Patent Grant
8304223
The present application is US national stage of international application PCT/EP2008/052880, which had an international filing date of Mar. 11, 2008, and which claimed priority to German application 102007014742.4, filed on Mar. 23, 2007. The international application was published in German under PCT Article 21(2) on Oct. 2, 2008. All prior applications are hereby incorporated by reference in their entirety.
The present invention relates to isoforms of pig liver esterase (γPLE), to vehicles containing the same and to their use in the production of enantiomerically enriched alcohols and esters.
Lipases and esterases can be used as efficient biocatalysts for preparing a multiplicity of optically active compounds. However, while a substantial number of lipases—in particular those of microbial origin—are commercially available, only very few esterases are available in industrial-scale quantities for the use in the resolution of racemates [Bornscheuer, U. T. and Kazlauskas R. J., Hydrolases in Organic Synthesis (2005), 2nd ed, Wiley-VCH, Weinheim].
Of particular interest here is pig liver esterase owing to its interesting catalytic properties in organic synthesis [Faber, K., Biotransformations in Organic Chemistry (2004), 5th ed. Springer, Berlin; Jones, J. B. Pure Appl. Chem, (1990), 62, 1445-1448, Jones et. al. Can. J. Chem. (1985), 63, 452-456; Lam, L. K. P. et. al., J. Org. Chem. (1986), 51, 2047-2050).
Although it has been demonstrated that esterase extracts from pig liver tissue can partly convert substrates with good stereoselectivity, the use of such extracts has a number of disadvantages, however. A particular problem with respect to stereoselectivities can be considered that of the presence of further hydrolases, in addition to fluctuations of the proportion of esterase between various batches (Seebach, D. et. al, 25 Chimia (1986), 40, 315-318). There is the additional problem that the conventional extracts consist of a plurality of isoenzymes (Farb, D., et. al, Arch. Biochem. Biophys. (1980) 203, 214-226), whose substrate specificities differ considerably in some cases. Heymann, E. and Junge, W. (Eur. J. Biochem. (1979), 95, 509-518; Eur. J. Biochem. (1979), 95, 519-525) performed a complicated electrophoretic separation, thereby isolating fractions which preferably cleave butyrylcholine, proline-β-naphthylamide and methyl butyrate. In contrast, other studies (for example Lam, L. K. P., et. al, J. Am. Chem. Soc. (1988) 110, 4409-4411) merely show individual fractions having different activities but not different specificities.
Although the cloning of putative pig liver esterase genes has been known for some time (Takahashi, T, et. al., J. Biol. Chem. (1989), 264, 11565-11571; FEBS Lett. (1991), 280, 297-300; FEBS Lett. (1991), 293, 37-41; David, L. et. al, Eur. J. Biochem. (1998) 257, 142-148), functional, recombinant expression of an active pig liver esterase has been described previously only in Pichia pastoris (Lange, S. et al., ChemBioChem (2001), 2, 576-582) and E. coli (DE 10061864).
The literature likewise describes additions to the medium during expression in E. coli. Addition of ethanol up to 3% (v/v) to the medium induces the formation of endogenous E. coli chaperones, enzymes which assist the folding process and normally support correct folding (Thomas, J G, Protein Expression and Purif (1997), 11, 289-296). However, expression of pig liver esterase in E. coli Origami, with the addition of 3% (v/v) ethanol to the medium, produced no detectable active esterase expression in E. coli but only inclusion bodies.
DE10061864 proposes coexpression of particular chaperones and γPLE. In this way it was possible to generate for the first time active γPLE from E. coli.
In summary it may be said that, although expression of native pig liver esterase from E. coli is possible, it has not yet been established on an industrial scale. It is furthermore useful and necessary to improve the (substrate) activities of pig liver esterases, in order to additionally obtain improved systems which can be employed preferably on industrial scale within the framework of biosynthetic preparation of chemical intermediates.
It was therefore an object of the present invention to specify novel esterases which are improved over the prior art. It was intended to provide novel esterases having improved activity and/or selectivity and/or stability. These esterases should be superior to those of the prior art in particular with regard to space/time yield and enantioselectivity during conversion and altered or extended substrate specificity.
This object is achieved according to the claims.
Providing esterases according to Seq. ID No. 2, having at least one mutation, selected from the group consisting of:
surprisingly results in species which fulfill the objects mentioned. More specifically, mutations at these sites can modify and improve substrate specificity, enantioselectivity and/or activity of the native γPLE. The position here naturally refers to the first amino acid of Seq. ID No. 2.
Preference is given to esterases according to Seq. ID Nos. 4, 6, 8 and 10. These have superior activity and/or selectivity and/or stability over native γPLE. The esterases of the invention are distinguished in particular with regard to activities, enantioselectivities and other substrate specificities.
In another embodiment, the present invention relates to isolated nucleic acid coding for an esterase of the invention. Preferred nucleic acid sequences are those of Seq. ID Nos. 3, 5, 7 and 9 or their complementary form.
In a further development, the present invention relates to genes, recombinant expression systems (for example microorganisms) or recombinant plasmids/vectors having one or more of the nucleic acids of the invention.
