresponding amides opens up an attractive route for synthesizing amino acids since 2-aminoamides can be hydrolyzed readily (WO 2001060789). This process proceeds under mild conditions and with a very high degree of selectivity and without the formation of byproducts such as salts, as accrue in connection with chemical hydrolysis.
Alternatively, amides can also be converted with alkali metal or alkaline earth metal hydroxides into the corresponding salts of the acids. This approach is particularly preferred when using calcium hydroxide for converting 4-methylthio-α-hydroxybutyramide (MHA-amide), since the calcium salt of MHA can be used directly as a feedstuff additive, as a product form which is an alternative to methionine or MHA.
However, for producing a commodity product such as DL-methionine, it is not sufficient to make available a high-activity biocatalyst. In order to increase the activity, it is necessary to establish a system for expressing the genes which are to be amplified. One possibility which presents itself is heterologous expression, for example, and in particular, in Escherichia coli, Bacillus, Pseudomonas, Pichia, Sacharomyces or Aspergillus, since these microorganisms exhibit rapid growth, achieve very high cell densities and are available molecular biological tools which permit very high expression levels (Lee S Y (1996) High cell-density culture of Escherichia coli. TIBTECH 14:98-105; Riesenberg D, Guthke R (1999) High-cell-density cultivation of microorganisms. Appl Microbiol Biotechnol 51:422-430).
It is known that at least 3 genes have to be coexpressed for nitrile hydratases to be expressed heterologously. In addition to two structural genes, a corresponding auxiliary protein has to be amplified both for iron-dependent and for cobalt-dependent enzymes (Nojiri M. et al., (1999) Functional expression of Nitrile hydratases in Escherichia coli: Requirement of a nitrile hydratase activator and a post-translational modification of a ligand cysteine. J Biochem 125: 696-704 and Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli: activity requires a novel downstream protein, Wu, S.; Fallon, R. D.; Payne, M. S. Applied Microbiology and Biotechnology (1997), 48(6), 704-708).
In addition to these 3 genes, a further gene, which encodes a cobalt transporter, was found, alongside the structural genes and the auxiliary protein gene, in a gene cluster in Rhodococcus rhodochrous J1 (A novel transporter involved in cobalt uptake, Komeda, Hidenobu et al., Proceedings of the National Academy of Sciences of the United States of America (1997), 94(1), 36-41). Over-expression in both Rhodococcus and in E. coli leads to an increased uptake of Co2+ ions from the culture medium. In addition, it was shown that, when the cobalt transporter is coexpressed together with the 3 other proteins, it is possible to achieve the same nitrile hydratase activity at a concentration of Co in the medium which is lower than when the structural genes and the auxiliary protein are expressed on their own. However, according to Komeda et al., this effect only occurs in Rhodococcus at concentrations of less than 42 μM.
EP 0 362 829 discloses the fermentation of Rhodococcus rhodochrous in the presence of cobalt salts.
The object of the invention is to make available nitrile hydratases which possess high activity and which, in particular, convert α-aminonitriles into amides.
The invention relates to the following:

    1. Isolated polynucleotide clusters, from Rhodococcus, especially from Rhodococcus opacus which contain four nucleotide sequences which encode four polypeptides which possess amino acid sequences which are in each case at least 90% identical to the amino acid sequences contained in the sequences SEQ ID NO:2 to SEQ ID NO:5, with the polypeptides possessing the activities of a nitrile hydratase, composed of an α subunit and a β subunit, of the auxiliary protein P15K and of a cobalt transporter.
    2. Polynucleotides, selected from the group:
    
        a) polynucleotide consisting of positions 1 to 708 in the nucleotide sequence SEQ ID NO:1 or in the nucleotide sequence which is complementary thereto,
        b) polynucleotide possessing a nucleotide sequence which corresponds to the sequence from a) within the bounds of the degeneracy of the genetic code,
        c) polynucleotide which hybridizes, under stringent conditions, with the complementary sequences a) or b), and
        d) polynucleotide possessing a nucleotide sequence from a), b) or c) which contains functionally neutral sense mutations,
        with the polynucleotides encoding the β subunit of the nitrile hydratase.
    
    
    3. Polynucleotides, selected from the group:
    
        a) polynucleotide comprising positions 710 to 1327 in the nucleotide sequence SEQ ID NO:1 or in the nucleotide sequence which is complementary thereto,
        b) polynucleotide possessing a nucleotide sequence which corresponds to the sequence from a) within the bounds of the degeneracy of the genetic code,
        c) polynucleotide which hybridizes, under stringent conditions, with the complementary sequences a) or b), and
        d) polynucleotide possessing a nucleotide sequence from a), b) or c) which contains functionally neutral sense mutations,
        with the polynucleotides encoding the α subunit of the nitrile hydratase.
    
    
    4. Polynucleotides, selected from the group:
    
        a) polynucleotide comprising positions 1324 to 1737 in the nucleotide sequence SEQ ID NO:1 or in the nucleotide sequence which is complementary thereto,
        b) polynucleotide possessing a nucleotide sequence which corresponds to the sequence from a) within the bounds of the degeneracy of the genetic code,
        c) polynucleotide which hybridizes, under stringent conditions, with the complementary sequences a) or b), and
        d) polynucleotide possessing a nucleotide sequence from a), b) or c) which contains functionally neutral sense mutations,
        with the polynucleotides encoding the auxiliary protein P15K.
    
