AU2006261217A1 - Selection of phosphatase-coding nucleic acid molecules - Google Patents

Abstract

The invention relates to methods for selecting nucleic acid molecules coding for proteins having phosphatase activity from a library of nucleic acid molecules that are to be analyzed.

Description

PARK IP TRANSLATIONS CertificatLion Park IP Translations TRANS IAIO'OS DECLA,/RAIO N: December 1 2, POO I, P~eggy F. WrightC, hereb dl: ecla re: That. ii pos sos s adva nced k:nowldg ok Mec 1 Ch Ge :ma: and EnglIis h languages. The atLacheci L.ransIa Lion AI the patent applI.icatLion with I[he tLitle 10 "'Sceewior of Pho s haLa so Act Lvi Ly" a nd the abstraMl -The oresent- invention relawLs to methods f or selecting nucleic01 0 aci.d molc uUles codi ng for proteins ha ving a phosrpha Lase acLiviQ fro ran library of: nuleeic acid mroecules to be anal yzed. " has Men ura sla Led by me and: o the best af: my knowl Iedge anrd belief K Lao eU:aI~e1. ret IeeLs Lhec menueuinq and M.n ention oft he or jiinal Lexh. Peggy F. Wright 850 Seve uu IIl AV'. 511 V1001 Yi(i\xv rk.NN.. 0 1 )9 Ruhne: 2 1 2-5~I8 70 IVa : 21I2-581I-887.5 Patent Attorneys, Attorneys at Law G leiss & G rosse European Patent Attorneys Intellectual Property and Technology Law European trek Dr. jur. Alf-Olav Gleiss* Cert. Eng. PA EPA Trainer Grosse Cert. Eng. PA EPA Dr. Andreas Schrell' Cert. Biolog PA EPA Torsten Armin KrUger* RA Nils Heide* RA Armin Eugen Stockinger RA PA TENT APPLICATION Dr. Frank Kapries RA Dr. Hartmut Schwahn* Dipl. Biolog PA Address Gleiss & Grosse Patent Attorneys, Attorneys at Law Leitzstrasse 45 Selection of Phosphatase Activity D-70469 Stuttgart Telephone +49 (0)711 99 3 11-0 Facsimile +49 (0)711 99 3 11-200 E-mail office()_qleiss-qrosse.com Gleiss Grosse Schrell & Partners Patent Attorneys, Attorneys at Law AG Stuttgart PR 167 In cooperation with Eucodis GmbH Shanghai Zhi Xin Patent Agency Brunner Strasse 59 Ltd. Shanghai, China 1235 Vienna PA Patent Attorney European Trademark and Design Attorney EPA European Patent Attorney European Trademark and Design Attorney RA Attorney at Law also admitted at DPMA. EPO, OHIM Partner of the partnership company Al Selection of Phosphatase Activity Description The present invention relates to methods for selecting nucleic acid molecules coding for proteins having a phosphatase activity from a library of nucleic acid 5 molecules to be investigated. Phosphatases are proteins having a phosphatase activity. A protein is a phosphatase, i.e., has a phosphatase activity, when it is capable of splitting off inorganic phosphate from phosphate-containing substrates. Examples of known phosphatases that are of economic interest include the plant phytases. 10 Up to 80% of the phosphorus in foods of plant origin cannot be metabolized by animals, specifically monogastric animals such as swine and poultry. Consequently, millions of tons of phosphorus enter the environment, causing major problems such as algal blooms, oxygen depletion and destruction of food chains. On the other hand, it is difficult in some cases to cover the phosphorus 15 needs of these animals with traditional feed. There is therefore a demand for animal feed having a phosphorus content that can be metabolized extensively. One possibility for increasing the amount of metabolizable phosphorus in feed is to supplement the feed with phytase. Phytases are enzymes that hydrolyze the phosphorus-containing phytic acid or phytate (salt of phytic acid) component 20 present in plant food sources so the inorganic phosphate is released. Phytic acid/phytate is a myo-inositol 1,2,3,4,5,6-hexakis-dihydrogen phosphate containing approx. 14 to 18% phosphorus plus usually 12 to 20% calcium. Phytate/phytic acid is usually present as a strongly negatively charged molecule, so its passage through biological membranes is difficult. A hydrate shell forms 25 around phytate/phytic acid and this also counteracts passage of the molecule 2 through a biological membrane and/or pores. For example, the hydrated' phytate molecule cannot penetrate through the periplasmic membrane of gram-negative bacteria. Phytase is the only known enzyme capable of initiating successive phosphate 5 hydrolysis at carbon atom 1, 3 or 6 in the inositol ring of phytate. 