An expression system means a system for recombinant expression of the nucleic acids of the invention and thereby for recombinant production of the polypeptides of the invention. Said production may preferably take place in microorganisms or other hosts transformed or transfected (the terms “transformation” and “transfection” are used synonymously according to the present invention) with corresponding nucleic acid sequences or vectors (see hereinbelow). Transformation and transfection may be carried out according to known methods, for example by calcium phosphate coprecipitation, lipofection, electroporation, PEG/DMSO method, particle bombardment or viral/bacteriophage infection. The cell of the invention may contain the recombinant nucleic acid in extrachromosomal or chromosomally integrated form. In other words: transfection/transformation may be stable or transient. Transfection and transformation protocols are known to the skilled worker (Chan and Cohen. 1979. High Frequency Transformation of Bacillus subtilis Protoplasts by Plasmid DNA. Mol Gen Genet. 168(1):111-5; Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation Norwich; Sambrook et al. 1989. Molecular Cloning. A Laboratory Manual. In: second ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. N.Y.; Irani and Rowe. 1997. Enhancement of transformation in Pseudomonas aeruginosa PAO1 by Mg2+ and heat. Biotechniques 22: 54-56; Balbas, P. and Bolivar, F. (1990), Design and construction of expression plasmid vectors in E. coli, Methods Enzymol. 185, 14-37; Rodriguez, R. L. and Denhardt, D. T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 205-225, Butterworth, Stoneham). For the general procedures (PCR, cloning, expression etc.), reference is also made to the following literature and the citations therein: Universal GenomeWalker™ Kit User Manual, Clontech, March 2000; Triglia T.; Peterson, M. G. and Kemp, D. J. (1988), A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences, Nucleic Acids Res. 16, 8186.
The host is preferably a recombinant microorganism of prokaryotic origin. Suitable host cells include cells of unicellular microorganisms such as bacterial cells. Microorganisms which may be mentioned in this regard are prokaryotes such as E. coli, Bacillus subtilis. Other bacteria which may be employed for expression of the nucleic acid sequences of the invention are those of the genera/species Lactobacillus, Bacillus, Rhodococus, Campylobacter, Caulobacter, Mycobacterium, Streptomyces, Neisseria, Ralstonia, Pseudomonas, and Agrobacterium. Preference is given to utilizing E. coli strains for this purpose. Very particular preference is given to: E. coli XL1 Blue, NM 522, JM101, JM109, JM105, RR1, DH5α, TOP 10-, HB101, BL21 codon plus, BL21 (DE3) codon plus, BL21, Rosetta, Rosetta-gami, MM294, W3110, DSM14459 (EP1444367), Origami. Corresponding strains are available in the prior art and may, at least partly, be obtained via the international depositary institutions such as ATCC or DMSZ.
It is likewise possible to employ eukaryotes such as mammalian cells, insect cells or plant cells, or organisms such as, for example, yeasts such as Hansenula polymorpha, Pichia sp., Saccharomyces cerevisiae, or fungi such as, for example, Aspergillus sp., for recombinant production of the polypeptides. Suitable eukaryotic cells include CHO cells, HeLa cells and others. Many of these cells can be obtained via depositary institutions such as ATCC or DMSZ.
The polypeptides of the invention may also be recombinantly prepared in a non-human host. The non-human host may be a cell or a multi- to polycellular organism. Suitable polycellular organisms include model systems familiar in molecular biology, such as Drosophila melanogaster, Zebrafisch or C. elegans. Transgenic non-human animals may be produced by methods known in the prior art. The transgenic non-human animal of the invention may preferably have different genetic constitutions. It may (i) overexpress the gene of a nucleic acid sequence of the invention constitutively or inducibly, (ii) contain an inactivated form of the endogenous gene of a nucleic acid sequence of the invention, (iii) contain a mutated gene of a nucleic acid sequence of the invention, which gene replaces completely or partly the endogenous gene of a nucleic acid sequence of the invention, (iv) have conditional and tissue-specific overexpression or underexpression of the gene of a nucleic acid sequence of the invention, or (v) have a conditional and tissue-specific knockout of the gene of a nucleic acid sequence of the invention.
The transgenic animal preferably contains in addition an exogenous gene of a nucleic acid sequence of the invention under control of a promoter allowing overexpression. Alternatively, the endogenous gene of a nucleic acid sequence of the invention may be overexpressed by activating or/and replacing the endogenous promoter. Preferably, the endogenous promoter of the gene of a nucleic acid sequence of the invention has a genetic modification which results in increased expression of said gene. Genetic modification of the endogenous promoter comprises both a mutation of individual bases and deletion and insertion mutations. In a particularly preferred embodiment of the host of the invention, said host is a transgenic rodent, preferably a transgenic mouse, a transgenic rabbit, a transgenic rat, or a transgenic sheep, a transgenic cow, a transgenic goat or a transgenic pig. Mice have numerous advantages over other animals. They are easy to keep and their physiology is regarded as a model system for that of humans. The production of such genetically manipulated animals is sufficiently known to the skilled worker and is performed by conventional methods (see, for example, Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994), Manipulating the Mouse-Embryo; A Laboratory Manual, 2nd edition., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; WO91/08216). Alternatively or additionally, it is also possible to employ cell culture systems, in particular human cell culture systems, for the applications described for the non-human transgenic animal of the invention.
Another development of the invention relates to complete genes which have the nucleic acids of the invention. Gene means according to the application a section at the molecular level, which in principle may consist of two different regions:
More detailed definitions can be found at: http://de.wikipedia.org/wiki/Gen.
The coding nucleic acid sequences may be cloned into conventional plasmids/vectors and, after transfection of microorganisms or other host cells with such vectors, be expressed in cell culture. Suitable plasmids or vectors are in principle any embodiments available to the skilled worker for this purpose. Such plasmids and vectors may be found, for example, in Studier and coworkers (Studier, W. F.; Rosenberg A. H.; Dunn J. J.; Dubendroff J. W.; Use of the T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol. 1990, 185, 61-89) or the brochures of Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Other preferred plasmids and vectors may be found in: Glover, D. M. (1985), DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, R. L. and Denhardt, D. T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V., Systems for heterologous gene expression, Methods Enzymol. 1990, 185, 3-7; Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.