    
    5. Polynucleotides, selected from the group:
    
        a) polynucleotide comprising positions 2076 to 3146 in the nucleotide sequence SEQ ID NO:1 or in the nucleotide sequence which is complementary thereto,
        b) polynucleotide possessing a nucleotide sequence which corresponds to the sequence from a) within the bounds of the degeneracy of the genetic code,
        c) polynucleotide which hybridizes, under stringent conditions, with the complementary sequences a) or b), and
        d) polynucleotide possessing a nucleotide sequence from a), b) or c) which contains functionally neutral sense mutations,
        with the polynucleotides encoding a protein which has the activity of a cobalt transporter.
    
    
    6) Polypeptide according to 2) or 3) which contains the amino acid sequences SEQ ID NO:2 and SEQ ID NO:3, with the polypeptide exhibiting the activity of a nitrile hydratase.
    7) Polypeptide according to 4) which contains the amino acid sequence SEQ ID NO:6, with the polypeptide exhibiting the activity of the auxiliary protein P15 K.
    8) Polypeptide according to 5) which contains the amino acid sequence SEQ ID NO:5, with the polypeptide exhibiting the activity of a cobalt transporter.
    9) Probe or primer, which contains at least 20 consecutive nucleotides within positions 1 to 1327 from the nucleotide sequence SEQ ID NO:1 or its complementary form.
    10) Probe or primer, which contains at least 20 consecutive nucleotides within positions 1324 to 1737 from the nucleotide sequence SEQ ID NO:1 or its complementary form.
    11) Probe or primer, which contains at least 20 consecutive nucleotides within positions 2076 to 3146 from the nucleotide sequence SEQ ID NO:1 or its complementary form.
    12) Isolated polynucleotide according to 2) and 3) which hybridizes, under stringent conditions, with the complement comprising positions 1 to 1327 from SEQ ID NO:1, with the stringent conditions comprising washing in 5×SSC at a temperature of from 50 to 68° C.
    13) Isolated polynucleotide according to 4) which hybridizes, under stringent conditions, with the complement comprising positions 1324 to 1737 from SEQ ID NO:1, with the stringent conditions comprising washing in 5×SSC at a temperature of from 50 to 68° C.
    14) Isolated polynucleotide according to 5), which hybridizes, under stringent conditions, with the complement comprising positions 2076 to 3146 from SEQ ID NO:1, with the stringent conditions comprising washing in 5×SSC at a temperature of from 50 to 68° C.
    15) Vectors which contain (a) polynucleotide(s) selected from 1) to 5) and 12) to 14), or according to 2), 3) and 4) or according to 5).
    16) Vector pUD15, comprising the nucleotide sequence SEQ ID No. 24, containing the sequences according to 2), 3), and 6) from SEQ ID NO:1, with the start codon gtg having been changed to atg.
    17) Vector pUD16, comprising the nucleotide sequence SEQ ID NO:25, containing the sequence according to 5), with the start codon ttg having been changed to atg.
    18) Host cell, which is transformed or transfected by introducing a polynucleotide according to 1) to 5) and 12) to 14). The host cell can be a eukaryote or prokaryote which is known to have an expression system of adequate stability.
    19) Host cell, which is transformed by introducing a vector according to 15) to 17).
    20) Transformed host cell according to 18) or 19), with the host cell being a bacterium of the family Enterobacteriaceae, in particular Escherichia. 

Vector DNA can be introduced into eukaryotic or prokaryotic cells using known transformation or transfection techniques.

“Transformation”, “transfection”, “conjugation” and “transduction” refer to procedures for introducing foreign DNA which are known in the prior art.

The invention also relates to polynucleotides which are essentially composed of a polynucleotide sequence which can be obtained by using hybridization to screen an appropriate Rhodococcus opacus gene library which contains the complete gene, or parts thereof, with a probe which contains the sequences of the polynucleotides according to the invention from SEQ ID No:1, or fragments thereof, and isolating said polynucleotide sequence.

Polynucleotides which contain the sequences according to the invention are suitable for use as hybridization probes for RNA, cDNA and DNA, for the purpose of isolating nucleic acids or polynucleotides or full-length genes which encode the proteins according to the invention or for the purpose of isolating nucleic acids or polynucleotides or genes whose sequences exhibit a high degree of similarity with those of the genes according to the invention. They can also be applied, as probes, on what are termed arrays, microarrays or DNA chips for the purpose of detecting and determining the corresponding polynucleotides or sequences, such as RNA or cDNA, which are derived therefrom.