6-Phytases (e.g., AppA) catalyze phosphate hydrolysis beginning at carbon number 6, while 3-phytases (glucose-1 -phosphatase, releasing only one orthophosphate group) begins at carbon atom number 3 accordingly. Cleavage of the phosphate group by phytase leads to the release of divalent metal ions such as calcium, iron, zinc 10 and others. Phytases occur in a variety of different microorganisms and plants as well as in the tissues of some animals. Phytases from gram-negative bacteria are intracellular proteins usually located in the periplasmic area. However, phytases from gram-positive bacteria and yeasts are extracellular proteins. All known 15 phytases are monomeric proteins except for phytase B (PhyB) from Aspergillus niger, which is a tetramer. The molecular weights of these enzymes vary greatly, but are usually between 38 and 100 kDa. Phytases from fungi and yeasts have a higher molecular weight because of glycosylation. However, glycosylation has no effect on the specific activity and thermal stability of phytase. 20 Known phytases are not very similar structurally, nor do they split off inorganic phosphate by the same mechanism. Phytases can be classified in three main groups, namely acidic, neutral and alkaline phosphatases, based on the pH dependence of their enzyme activity. Primarily phytases from the group of acid phosphatases may be considered for the support of phosphate metabolism in 25 animal feeds for monogastric animals. TN: "hydriert" is mostly used in the sense of "hydrogenated" but "hydrated" is a less common meaning. 3 The phylogenetic analysis of the amino acid sequences of various phytases also yields three main classes. This classification is in good agreement with the classification of phytases according to biochemical or catalytic properties: Class I - histidine acidic phosphatase phytase (HAP) 5 One of the main classes is the HAP family, the members of which include PhyA and PhyB from Aspergillus niger and AppA from Escherichia coli. These have a broad substrate specificity and are capable of hydrolyzing metal-free phytate in an acidic pH range. They have in common the highly preserved motif RHGXRXP. This class can be further subdivided into three groups: PhyA, PhyB and PhyC, 10 depending on the homology of the amino acid sequence and biochemical properties, such as optimum pH or the positional specificity of phytate hydrolysis. Class II - p-propeller phytases (BPP) or alkaline phytases These include, for example, PhyC from Bacillus subtilis and PhyA from Bacillus amyloliquefaciens FZB45, Bacillus phytase (P-propeller) catalyzes the hydrolysis 15 of phytic acid into inositol and orthophosphate in the presence of divalent elements such as calcium, iron or zinc. This class differs from the HAP class in a number of aspects, such as optimum pH, molecular weight, tertiary structure, substrate specificity and the need for the presence of calcium ions in catalysis. Alkaline phytases from Bacillus or plant seeds can be classified as an additional 20 group PhyD on the basis of these biochemical differences and the phylogenetic data. Class Ill - purple acid phosphatases (PAP) This class includes for example GmPhy from soybean, Glycine max L. GmPhy was recently isolated from the cotyledons of sprouting soybeans. The active 25 fragment of GmPhy has the motif of a purple acid phosphatase (PAP). The estimated molecular weight is 70 to 72 kDa, like other plant-based PAP. However, GmPhy is the only known PAP with a significant phytase activity. 4 Comparison of the amino acid sequence yielded a 33% homology between GmPhy and PAP from kidney beans. Phytases release inorganic phosphate from indigestible phytic acid, so phosphate can be absorbed from this animal feed. Not only does this reduce 5 phosphate waste but also there is no longer a need for otherwise supplementing such animal feeds with metabolizable phosphate. However, degradation of phytic acid and/or phytate in feeds does not serve merely to supply metabolizable phosphate. Phytic acid or phytate, such as that occurring in the boundary layers of the cereal grain, is considered to be a 10 chelating agent for metal ions and thus also prevents absorption of important metal ions from feeds such as calcium, iron, magnesium, phosphorus or zinc. The relationship between a mineral deficiency induced by certain whole-grain products and the occurrence of phytic acid and/or phytate in these whole-grain products in humans has been discussed. It would therefore be desirable to 15 reduce the amount of phytic acid and/or phytate in whole-grain products. This can be accomplished by corresponding use of phytase in the production of whole-grain products or through the corresponding addition of phytase to the whole-grain products. On the other hand, the use of known phytases in feeds is limited by the high cost 20 of obtaining phytases. The applicability of known phytases in animal feeds is also limited by the fact that they are inactivated at the high temperatures used for pelletization of the feeds (usually more than 800C). In addition, known phytases lose most of their activity during storage of the feeds. A number of known phytases are unsuitable just because they are only stable and/or active in a 25 narrow pH range that does not usually occur in the digestive tract. A number of the known phytases also lack resistance to degradation by the proteases that occur in the digestive tract. Furthermore, phytic acid or phytate as a phosphorus source is present in the digestive tract of these animals mainly as a calcium phytate complex, especially when the feed has a high calcium content (approx. 