Plasmids which may be used for cloning the nucleic acid sequence of the invention or a gene construct containing them into the host organism in a very preferred manner are: pUC18 (Roche Biochemicals), pKK-177-3H (Roche Biochemicals), pBTac2 (Roche Biochemicals), pKK223-3 (Amersham Pharmacia Biotech), pKK-233-3 (Stratagene) or pET (Novagen). Suitable vectors are also, for example, pET-21a(+) for E. coli, but other expression vectors for prokaryotic unicellular organism and vectors for eukaryotes may also be used. Examples of vectors which have proved suitable for yeasts are the pREP vector and the pINT vector. Baculovirus vectors such as those in EP127839 or EP549721, for example, have been disclosed for expression in insect cells, and SV40 vectors, for example, are suitable for expression in mammalian cells and are generally available. Particular preference is given to vectors for unicellular eukaryotic organisms, in particular from the group of pET vectors, for transformation of E. coli cells, in particular E. coli Origami.
In a particularly preferred embodiment, the nucleic acid sequence of the invention, which has been introduced into the vector, is additionally fused to a histidine tag provided by said vector. Preference is given to cloning the introduced nucleic acid sequence into the preferred pET vector in such a way that transcription is under the control of the IPTG-regulatable promoter present in the vector. Alternatively, preference is also given to employing rhamnose-regulatable promoters.
Aside from the usual markers such as, for example, antibiotic resistance genes, the vectors may contain further functional nucleotide sequences for regulating, in particular repressing or inducing, the expression of the ADH gene and/or a reporter gene. Preference is given to utilizing promoters which are regulatable weak promoters such as the rha promoter or the nmtl promoter, for example, or regulatable strong promoters such as the lac, ara, lambda, pL, T7 or T3-promoter, for example. The coding DNA fragments must be transcribable from a promoter in the vectors. Other examples of established promoters are the Baculovirus polyhedrin promoter for expression in insect cells (see, for example, EP127839) and the early SV40 promoter, and LTR promoters, for example of MMTV (Mouse Mammary Tumour Virus; Lee et al. (1981) Nature, 294 (5838), 228-232).
Accordingly, the genes, vectors/plasmids of the invention may contain further functional sequence regions such as, for example, an origin of replication, operators or termination signals.
In a particularly advantageous embodiment, the present invention relates to rec microorganisms which, in addition to the nucleic acid sequence of the invention, also contain one or more cloned chaperone genes. Preferred suitable chaperones are GroEL and GroES, preferably in an E. coli Origami strain. Surprisingly, expression of the active enzyme was achieved in the presence of these two folding helper proteins, although other alternative chaperone systems such as, for example, the endogenous E. coli chaperones, induced by the addition of ethanol or other coexpressed chaperones such as DnaK, DnaJ and GrpE, were not successful (DE 10061864).
To a person skilled in the art, it comes as a surprise that equivalent coexpression of the chaperone systems Dnak, DnaJ, GrpE and GroEL, GroES, or coexpression of GroEL or GroES alone, together with pig liver esterase in E. coli Origami, only results in expression in the form of inclusion bodies and not in a detectable activity in E. coli crude cell extract. Preference is therefore in any case given to the chaperone system GroEL/GroES being the preferred induced/expressed system, even if other chaperone systems are present in the host organism at the same time.
Functional expression of eukaryotic proteins in E. coli represents a formidable challenge, in particular if said proteins are posttranslationally glycosylated proteins. In the case of recombinant expression of pig liver esterase in E. coli, the use of the special chaperone system GroEL, GroES apparently cancels out the lack of posttranslational glycosylation. Reference is made to DE102006031600 with regard to this development and its embodiment.
In a further development, the present invention concerns the use of the (rec)polypeptides of the invention for preparing enantiomerically enriched alcohols, carboxylic acids and esters, in particular from the mesoforms of the aforementioned compounds, such as, for example, optionally substituted dicarboxylic esters such as malonic diesters.
It is possible to use the enzymes in immobilized form (Sharma B. P.; Bailey L. F. and Messing R. A. (1982), Immobilisierte Biomaterialien—Techniken and Anwendungen [Immobilized biomaterials—Techniques and applications], Angew. Chem. 94, 836-852). Immobilization is advantageously achieved by lyophilization (Paradkar, V. M.; Dordick, J. S. (1994), Aqueous-Like Activity of α-Chymotrypsin Dissolved in Nearly Anhydrous Organic Solvents, J. Am. Chem. Soc. 116, 5009-5010; Mori, T.; Okahata, Y. (1997), A variety of lipi-coated glycoside hydrolases as effective glycosyl transfer catalysts in homogeneous organic solvents, Tetrahedron Lett. 38, 1971-1974; Otamiri, M.; Adlercreutz, P.; Matthiasson, B. (1992), Complex formation between chymotrypsin and ethyl cellulose as a means to solubilize the enzyme in active form in toluene, Biocatalysis 6, 291-305). Very particular preference is given to lyophilization in the presence of surfactants such as Aerosol OT or polyvinylpyrrolidone or polyethylene glycol (PEG) or Brij 52 (diethylene glycol monocetyl ether) (Kamiya, N.; Okazaki, S.-Y.; Goto, M. (1997), Surfactant-horseradish peroxidase complex catalytically active in anhydrous benzene, Biotechnol. Tech. 11, 375-378).
Most preference is given to immobilization to Eupergit®, in particular Eupergit C® and Eupergit 250L® (Röhm) (for an overview, see: E. Katchalski-Katzir, D. M. Kraemer, J. Mol. Catal. B: Enzym. 2000, 10, 157). Preference is likewise given to immobilization to Ni-NTA in combination with the polypeptide modified by attaching a His tag (hexahistidine) (Petty, K. J. (1996), Metal-chelate affinity chromatography In: Ausubel, F. M. et al. eds. Current Protocols in Molecular Biology, Vol. 2, New York: John Wiley and Sons).
The use as CLECs is also conceivable (St. Clair, N.; Wang, Y.-F.; Margolin, A. L. (2000), Cofactor-bound cross-linked enzyme crystals (CLEC) of alcohol dehydrogenase, Angew. Chem. Int. Ed. 39, 380-383).