Polynucleotides which contain the sequences according to the invention are also suitable for use as primers which can be used, together with the polymerase chain reaction (PCR), to prepare DNA from genes which encode the proteins according to the invention.
These oligonucleotides, which serve as probes or primers, contain at least 25 or 30, preferably at least 20, very particularly preferably at least 15, consecutive nucleotides. Oligonucleotides having a length of at least 40 or 50 nucleotides are likewise suitable. Where appropriate, oligonucleotides having a length of at least 100, 150, 200, 250 or 300 nucleotides are also suitable.
“Isolated” means taken out of its natural environment. In general, “polynucleotide” refers to polyribonucleotides and polydeoxyribonucleotides, with it being possible for the RNA or DNA to be unmodified or modified.
The polynucleotides according to the invention encompass polynucleotides as depicted in SEQ ID No. 1, or fragments contained therein, and also polynucleotides which are at least 90%, 93%, 95%, 97% or 99% identical to the polynucleotides as depicted in SEQ ID NO:1 or fragments contained therein.
“Polypeptides” are understood as being peptides or proteins which contain two or more amino acids which are linked by way of peptide bonds.
The polypeptides according to the invention encompass a polypeptide as depicted in the sequences SEQ ID NO:2 to SEQ ID NO:4 and SEQ ID NO:6 and also polypeptides which are at least 90%, and particularly preferably at least 91%, 95%, 97% or 99%, identical to the polypeptides as depicted in the sequences SEQ ID NO:2 to SEQ ID NO: 4 and SEQ ID NO:6.
The SEQ ID NO:1 polynucleotides contain several individual sequences which encode different proteins. The sequences for the α subunit and the auxiliary protein P15K overlap each other.
The genes which encode the α subunit and the β subunit of nitrile hydratase have to be expressed jointly in order to obtain an active protein.
SEQ ID NO:2 depicts the amino acid sequence of the β subunit, and SEQ ID NO:3 depicts that of the α subunit, of the protein which exhibits nitrile hydratase activity.
SEQ ID NO:2 is derived from positions 1 to 708, and SEQ ID NO:3 is derived from positions 710 to 1327, of the nucleotide sequence SEQ ID NO:1.
The amino acid sequence of the auxiliary protein P15K is to be found in SEQ ID NO:6, corresponding to positions 1324 to 1737 in the nucleotide sequence SEQ ID NO:1.
The auxiliary protein activates the nitrile hydratase and has to be present, together with this enzyme, in the microorganism which is forming the nitryl hydratase.
SEQ ID NO:4 stands for the amino acid sequence of the cobalt transporter and is derived from positions 2076 to 3146 of the nucleotide sequence SEQ ID NO:1.
PatentIN Version 3.1 translates the start codon ttg in SEQ ID NO:4 as leucine and translates the start codon gtg in SEQ ID NO:6 as valine. The correct amino acid is methionine.
It has been found that the nitrile hydratase activity in E. coli is increased many times over by coexpressing the cobalt transporter. This is also the case when high concentrations of cobalt are used in the medium, with these concentrations being orders of size above the concentrations which occur naturally. Surprisingly, coexpressing the cobalt transporter does not lead to any poisoning of the organism but only to a slightly increased sensitivity of the cells towards high cobalt concentrations in the medium.
In order to isolate the gene cluster according to the invention, a gene library of this microorganism is generally first of all prepared in Escherichia coli (E. coli). The preparation of gene libraries is described in well-known textbooks and manuals. Examples which may be mentioned are the textbook by Winnacker: Gene und Klone, Eine Einführung in die Gentechnologie [Genes and clones, an introduction to recombinant DNA technology] (Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A very well known gene library is that of the E. coli K-12 strain W3110, which was prepared by Kohara et al. (Cell 50, 495-508 (1987)) in λ vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was prepared in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575) using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164).
It is also possible to use plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268) for preparing a gene library in E. coli. Suitable hosts are, in particular, E. coli strains which are restriction defective and recombination defective. An example of these strains is the strain DH5αmcr, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragments which have been cloned using cosmids can then in turn be subcloned into common vectors which are suitable for sequencing and then sequenced as described, for example, in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).
The resulting DNA sequences can then be investigated using known algorithms or sequence analysis programs such as that of Staden (Nucleic Acids Research 14, 217-232 (1986)), that of von Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).
Coding DNA sequences which ensue from the sequences contained in from SEQ ID No. 1 as a result of the degeneracy of the genetic code likewise form part of the subject matter of the invention. In the same way, DNA sequences which hybridize with these sequences, or parts thereof, form part of the subject matter of the invention. Furthermore, conservative amino acid substitutions, such as the replacement of glycine with alanine, or of aspartic acid with glutamic acid, in proteins are known in the field as sense mutations which do not lead to any fundamental change in the activity of the protein, i.e. are functionally neutral. It is furthermore known that changes at the N terminus and/or C terminus of a protein may not significantly impair the function of the protein or may even stabilize it. The skilled person can find information in this regard in, inter alia, Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), O'Regan et al. (Gene 77:237-251 (1989)), Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), and in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology.
Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which ensue from SEQ ID NO: 1 also form part of the subject matter of the invention. These oligonucleotides typically have a length of at least 15 nucleotides.
The skilled person can find instructions for identifying DNA sequences by means of hybridization in, inter alia, the manual “The DIG System Users Guide for Filter Hybridization” published by the company Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). The hybridization takes place under stringent conditions, i.e. the only hybrids to be formed are those in which the probe and target sequence, i.e. the polynucleotides treated with the probe, are at least 90% identical. It is known that the stringency of the hybridization, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is preferably carried out at a stringency which is relatively low as compared with that of the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).
For example, a 5×SSC buffer can be used, at a temperature of approx. 50° C.-68° C., for the hybridization reaction. Under these conditions, probes can also hybridize with polynucleotides which exhibit less than 70% identity with the sequence of the probe. These hybrids are less stable and are removed by washing under stringent conditions. This can, for example, be achieved by lowering the salt concentration down to 2×SSC and, where appropriate, subsequently to 0.5×SSC (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995), with the temperature being set at approx. 50° C.-68° C. It is possible, where appropriate, to lower the salt concentration down to 0.1×SSC. By increasing the hybridization temperature stepwise from 50° C. to 68° C. in steps of approx. 1-2° C., it is possible to isolate polynucleotide fragments which possess, for example, at least 90% to 95% identity with the sequence of the probe which is employed. Further hybridization instructions can be obtained on the market in the form of kits (e.g. DIG Easy Hyb supplied by Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).
The skilled person can find instructions for amplifying DNA sequences using the polymerase chain reaction (PCR) in, inter alia, the manual by Gait: Oligonucleotide synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
In general, the approach is to clone a gene which is expressed at a high level into a vector having a low copy number and to clone genes which are expressed more weakly into a vector with a higher copy number and/or a strong promoter. The host cells are transformed with these vectors such that they then in each case contain at least one additional copy, as compared with the starting organism, of the nucleotide sequences which encode the formation of nitrile hydratase or of the other proteins.
It has been found to be advantageous to express the cobalt transporter-encoding gene at a lower level, for example using a vector of low copy number, i.e. at least one copy less, than that for the polynucleotide sequences which encode the α and β subunits and the P15K auxiliary protein. Differential expression of said genes can also be achieved by using promoters of differing strength.
The nucleotides encoding the α and β subunits, on the one hand, and the auxiliary protein, on the other hand, are preferably located jointly on one vector and either share a common promoter or have two separate promoters.
The transformed or recombinant microorganisms which have been prepared in this way likewise form part of the subject matter of the invention.
It has been found that amplifying the genes encoding the nitrile hydratase, the P15K auxiliary protein and the cobalt transporter in microorganisms leads to an increased production of the nitrile hydratase or else to an increased activity of the nitrile hydratase.
In this connection, the term “amplification” describes the increase in the intracellular activity, in a micro-organism, of one or more enzymes, which are encoded by the corresponding DNA, which is acheived by, for example, increasing the copy number of the gene or genes, using a strong promoter or using a gene which encodes a corresponding enzyme having a high activity, and, where appropriate, combining these measures.
In order to achieve overexpression, the promoter and regulatory region or the ribosomal binding site, which is located upstream of the structural gene, can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same manner. In addition, it is possible, by using inducible promoters, to increase expression during the course of the fermentative amino acid production. Expression is likewise improved by measures taken to extend the lifetime of the mRNA.
In addition, the enzyme activity is also augmented by preventing the enzyme protein from being broken down. The genes or gene constructs can either be present in plasmids having differing copy numbers or be integrated, and amplified, in the chromosome. Alternatively, it is also possible to achieve overexpression of the genes concerned by altering the composition of the medium and the conduct of the culture.
In general, the amplification, in particular over-expression, measures which are taken increase the activity or concentration of the corresponding protein by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, and maximally up to 1000% or 2000%, based on that of the wild-type protein or on the activity or concentration of the protein in microorganisms which are not transformed with the nucleotide sequences according to the invention.
The invention also relates to the provision of vectors which are in general autonomously replicatable in the selected host strains, which are compatible with each other and which contain at least nucleotide sequences as claimed in claims 2, 3 and 4 or a nucleotide sequence as claimed in claim 4.
Vector DNA can be introduced into eukaryotic or prokaryotic cells using known transformation techniques.
The host organisms employed are preferably microorganisms, such as Pseudomonas, Pichia, various yeasts, Saccaromyces, Aspergillus or the family Streptomyces, in particular E. coli, for which expression systems are existing. Microorganisms of the genus Rhodococcus are also suitable.
The invention also relates to a process for preparing nitrile hydratase originating from Rhodococcus, especially Rhodococcus opacus or microorganisms comprising this enzyme, in which

    a) a transformed microorganism, which comprises over-expressed genes having the nucleotide sequences as claimed in claims 1 to 4, is fermented in the presence of from 0.15 to 4 mM (mmol/l) Co2+, in particular from 0.3 to 4 mM, under conditions which lead to the formation of the nitrile hydratase,
    b) this enzyme is allowed to accumulate in the micro-organism, and
    c) this enzyme is isolated from the cells, or
    d) the microorganisms are harvested and isolated as resting cells which comprise the enzyme.


The recombinantly produced nitrile hydratase converts α-aminonitriles with an activity of >50 U/mg of dry biomass.
The fermentation is preferably carried out in the presence of from 0.5 to 3.5 mM Co2+, in particular of from 0.7 to 3 mM, which is preferably added to the fermentation broth as soluble salt.
The microorganisms which are used in accordance with the invention can be cultured continuously or discontinuously, in a batch process or a fed batch process or a repeated fed batch process. A summary of known culturing methods is given in the textbook by Chmiel (Bioprozeβtechnik 1. Einfuhrüng in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium which is to be used must suitably satisfy the requirements of the given strains.
Descriptions of media for culturing different micro-organisms are to be found in the manual “Manual of Methods for General Bacteriology” published by the American Society for Bacteriology (Washington D.C., USA, 1981).
Carbon sources which can be used are sugars and carbo-hydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids, such as palmitic acid, stearic acid and linoleic acid, alcohols, such as glycerol and ethanol, and organic acids, such as acetic acid. These substances can be used individually or as mixtures.
Nitrogen sources which can be used are nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually or as mixtures.
The phosphorus sources used can be phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium has furthermore to contain metal salts, such as magnesium sulfate or iron sulfate, which are required for growth. Finally, it is possible to employ essential growth substances, such as amino acids and vitamins, in addition to the abovementioned substances. The abovementioned added substances can be added to the culture in the form of a once-only mixture or fed in, in a suitable manner, during the culture.
Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds, such as phosphoric acid or sulfuric acid, are used, in a suitable manner, for controlling the pH of the culture. Antifoamants, such as fatty acid polyglycol esters, can be used for controlling foam formation. Suitable substances which act selectively, such as antibiotics, can be added to the medium, in order to maintain the stability of plasmids. Oxygen and oxygen-containing gas mixtures, such as air, are passed into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 10° C. to 40° C. and preferably from 10° C. to 30° C. The culture is preferably continued at least until it has passed through the logarithmic phase of growth. This objective is normally achieved within from 10 hours to 70 hours.
The invention also relates to a process for enzymically preparing amides from nitrites, which comprises the following steps:

    a) converting a nitrile group-containing compound using a Rhodococcus, especially Rhodococcus opacus enzyme which exhibits nitrile hydratase activity, and
    b) where appropriate, separating off the amide.


In one process variant, the cells are harvested, washed and taken up, as a suspension, in a buffer at a pH of 5-9, in particular of from 6.8 to 7.9. The concentration of the resting cells is generally 1-25%, in particular from 1.5 to 15% (wet weight/v). The cells can be permeabilized using physical or chemical methods, for example toluene as described in Wilms et al., J. Biotechnol., Vol 86 (2001), 19-30, such that the nitrile compounds which are to be transformed can penetrate through the cell wall and the resulting amide can escape.
The biocatalyst (whole-cell catalyst) is outstandingly stable, such that product concentrations of more than 100 g/l can be achieved.
It is also possible to use known methods to separate off the nitrile hydratase according to the invention from the cells and, where appropriate, purify it and use it for converting the nitrites.
The invention also relates to a process which is characterized in that compounds of the general formulae




in which:

    X: is OH, H, alkyl having from 1 to 4 C atoms, aryl, or, in particular, NH2;
    R: is H, saturated alkyl radical having from 1 to 12 C atoms, branched or unbranched, optionally substituted by NH2,
    
        alkenyl radicals having from 1 to 12 C atoms, branched or unbranched, cycloalkyl groups having from 3 to 6 C atoms,
        alkylthio group-substituted alkylene radicals, where alkyl in this case corresponds to a C1 to C3 radical and alkylene corresponds to a bivalent C3 to C8 radical,
    
    
    R′: is H, or alkyl having from 1 to 3 C atoms,
    R′′: is a mononuclear or binuclear aromatic ring, which possesses from 6 to 12 C atoms and which is optionally substituted by one or two alkyl groups (C1-C3) or Cl or F.
    
        Alkylnitrile having from 1 to 6 C atoms.

are converted into the corresponding amides.

    
    


The following nitriles are preferably converted:
saturated mononitriles:
acetonitrile, propionitrile, butyronitrile, isobutyronitrile, valeronitrile, isovaleronitrile and capronitrile,
saturated dinitriles:
malonitrile, succinonitrile, glutaronitrile and adiponitrile,
aromatic unsubstituted and substituted mononitriles and dinitriles:
benzonitrile, 2,6-difluorobenzonitrile, phthalonitrile, isophthalonitrile and terephthalonitrile,
α-aminonitriles:
α-aminopropionitrile, α-aminomethylthiobutyronitrile, α-aminobutyronitrile, aminoacetonitrile, all nitrites derived from natural amino acids, α-amino-3,3-dimethylpropionitrile and α-amino-2,3-dimethylpropionitrile
nitriles containing carboxyl groups:
cyanoacetic acid
β-aminonitriles:
3-aminopropionitrile
unsaturated nitrites:
acrylonitrile, methacrylonitrile, allyl cyanide and crotonormitrile
α-hydroxynitriles:
α-hydroxy-n-propionitrile, α-hydroxy-n-butyronitrile, α-hydroxyisobutyronitrile, α-hydroxy-n-hexanonitrile, α-hydroxy-n-heptyronitrile, α-hydroxy-n-octanonitrile, α,γ-dihydroxy-β,β-dimethylbutyronitrile, acrolein cyanohydrin, methacrylaldehyde cyanohydrin, 3-chlorolactonitrile, 4-methylthio-α-hydroxybutyronitrile and α-hydroxy-α-phenylpropionyl.
The concentration, in the reaction solution, of the nitrites to be converted is not restricted to specific ranges.
In order to avoid the enzyme activity being inhibited by the substrate, the concentration of the nitrile is in general kept to from 0,001 to 10 w/w %, in particular from 0.1 to 2 w/w %, based on the quantity of the biocatalyst as dried cell mass. All of the substrate can be added at the beginning of the reaction or the substrate can be added continuously or discontinuously during the reaction.
A solubilizer can be added if the solubility of the nitrile compound in the aqueous reaction system is too low.
However, as an alternative, the reaction can also be carried out in a water/organic solvent two-phase system.
When cells of the microorganism are used as enzymically active material, the ratio of the quantity of the cells employed to the substrate quantity is preferably from 0.001 to 8 w/w % as dried cell mass.
The dry weight of the cell mass is determined using an MA45 Moisture Analyser (Sartorius).
It is also possible to use well-known techniques to immobilize the isolated enzyme and then to employ the enzyme in this form.
The reaction is generally carried out at temperatures of from −5° C. to 50° C., in particular of from 0° C. to 30° C., and over a period of from 0.1 to 100 hours.
The pH of the reaction mixture which is to be maintained is not restricted to specific values as long as the enzymic activity is not impaired. After the reaction, the amide which has been formed can, in a known manner, be separated out of the reaction solution and purified.
The invention also relates to a process in which the amide, or the solution containing the amide, is, for example, separated off from the cells of the biomass and the amide is either hydrolyzed to give the corresponding acid or converted into the corresponding salts of the acid in the added presence of alkali metal or alkaline earth metal hydroxides. Preference is given to MHA-amide being hydrolyzed with calcium hydroxide and the corresponding calcium salt being isolated.