These measures may succeed in generating from (rec) polypeptides which are rendered unstable by organic solvents polypeptides which may work in mixtures of aqueous and organic solvents or wholly in organics.
Esters or carboxylic acids and alcohols are converted using the polypeptides of the invention preferably as follows. The polypeptides are added in the desired form (free, immobilized, in host organisms or in a randomly disrupted form) to the appropriate medium, preferably the aqueous solution. The substrate is added to this mixture, while maintaining the optimal temperature range and the optimal pH range. After the conversion has been completed, the alcohol or ester obtained may be isolated from the reaction mixture by methods known to the skilled worker (crystallization, extraction, chromatography).
Enzymatic conversion of esters or carboxylic acids and alcohols to enantiomerically enriched alcohols, carboxylic acids and esters by means of esterases is known in principle to a person skilled in the art (see references cited at the outset; diagram 2). Of particular interest in this context are the conversions of mesoforms of the aforementioned derivatives. Here the conversion of the invention is beneficial in that an enantiomerically enriched product can be obtained with a yield of 100%, while normal resolutions of the racemates can only produce a yield of up to 50% of the particular enantiomers. Thus, for example, using particular substituted malonic diesters can produce enantiomerically enriched products which are valuable intermediates in chemical synthesis. In this way it is possible to efficiently and easily produce from optionally N-protected amino malonic diesters the corresponding enantiomerically enriched asymmetric aminocarboxylic monoesters. The reaction sequence depicted in diagram 1 below is also interesting.
Suitable radicals for this reaction can be found in the list below.
The measures of amidation and of C1 degradation are sufficiently known to the skilled worker (Organikum, VEB Deutscher Verlag der Wissenschaften, Berlin 1986, pp. 388ff, pp. 571ff).
Examples of enantiomerically enriched alcohols which may be prepared from the corresponding chiral esters according to I, or VI, or which may be used to prepare enantiomerically enriched esters are likewise familiar to the skilled worker. They can be summarized, for example, by the following general formula,
in which R, R′ and R″ are different from one another, in particular
H, (C1-C8)-alkyl, (C1-C8)-alkoxy, HO—(C1-C8)-alkyl, (C2-C8)-alkoxyalkyl, (C6-C18)-aryl, (C7-C19)-aralkyl, (C3-C18)-heteroaryl, (C4-C19)-heteroaralkyl, (C1-C8)-alkyl-(C6-C18)-aryl, (C1-C8)-alkyl-(C3-C18)-heteroaryl, (C3-C8)-cycloalkyl, (C1-C8)-alkyl-(C3-C8)-cycloalkyl, (C3-C8)-cycloalkyl-(C1-C8)-alkyl or R and R′ and/or R and R″ and/or R′ and R″ form a (C3-C5)-alkylene bridge.
Examples of enantiomerically enriched esters or acids may be assigned to the following general formulas,
in which R, R′ and R″ are identical or different from one another, in particular
H, (C1-C8)-alkyl, (C1-C8)-alkoxy, HO—(C1-C8)-alkyl, (C2-C8)-alkoxyalkyl, (C6-C18)-aryl, (C7-C19)-aralkyl, (C3-C18)-heteroaryl, (C4-C19)-heteroaralkyl, (C1-C8)-alkyl-(C6-C18)-aryl, (C1-C8)-alkyl-(C3-C18)-heteroaryl, (C3-C8)-cycloalkyl, (C1-C8)-alkyl-(C3-C8)-cycloalkyl, (C3-C8)-cycloalkyl-(C1-C8)-alkyl or R and R′ or R and R″ or R′ and R″ form a (C3-C5)-alkylene bridge, and R1, R2 and R3 are different from one another, in particular
H, (C1-C8)-alkyl, (C1-C8)-alkoxy, HO—(C1-C8)-alkyl, (C2-C8)-alkoxyalkyl, cyclopentadienyl, (C6-C18)-aryl, (C7-C19)-aralkyl, (C3-C18)-heteroaryl, (C4-C19)-heteroaralkyl, (C1-C8)-alkyl-(C6-C18)-aryl, (C1-C8)-alkyl-(C3-C18)-heteroaryl, (C3-C8)-cycloalkyl, (C1-C8)-alkyl-(C3-C8)-cycloalkyl, (C3-C8)-cycloalkyl-(C1-C8)-alkyl or R1 and R2 and/or R1 and R3 and/or R2 and R3 form a (C3-C5)-alkylene bridge.
Suitable for the use according to the invention are aqueous solvents which are suitably buffered. However, it is also possible to perform the reaction using a pH-stat instrument [company: Schott A G, Mainz, Germany, brand TitroLine alpha].
Preference is given to carrying out the conversion at a temperature of between 0° C. and 85° C., particularly preferably between 30 and 80° C., very particularly preferably at around 50° C. The skilled worker also has a free choice of pH of the reaction, and the reaction may be carried out both at a fixed pH and with the pH being varied within a pH interval. The pH is chosen in particular with regard to an optimal reaction result according to the present object. Preference is given to carrying out the reaction at a pH at from pH 5 to 9, preferably pH 6 to 8 and particularly preferably pH 6.5 to 7.5.
As already mentioned above, the polypeptide concerned may be applied in the native form by way of homogeneously purified compounds, or as a recombinantly produced enzyme. Furthermore, the (rec)polypeptide may also be employed as a component of an intact guest organism or in connection with the disrupted cell mass of the host organism, which may have any degree of purity.
If the substrate used is converted to the desired product in cell culture, for example by employing a suitable host, a suitable nutrient medium is used depending on the host organism used or the cell culture used. Suitable media for the host cells are generally known and commercially available. Moreover, the cell cultures may be supplemented with usual additives such as, for example, antibiotics, growth promoters such as, for example, sera (fetal calf serum, etc.), and similar known supplements.
Further optimal reaction conditions can be found in DE102006031600.