EXAMPLES
Example 1
Cloning the Rhodococcus opacus Nitrile Hydratase
Rhodococcus opacus chromosomal DNA was digested with the restriction enzymes PinAI, PstI and XmaI (Roche) and the fragments were separated on a 0.8% agarose gel. Standard methods (e.g. in Sambrook et al.: Molecular Cloning, A Laboratory Manual, Cold Spring Habor Laboratory Press, 1989) were used to carry out a Southern blot onto a positively charged nylon membrane (Hybond-N+, Amersham). Hybridization was carried out with a DIG-labeled probe in accordance with the manufacturer's (Roche) instructions. The probe was prepared by means of PCR using the degenerate primers 1F and 1R and employing genomic DNA as a template. The primers were derived from homologous regions of the β subunit, with these regions being determined by aligning the sequences of various NHases. Their sequences were obtained from databases. In order to isolate a detected PinAI fragment of approx. 2.2 kb in size, PinAI-cut DNA fragments of between 2 and 2.5 kb were purified by means of preparative gel electrophoresis and ligated into the XmaI-cut vector pUC18 (Promega), and the ligation mixture was transformed into E. coli JM109 (Promega). Positive transformants were identified by means of colony hybridization using the same probe. The clones which were obtained in this way contained a 2206 nt insert comprising the gene for the β subunit, and the majority of the gene for the α subunit, of the nitrile hydratase.
In order to obtain the missing sequence, the above-described method, employing the primers 2F and 2R, was used to prepare a new probe which hybridized at the 3′ end of the cloned PinAI fragment. The PinAI fragment cloned into pUC18 served as template. Prior to hybridizing with this probe, the color signals, and the first probe, were first of all removed from the above-described membrane in accordance with the manufacturer's (Roche) instructions. A PstI band of approx. 2 kb in size was detected on this membrane using the second probe. As described above, the corresponding DNA fragment was cloned into the vector pUC18, which had been opened with PstI, and the product was transformed into E. coli JM109; positive clones were then identified by means of colony hybridization. The PstI fragment is 1883 nt in size and contains a (3′) part of the gene for the α subunit of the nitrile hydratase, the gene for the auxiliary protein P15K and a (5′) part of the gene for the cobalt transporter.
In order to clone a DNA fragment containing the missing sequence of the cobalt transporter gene, the primers 3F and 3R, and the PstI fragment cloned in pUC18, with this fragment serving as template, were used to prepare a new probe which hybridized at the 3′ end of the cloned PstI fragment. This probe was used to detect an XmaI band of approx. 1.7 kb in size on the same membrane, from which color signals and the second probe had in turn been previously removed. The corresponding DNA fragment was cloned into the pUC18 vector, which had been opened with XmaI, and the product was transformed into E. coli JM109; positive clones were identified by means of colony hybridization. A probe which had been amplified using the primers 4F and 3R was used for this purpose. The XmaI fragment is 1747 nt in size and contains a (3′) part of the gene for the cobalt transporter.
The continuous sequence of the gene cluster, which contains the polynucleotides encoding the α and β subunits of the nitrile hydratase, the auxiliary protein P15K and the cobalt transporter, is depicted in SEQ ID NO:1.
Example 2
Constructing the Expression Vectors
The structural genes were cloned into an expression vector which is known for being used in E. coli and in which the inserted genes are under the control of a rhamnose promoter. A second rhamnose promoter was inserted in addition. To achieve this, the gene for the β subunit was amplified using the primers 5F and 5R, which inserted cleavage sites for the restriction enzymes NdeI, BamHI and HindIII. The second rhamnose promoter was amplified using the primers 6F and 6R, which inserted the cleavage sites for the restriction enzymes BamHI, NcoI and HindIII. The gene for the α subunit was amplified using the primers 7F and 7R, which inserted cleavage sites for the restriction enzymes NcoI, KpnI and HindIII. The gene for the P15K protein was amplified using the primers 8F and 8R, which inserted the cleavage sites for the restriction enzymes KpnI and HindIII and altered the start codon from GTG to ATG. The expression vector which was constructed in this way is designated pUD 15.
The restriction map is given in FIG. 1, while the sequence is given in SEQ ID NO:24.
The gene for the cobalt transporter was cloned into another E. coli expression vector in which the inserted genes are also under the control of the rhamnose promoter. For this, the cobalt transporter gene was amplified using the primers 9F and 9R, which inserted the cleavage sites for the restriction enzymes NdeI and HindIII and altered the start codon from TTG to ATG. The expression vector which was constructed in this way is designated pUD 16.
The restriction map is given in FIG. 2, while the sequence is given in SEQ ID NO:25.
The expression plasmids were transformed into the E. coli strain DSM 14459, which is deposited in the Deutschen Sammlung von Mikroorganismen and Zellkulturen [German collection of microorganisms and cell cultures] GmbH (DSMZ).
Primers:









1F
5′-ATG AAY GGH ATY TTC GA-3′



 


1R
5′-ATC CAG TGY YHG TAG TA-3′


 


2F
5′-CGA AGA CAT GAT CGT CGT G-3′


 


2R
5′-ACC GGT CCC ACA CCG A-3′


 


3F
5′-TCG AGG AGA TCG GAG G-3′


 


3R
5′-GTA TCG AAG GTG CTC ATC-3′


 


4F
5′-CGC GGG CTG GGT GAA-3′


 


5F
5′-CGG CGG AAT TCA AGA AGG AGA CCC GCA TAT GAA



CGG-3′


 


5R
5′-GGT GCA AGC TTGGAT CCT GTC AGA TTC CTC GAG



TAG-3′


 


6F
5′-GCG AAG GAT CCT GCA TGC ATC GAA ATT AAT



ACG-3′


 


6R
5′-CAT CAA GCT TTT CGC CAT GGC TAT ATC TCC



TTC-3′


 


7F
5′-CTG ACA GGA TCC AAG AAG GAG ATA TAG CCA TGG



CCG A-3′


 


7R
5′-GTT GCA AGC TTG GTA CCG CTC AAG ACA TCG CCT



CCC T-3′


 


8F
5′-GTG GGT ACC AAG AAG GAG GCG ATC ATA TGA GCA



CGC-3′


 


8R
5′-GCG GAC GAG TAG CGA AGC TTG TTA GTT CAC



CG-3′


 


9F
5′-TCA AAG CTT GAA GGA GAT ATA CAT ATG ACG ATT



ACT-3′


 


9R
5′-GTC AAG CTT GGT ACC GAC ATC TCA CAC CTT



CGA-3′






The genes are located on the segments:


















pUD15:
gene for the β subunit:
nt
 25-732




gene for the α subunit:
nt
 949-1566




P15K gene:
nt
1592-2005



pUD16:
gene for the cobalt
nt
 25-1095




transporter:










Example 3
Heterologous Expression of the Nitrile Hydratase in E. coli DSM 14559
DSM 14559 was deposited in connection with DE 101 55 928.
The pUD15-transformed cells were grown, at 37° C. and while shaking, in LB medium (LB broth in accordance with Miller, VWR) which contained 1 mM CoCl2 and 100 μg of ampicillin/ml. The cells transformed with pUD15 and pUD16 were grown in an analogous manner but with the medium additionally containing 50 μg of chloramphenicol/ml. After that, the cells were inoculated over into the same medium 3 times after they had reached an OD600 of at least 2. After 12-16 hours, a quantity of the last preliminary culture was inoculated over into a main culture such that this latter had an OD600 of 0.1. While the culture medium for the main culture corresponded to that for the preliminary culture, it additionally contained 2 g of L-rhamnose/l. The cells were harvested after 22 hours.
Example 4
Determining the Enzymic Activity
The cells were grown as described in example 3, separated off from the culture medium by centrifugation and resuspended in standard buffer (50 mM potassium phosphate buffer, pH 7.5). 50 μl of this cell suspension were added to 700 μl of the standard buffer and 250 μl of a 200 mM solution of the nitrile in standard buffer were added to start the reaction. In this connection, the concentration of the cells in the cell suspension was gauged such that 5-30% of the nitrile had reacted after 10 min at 20° C. After 10 min at 20° C., the reaction was stopped by adding 20 μl of half-concentrated phosphoric acid and the cells were separated off by centrifugation.










HPLC analysis
















Column
Intersil ODS-3V


Mobile phase
Mixture composed of 10 mM potassium phosphate



buffer, pH 2.3, and acetonitrile in



a ratio of 85:15 in the case of methionine



nitrile, MHA-nitrile and acetocyanohydrin



and of 99:1 in the case of all the other



substrates


Flow rate
1 ml/min


Detection
UV at 200 nm









The activity of one unit (U) was defined as the quantity of enzyme which converts 1 μmol of N-formylvaline nitrile to the amide in one minute. The specific activity is given in U per mg of dry biomass (U/mg of DBM).
This is measured using a model MA45 Moisture Analyser (Sartorius).
Example 5
Coexpressing the genes encoding the nitrile hydratase α subunit, the β subunit and the p15K protein.
The expression was carried out as described in example 3 using the transformed E. coli strain DSM 14459, which harbored plasmid pUD15. The specific activity of the cells was 23 U/mg of DBM.
Example 6
Coexpressing the genes encoding the nitrile hydratase α subunit, the β subunit, the p15K protein and the cobalt transporter.
The expression was carried out as described in example 3 using the transformed E. coli strain DSM 14459 harboring plasmids pUD15 and pUD16. The specific activity of the cells was 81 U/mg of DBM.
Example 7
Substrate Specificity
Various nitriles were converted, in analogy with example 3, using resting transformed E. coli DSM 14459 cells which harbored plasmid pUD15. The specific activity which was obtained with N-formylvaline nitrile was set as being equal to 100%. The other activities were given in relation to it. The results are depicted in FIG. 3.
Example 8
Growth of Transformed E. coli DSM 14459 in the Presence of Co2+Salts
Transformed E. coli DSM 14459 cells, which harbored either only plasmid pUD15 or pUD15 and pUD16, were grown as described in example 3. At the same time, the cobalt concentration in the medium was varied from 0.5 to 2 mM. After 24 hours, the optical densities of the cultures were measured at 600 nm.