Another application relates to the preparation of a polypeptide having improved activity and/or selectivity and/or stability over SEQ. ID. NO: 2 polypeptide by
The procedure of improving the nucleic acid sequences of the invention or the polypeptides encoded by them by means of mutagenesis methods is well known to a skilled worker. Suitable mutagenesis methods are any methods available for this purpose to the skilled worker. These are, in particular, saturation mutagenesis, random mutagenesis, in vitro recombination methods, and site directed mutagenesis (Eigen, M. and Gardiner, W., Evolutionary molecular engineering based on RNA replication, Pure Appl. Chem. 1984, 56, 967-978; Chen, K. and Arnold, F., Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media. Bio/Technology 1991, 9, 1073-1077; Horwitz, M. and Loeb, L., Promoters Selected From Random DNA-Sequences, Proc Natl Acad Sci USA 83, 1986, 7405-7409; Dube, D. and L. Loeb, Mutants Generated By The Insertion Of Random Oligonucleotides Into The Active-Site Of The Beta-Lactamase Gene, Biochemistry 1989, 28, 5703-5707; Stemmer, P. C., Rapid evolution of a protein in vitro by DNA shuffling, Nature 1994, 370, 389-391 and Stemmer, P. C., DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91, 1994, 10747-10751).
The new nucleic acid sequences obtained are cloned into a host organism by the methods indicated hereinbelow (for references see below), and the polypeptides expressed in this way are detected using suitable screening methods and subsequently isolated. Suitable for detection are in principle any possible detection reactions for the molecules produced by this polypeptide. Particularly suitable for this are photometric assays for NADH produced or consumed, HPLC or GC methods for detecting the alcohols produced by this enzyme. Moreover, detection methods by means of gel electrophoresis or by means of antibodies are also suitable for detecting new polypeptides which have been modified by genetic engineering methods.
Optically enriched (enantiomerically enriched, enantiomer-enriched?) compounds mean for the purpose of the invention the presence of one optical antipode in the mixture with the other one at >50 mol %.
The term nucleic acid sequences means all kinds of single-stranded or double-stranded DNA as well as RNA or mixtures thereof. Accordingly, the nucleic acid sequence of the invention may be a DNA molecule or an RNA molecule. Preference is given to the nucleic acid molecule being a cDNA molecule or an mRNA molecule. According to the invention, the DNA molecule may also be a genomic DNA molecule. The invention furthermore comprises embodiments in which the DNA molecule is a PNA molecule or another derivative of a DNA molecule.
The term “complementary” means according to the invention that complementarity extends across the entire region of the nucleic acid molecule of the invention, without any gaps. In other words: preference is given according to the invention to 100% complementarity extending across the entire region of the sequence of the invention, i.e. from the 5′ end depicted to the 3′ end depicted.
Improving the activity and/or selectivity and/or stability means according to the invention that the polypeptides are more active and/or more selective and/or, under the reaction conditions used, more stabile. While activity and stability of the enzymes naturally should be as high as possible for industrial application, improvement with respect to selectivity refers to the situation in which substrate selectivity decreases but enantioselectivity of the enzymes increases. The same applies mutatis mutandis to the expression not substantially reduced, used in this context.
Of the claimed protein sequences and the nucleic acid sequences, the invention also comprises those sequences which are more than 97%, preferably more than 97.5%, 98% or 98.5%, more preferably more than 99% or 99.5%, homologous (excluding natural degeneration) to any of these sequences, as long as such a sequence retains its functionality or purpose. The expression “homology” (or identity), as used herein, may be defined by the equation H (%)=[1−V/X]×100, wherein H means homology, X is the total number of nucleobases/amino acids of the comparative sequence, and V is the number of different nucleobases/amino acids of the sequence to be considered, based on the comparative sequence. In any case, the term nucleic acid sequences which code for polypeptides includes any sequences that appear possible according to the degeneracy of the genetic code.
The expression “under stringent conditions” is understood herein as described in Sambrook et al. (Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York). Preferably, a hybridization is stringent according to the present invention, if, after washing with 1×SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) and 0.1% SDS (sodium dodecylsulfate) at 50° C., preferably at 55° C., more preferably at 62° C. and most preferably at 68° C., for 1 hour, and more preferably with 0.2×SSC and 0.1% SDS at 50° C., more preferably at 55° C., still more preferably at 62° C. and most preferably at 68° C., for 1 hour, a positive hybridization signal is still observed.
(C1-C8)-Alkyl radicals can be considered methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl or octyl, and any of their binding isomers.
A (C1-C20)-alkyl radical is, within the scope of the definition according to the invention, a corresponding radical having from 1 to up to 20 carbon atoms.
A (C3-C20)-alkyl radical is, within the scope of the definition according to the invention, a corresponding radical having from 3 to up to 20 carbon atoms.
The radical (C1-C8)-alkoxy corresponds to the radical (C1-C8)-alkyl with the proviso that the former is bound via an oxygen atom.
(C2-C8)-Alkoxyalkyl means radicals in which the alkyl chain is interrupted by at least one oxygen function, it not being possible for two oxygen atoms to be linked to one another. The number of carbon atoms indicates the total number of carbon atoms contained in the radical.
A (C3-C5)-alkylene bridge is a carbon chain having from three to five carbon atoms, which chain is bound via two different carbon atoms to the molecule concerned.
The radicals described above may be mono- or polysubstituted with halogens and/or (C1-C8)-alkoxycarbonyl and/or N-, O-, P-, S-, Si-atom-containing radicals. The latter are in particular alkyl radicals of the above kind, whose chain has one or more of said heteroatoms or which are bound via one of these heteroatoms to the molecule.
(C3-C8)-Cycloalkyl means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl radicals, etc. These may be substituted with one or more halogens and/or N-, O-, P-, S-, Si-atom-containing radicals, and/or may have N, O, P, S atoms in the ring, such as, for example, 1-, 2-, 3-, 4-piperidyl, 1-, 2-, 3-pyrrolidinyl, 2-, 3-tetrahydrofuryl, 2-, 3-, 4-morpholinyl.