E. coli harboring
E. coli harboring



pUD15
pUD15 and pUD16


















0.5 mM CoC12
2.808
2.524


1.0 mM CoC12
2.6955
2.173


2.0 mM CoC12
2.330
2.113









It is found that it is only possible to observe a slight influence on the growth of the cells even at high cobalt concentrations.
Example 9
Converting methionine nitrile using resting transformed E. coli DSM 14459 cells which harbor plasmid pUD15.
E. coli DSM 14459 cells which harbored plasmid pUD15 were grown, and centrifuged down, as described in example 3. 2.8 g of the cells, based on the wet weight, were resuspended in 47.2 ml of 50 mM potassium phosphate buffer, pH 7.5, and methionine nitrile was added continuously, at 20° C. and while agitating vigorously, at a rate which was such that the concentration did not exceed 15 g/l at any time during the reaction. The pH was maintained constant at 7.5. The reaction was monitored by means of HPLC, as described in example 4. After 320 min, 9.1 g of the nitrile had been completely converted into 10.4 g of amide. This corresponds to a concentration of 176 g/l.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1










Plasmid pUD15


















rhaP
rhamnose promoter



beta
gene for the nitrile hydratase β subunit



alpha
gene for the nitrile hydratase α subunit



P15K
gene for the auxiliary protein P15K



ori
origin of replication



bla
gene for resistance to ampicillin (β-lactamase)










FIG. 2










Plasmid pUD16


















rhaP
rhamnose promoter



CoTrans
gene for the cobalt transporter



ori
origin of replication



Cmr
gene for resistance to chloramphenicol










Relative specific activity when converting various nitriles in comparison with the activity when converting N-formylvaline nitrile.

## Claims

1. A process for enzymatically preparing an amide from a nitrile comprising:
a) converting a nitrile group-containing compound using a with transformed microorganism transformed with a vector expressing a nitrile hydratase enzyme having:
i) a beta subunit with an amino acid sequence that is at least 95% identical to that of SEQ ID NO:2; and
ii) an alpha subunit at least 95% identical to the sequence SEQ ID NO:3; and

b) purifying said amide.

2. The process of claim 1, wherein said beta subunit comprises the amino acid sequence of SEQ ID NO:2 and said alpha subunit comprises the amino acid sequence of SEQ ID NO:3.

3. The process of claim 1, wherein said beta subunit consists of the amino acid sequence of SEQ ID NO:2 and said alpha subunit consists of the amino acid sequence of SEQ ID NO:3.

4. The process of claim 1, wherein said nitrile hydratase converts α-aminonitriles with a specific activity of> 50 U/mg of dry biomass.

5. The process of claim 1, wherein resting cells which comprise said nitrile hydratase are used.

6. The process of claim 1, wherein a nitrile of the general formula




is converted into the corresponding amide and in which:
X is OH, H, an alkyl having from 1 to 4 carbon atoms or NH2;
R is H; a branched or unbranched saturated alkyl radical having from 1 to 12 carbon atoms and optionally substituted with NH2; a branched or unbranched alkenyl radical having from 1 to 12 carbon atoms; a cycloalkyl group having from 3 to 6 C atoms; an alkylthio group-substituted alkylene radical, wherein said alkyl is a C1 to C3 radical and said alkylene is a bivalent C3 to C8 radical;
R′ is H, or an alkyl having from 1 to 3 with carbon atoms, and which is optionally substituted by one or two C1 to C3 alkyl groups or Cl, Br or F, or a monovalent alkylnitrile radical having from 1 to 6 carbon atoms.

7. The process of claim 1, wherein said nitrile is methionine nitrile.

8. The process of claim 6, wherein said beta subunit comprises the amino acid sequence of SEQ ID NO:2 and said alpha subunit comprises the amino acid sequence of SEQ II) NO:3.

9. The process of claim 8, wherein said nitrile is methionine nitrile.

10. The process of claim 6, wherein said beta subunit consists of the amino acid sequence of SEQ ID NO:2 and said alpha subunit consists of the amino acid sequence of SEQ ID NO:3.

11. The process of claim 10, wherein said nitrile is metbionine nitrile.

12. The process of claim 6, wherein R and R1 are H.

13. The process of claim 12, wherein X is OH.

14. The process of claim 12, wherein X is an alkyl having from 1 to 4 carbon atoms.

15. The process of claim 12, wherein X is NH2.

16. The process of claim 6, wherein R is a branched or unbranched saturated alkyl radical having from 1 to 12 carbon atoms and optionally substituted with NH2.

17. The process of claim 16, wherein X is an alkyl having from I to 4 carbon atoms.

18. The process of claim 16, wherein X is NH2.

19. The process of claim 6, wherein R is a cycloalkyl group having from 3 to 6 C atoms.

20. The process of claim 19, wherein X is an alkyl having from 1 to 4 carbon atoms.