A (C3-C8)-cycloalkyl-(C1-C8)-alkyl radical refers to a cycloalkyl radical as depicted above which is bound via an alkyl radical as indicated above to the molecule.
(C1-C8)-Alkoxycarbonyl means for the purpose of the invention an alkyl radical as defined above having up to 8 carbon atoms, which is bound via an O(C═O) function.
(C1-C8)-Acyloxy means for the purpose of the invention an alkyl radical as defined above having up to 8 carbon atoms, which is bound via a (C═O)O function.
(C1-C8)-Acyl means for the purpose of the invention an alkyl radical as defined above having up to 8 carbon atoms, which is bound via a (C═O) function.
A (C6-C18)-aryl radical means an aromatic radical having from 6 to 18 carbon atoms. More specifically, it includes compounds such as phenyl, naphthyl, anthryl, phenanthryl, biphenyl radicals or systems of the above-described kind which are annealed to the molecule in question, such as, for example, indenyl systems, which may optionally be substituted with (C1-C8)-alkyl, (C1-C8)-alkoxy, (C2-C8)-alkoxyalkyl, NH(C1-C8)-alkyl, N((C1-C8)-alkyl)2, OH, O(C1-C8)-alkyl, NO2, NH(C1-C8)-acyl, N((C1-C8)-acyl)2, F, Cl, CF3, (C1-C8)-acyl, (C1-C8)-acyloxy, (C7-C19)-aralkyl radical, (C4-C19)-heteroaralkyl.
A (C7-C19)-aralkyl radical is a (C6-C8)-aryl radical which is bound to the molecule via a (C1-C8)-alkyl radical.
A (C3-C18)-heteroaryl radical, within the scope of the invention, refers to a five-, six- or seven-membered aromatic ring system of from 3 to 18 carbon atoms which has heteroatoms such as, for example, nitrogen, oxygen or sulfur in the ring. Such heteroaromatics are considered in particular radicals such as 1-, 2-, 3-furyl, such as 1-, 2-, 3-pyrrolyl, 1-, 2-, 3-thienyl, 2-, 3-, 4-pyridyl, 2-, 3-, 4-, 5-, 6-, 7-indolyl, 3-, 4-, 5-pyrazolyl, 2-, 4-, 5-imidazolyl, acridinyl, quinolinyl, phenanthridinyl, 2-, 4-, 5-, 6-pyrimidinyl. The heteroaromatics may be substituted in the same way as the (C6-C18)-aryl radicals mentioned above.
A (C4-C19)-heteroaralkyl means a heteroaromatic system corresponding to the (C7-C19)-aralkyl radical.
Suitable halogens (Hal) are fluorine, chlorine, bromine or iodine.
The term aqueous solvent means water or a solvent mixture which mainly consists of water and contains water-soluble organic solvents such as, for example, alcohols, in particular methanol or ethanol, or ethers, such as THF or dioxane, or other cosolvents such as DMSO.
The references cited in this document are considered to be also within the scope of the disclosure.
The protein sequences depicted in the sequence listing additionally contain a His-tag and a linker sequence on the C-terminus. The actual protein sequences which represent the active γPLEs are therefore the sequences depicted in the sequence listing, which are truncated by 21 amino acids on the C-terminus. The same applies to the nucleic acid sequences coding for said protein sequences.
Methods:
Isolation of mRNA and cDNA Synthesis
Fresh pig liver tissue (0.1 g) was treated with Trizol® reagent (TRIzol® Plus RNA Purification Kit, Invitrogen, Calif., USA), homogenized (10 min at RT; Ultraturrax T25, IKA-Labortechnik), and the RNA was isolated according to the manufacturer's instructions. The RNA concentration was determined spectrophotometrically. The cDNA synthesis was carried out by means of RT-PCR using oligo(dT)15 primers and MMLV reverse transcriptase with RNase H activity (Promega, Madison, Wis., USA) according to the manufacturer's protocol.
Amplification and Cloning of PLE Genes
The RT-PCR product was used for the amplification of PLE genes using two gene-specific primers based on the γPLE sequence (5′-CACCCATATGGGGCAGCCAGCCTCGC-3′ (Seq. ID No. 11), with the NdeI restriction cleavage site marked in italics, and 5′-CCGCTCGAGTCACTTTATCTTGGGTGGCTTCTTTGC-3′ (Seq. ID No. 12), with the XhoI restriction cleavage site marked in italics; start and stop codons are underlined). The forward primer moreover contains on its 5′ end the bases CACC which make possible subsequent cloning into a TOPO vector (see below). These primers already eliminate the 18-amino-acid signal sequence attached to the N terminus of the original porcine gene, and the 4-amino-acid C terminal ER (endoplasmic reticulum) retention signal, thereby facilitating subsequent expression in E. coli (Lange, S, et. al., ChemBioChem (2001), 2, 576-582). The PCR was carried out on a thermocycler (Techne Progene, Jepson Bolton Laboratory Equipment, Watford, United Kingdom). The PCR employed Pfu Plus Polymerase (Roboklon, Berlin, Germany) according to the manufacturer's instructions and the following temperature program: after denaturation for 5 minutes at 95° C., 30 cycles of 1 min at 95° C., 1 min at 60° C., 3 min at 72° C. were carried out, with a final 7 min at 72° C. The PCR products were fractionated in an agarose gel, purified and cloned into a TOPO/pET101 according to the manufacturer's protocol (Champion™ pET Directional TOPO® Expression Kit; Invitrogen, Carlsbad, Calif., USA). E. coli TOP10 cells [F− mcrA D(mrr-hsdRMSmcrBC) (F80lacZDM15) DlacX74 recA1 deoR araD139 D(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG] (Invitrogen) were transformed with the construct mixture and separated out on agar plates. The recombinant single clones obtained in this way were cultured separately, the plasmid DNA was isolated, identified by size determination or restriction mapping and used as template for PCR amplification of the PLE sequences. The amplified sequences were then sequenced (MWG-Biotech, Martinsried, Germany).
Construction of the Expression System
Plasmid DNA of the individual TOPO/pET101-PLE constructs were digested with NdeI and XhoI according to the manufacturer's protocol (New England Biolabs, Beverly, Mass., USA; Promega, Madison, Wis., USA), and the particular fragments of about 1694 bp in size were inserted into NdeI/XhoI-digested, agarose gel-purified pET15b vector (Novagen, Madison, Wis., USA), which adds an additional N-terminal His tag to the gene. The ligation products were used for transformation of E. coli DH5a strains (Novagen, Madison, Wis., USA) [supE44ΔlacU169 (Φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1relA1], and the plasmid was propagated by culturing the transformed strains. The plasmid was isolated from the recombinant strains and again sequenced for a check. The pET15b-PLE constructs thus obtained were used for transformation of E. coli Origami (DE 3) strains [Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC galE galK rpsL F′[lac+ lacIq pro] (DE3) gor522::Tn10 trxB (KanR, StrR, TetR)4] (Novagen, Madison, Wis., USA) which had previously been transformed with pGro7 plasmid (Chaperone Plasmid Set, TAKARA BIO Inc., Otsu, Shiga, Japan), which enables the chaperone GroEL+GroES to be expressed.
For details of expressing pig liver esterase with coexpression of the chaperone complex GroEL/ES, see: Böttcher, D., Brüsehaber, E., Doderer. K., Bornscheuer, U. T. (2007), Functional expression of the gamma-isoenzyme of pig liver carboxyl esterase in Escherichia coli, Appl. Microbiol. Biotechnol., (2007), 73(6), 1282-1289.
Expression of Recombinant PLE Isoenzymes in E. coli Origami with Coexpression of the GroEL/ES Chaperone Complex.
The chaperones were coexpressed with the PLE isoenzymes in 150 ml of LB medium containing 20 μg mL−1 chloramphenicol and 50 μg mL−1 ampicillin for plasmid selection. Chaperone expression was immediately initiated by adding 1 mg mL−1 L-arabinose. PLE production was induced at OD600=0.5 by adding 40 μM IPTG. The cells were removed by centrifugation after 24 h, resuspended in 10 ml of sodium phosphate buffer (50 mM, pH 7.5) and disrupted using ultrasound. Cell debris was removed by centrifugation, and the supernatant was used for further experiments (crude cell extract). Protein content and esterase activity were determined according to Bradford or using the pNPA assay.
Native Polyacrylamide Gel Electrophoresis and Activity Staining with Fast Red.
A mixture containing the crude extract of recombinantly produced PLE (5-15 μL, corresponding to 0.05-0.15 U of pNPA analysis) was mixed with buffer solution (20% (w/v) glycerol; 0.0025% (w/v) bromophenol blue in dH2O) (5-10 μL). Aliquots thereof were separated on a native polyacrylamide gel (7.5%). For activity staining, the gel was incubated in a mixture of freshly prepared α-naphthyl acetate and Fast Red. Formation of a red complex between α-naphthol generated and Fast Red indicated hydrolytic activity of the esterase. (Krebsfänger, N., et. al., (1998) Enzyme Microb. Technol, 22, 641-646). The gel was then stained with Coomassie Brilliant Blue.
Esterase Activity
Esterase activity was determined spectrophotometrically in sodium phosphate buffer (50 mM) by means of p-nitrophenyl acetate (10 mM, dissolved in dimethyl sulfoxide) as substrate. The amount of p-nitrophenol generated was determined at a wavelength of 410 nm (ε=15*103 M−1 cm−1) at RT and pH 7.5. 1 unit (U) is defined as the amount of enzyme capable of converting 1 μM p-nitrophenol per minute under analytical conditions (Krebsfänger, N., et. al., (1998) Enzyme Microb. Technol, 22, 641-646).
Esterase substrate specificity was analyzed at constant pH. A known amount of esterase was added to an emulsion (20 mL) containing an ester substrate (5% (v/v); tributyrin, ethyl acetate, triolein or methyl butyrate) and gum arabic (2% (w/v)) at 37° C. The acid liberated was counter titrated automatically in a pH statiometer (Schott, Mainz, Germany) with 0.01 N NaOH in order to maintain a constant pH of 7.5. 1 unit (U) is defined as the amount of enzyme capable of generating 1 μM acid per minute under analytical conditions.
Stereoselectivity of Enzymatic Hydrolysis of Acetates of Secondary Alcohols
(see DE10258327A1)
Hydrolysis was carried out in 1.5 ml reaction vessels in a thermomixer (Thermomixer comfort Eppendorf, Hamburg, Germany) at 37° C. 0.5 U esterase crude extract (based on the pNPA assay) were used for 1 ml of substrate solution (10 mM in sodium phosphate buffer pH 7.5, 50 mM). The reaction was stopped by extracting the mixture with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate.
Enantiomeric purity and conversion were determined by gas chromatography. Enantioselectivity of the variant enzymes was calculated according to Chen et al. (C. S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. Chem. Soc. 1982, 104, 7294.).
For a more detailed description of the synthesis of the substrates and the retention times in GC analysis, see Musidlowska-Persson, A. and Bornscheuer, U. T., “Substrate Specificity of the γ-isoenzyme of recombinant pig liver esterase towards acetates of secondary alcohols” J. Mol. Catal. B. Enzym. 2002, 19-20, 129-133.
Stereoselectivity of the Enzymatic Hydrolysis of cis-3,5-diacetoxycyclopent-1-ene
The experimental procedure was identical to the resolution of the racemates of secondary alcohols, using 0.5 units (pNPA) of PLE crude extract in 1 ml of reaction mixture containing 10 mM substrate in sodium phosphate buffer pH 7.5 50 mM. The reaction was carried out at 37° C., and samples were taken at the times indicated. Conversion and product enantiomeric excesses were analyzed by gas chromatography. The analysis employed a Hydrodex®-b-3P (heptakis-(2,6-di-O-methyl-3-O-pentyl-b-cyclodextrin) (25 m, 0.25 mm)) GC column (Machery Nagel, Düren, Germany) in a C-R5A Chromatopac/Integrator GC instrument (Shimadzu, Duisburg, Germany). The retention times for isothermal fractionation at a column temperature of 110° C. were: cis-3,5-diacetoxy-cyclopent-1-ene: 21.8 min, 3(S)-acetoxy-5(R)-hydroxy-cyclopent-1-ene: 18.2 min, 3(R)-acetoxy-5(S)-hydroxy-cyclopent-1-ene: 16.0 min.
Results:
Isolation of mRNA from Pig Liver and RT-PCR
The quality of the isolated RNA was checked on an agarose gel (figure not shown), and said RNA was quantified by spectrophotometry (2.6 μg/μl); thus, 260 μg of RNA were obtained from 0.1 g of tissue used. After transcription into cDNA by RT-PCR, the quality of said cDNA was checked by using it as template for amplification of the household gene β-actin.
Amplification and Cloning of PLE Genes
The cDNA was used for the amplification of PLE genes, using primers based on the sequence of γPLE. As
Sequencing Results
Sequencing of the new PLE genes revealed that four new gene sequences were obtained which are distinct with respect to each other and with respect to γ-PLE. The sites deviating from the γ-PLE sequence are depicted in the alignment of amino acid sequences of
PLE 2: 6 Nucleotide substitutions (3 amino acid substitutions) [isoenzyme 4]
PLE 3: 35 Nucleotide substitutions (20 amino acid substitutions) [isoenzyme 24]
PLE 4: 34 Nucleotide substitutions (20 amino acid substitutions) [isoenzyme 39]
PLE 5: 34 Nucleotide substitutions (21 amino acid substitutions) [isoenzyme 41]
Expression of the Proteins
The five genes found were subcloned into a pET15b vector. E. coli Origami was transformed with these constructs and with the pGro7 plasmid which codes for the two chaperone parts, GroES and GroEL. Successful overexpression of PLE in E. coli had been described for this expression system (DE 10061864). The strains were cultured on a small scale (50 ml/150 ml), and protein expression of chaperone and PLE construct was induced. In addition, γPLE and E. coli Origami with pGro7 without the pET vector were cocultured for comparison and protein expression was induced. The supernatants were disrupted (Volume: 3 ml/9 ml), and the crude extracts containing the soluble intracellular proteins were obtained by centrifugation and used for subsequent experiments.
The protein content in all crude extracts was about 7-9 mg/ml (of which a large part of the protein formed corresponds to the chaperones); the final volume of the crude extract was 3 or 9 ml, depending on the size of the culture.
Native Gel and Fast Red Staining
Esterase activity was checked by applying the crude extracts to a native gel which was then incubated in Fast Red solution with α-naphthyl acetate as substrate. This was followed by Coomassie staining (
Active esterase bands are visible with PLE2, PLE3, PLE4, PLE5 and γPLE, which strangely have different sizes and are very heavily smeared. This may be due to the method, since native gels do not run as clearly as denaturing SDS gels; however, it is also possible that some crude extracts contain both trimeric and tetrameric PLE constructs, both of which are active.
Clone 12 does not show any activity. Likewise, as expected, no esterase activity is detectable in the crude extract of E. coli Origami without the pET15b construct.
The protein bands in Coomassie staining clearly indicate that a lot of chaperone was overexpressed in addition to γPLE. The protein contents determined by means of Bradford thus comprise mainly the chaperone and do not give any information about the γPLE content. The extent of overexpression cannot be assessed precisely anymore on the basis of the Coomassie staining carried out after Fast Red. Compared to the E. coli wild type with chaperone there is no additional protein band visible in the region of the active bands. However, frequently the protein can no longer be stained with Coomassie after activity staining. However, it could also mean that overexpression is only very low but still provides protein with good activity.
Activity with p-Nitrophenyl Acetate
Esterase activity was checked first by using the pNPA assay. It produced the results listed in table 1.
E. coli Origami + pGro7
All PLEs show activity with pNPA, which is for some of the new esterases even twice to four times as high as for γ-PLE.
Activity with Achiral Esters
The activity of the new PLE variants with achiral esters was examined using pH stat. After purification of the enzymes, specific activities were determined which are listed in table 2.
Resolution of the Racemates of Acetates of Secondary Alcohols
Hydrolysis of the following racemic acetates was investigated:
Gas chromatographic studies produced the results depicted in tables 3 to 6. The data obtained for the commercial PLE preparations by A. Musidlowska (Musidlowska-Persson, A. and Bornscheuer, U. T., J. Mol. Catal. B. Enzym. 2002, 19-20, 129-133.) have been added for comparison.
Influence of Inhibitors
The inhibitability of the new PLE variants was determined by treating the crude extracts with any of three esterase inhibitors. The influence of phenylmethylsulfonyl fluoride, sodium fluoride and physostigmine was investigated. Samples were taken at certain points in time and the remaining esterase activity was determined by the pNPA assay. Table 8 depicts the results.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2007 014 742 | Mar 2007 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/052880 | 3/11/2008 | WO | 00 | 9/21/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/116745 | 10/2/2008 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7229817 | Bornscheuer et al. | Jun 2007 | B2 |
| Number | Date | Country |
|---|---|---|
| 100 61 864 | Jul 2002 | DE |
| 102 58 327 | Jun 2004 | DE |
| WO 2004055177 | Jul 2004 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20110207186 A1 | Aug 2011 | US |