3 5 to 40 g/kg). However, this calcium phytate complex cannot serve as a substrate from many known phytases. There is therefore a demand for new phosphatases, especially phytases, having improved biochemical and catalytic properties, so that they are more suitable for 5 use in animal feed and human food industries. Proteins with a phytase activity are expressed in a number of different organisms. Not all naturally occurring phytases have been characterized or isolated so far and not all genes that code for proteins having a phytase activity are known. Therefore, there is a demand for providing selection methods with which genes that code for a certain selected 10 phosphatase activity, especially a phytase activity, can be detected and selected in an especially effective manner. Known selection methods which have as their goal discovering novel genes that code for a certain function, e.g., an enzyme activity, are usually based on liquid batches in the aqueous phase, so-called liquid assays. Assays for selection of 15 phosphatase, especially phytase coding genes, are based on the formation of a phosphorous molybdate complex, for example, and the color reaction associated with it. Hoenig et al. (J. Biochem. Biophys. Methods, 1989, 19 (2-3): 249-251) describe an assay based on malachite green; Greiner et al. (Arch. Biochem. Biophys. 1997, 341 (2): 201-206) describe the determination of phytase activity 20 with a reagent solution containing heptamolybdate/ammonium vanadate. According to Heinonen and Lathi (Anal. Biochem. 1981, 113 (2): 313-317) phytase activity can be detected by reduction of the phosphorous molybdate complex that is formed. These known detection methods may be sufficient for a small number of samples to be investigated, but the known assays are not 25 suitable if an entire library is to be analyzed, necessitating the use of several million different clones. This would be extremely time-consuming or not even feasible given the large number of different clones, so there is also a demand for a selection method which can be performed with a large number of samples to be investigated and can also be used in particular for an automated application. 30 Therefore this should be a rapid and simple selection assay. 6 Against this background, the technical problem on which the present invention is based is essentially that of providing a method for selection of a nucleic acid molecule that codes for a selective phosphatase activity. This method should especially be easy to perform with known means and should permit an especially 5 good and rapid selection, even when a large number of samples must be analyzed. The basic technical problem is solved by a method for selection of a nucleic acid molecule that codes for a protein having a selected phosphatase activity, such that the nucleic acid molecule is selected from a group of nucleic acid molecules 10 to be analyzed. The group is preferably a bank or a library. The inventive method contains the following steps, in particular comprising: a) the nucleic acid molecule to be analyzed is made available; b) the nucleic acid molecule to be analyzed and preferably also a nucleic acid molecule coding for the Kil protein phage lambda (X) is inserted into the 15 genome of a host cell that has a phosphatase deficiency and/or in which no phosphatase activity is expressed; c) the host cell in which the nucleic acid molecule to be analyzed and preferably the nucleic acid molecule coding for the Kil protein of phage lambda was introduced is cultured in a deficiency medium for inorganic phosphate in 20 which the deficiency medium preferably contains the substrate of the selected phosphatase activity as a single phosphate source and the substrate of the selected phosphate activity is present in the deficiency medium preferably in a sufficient amount to allow growth of the host cell culture in principle or to cover the phosphate needs of the host cells; 25 d) the growth of the host cells in the medium is demonstrated, such that in the presence of the nucleic acid molecule that codes for the selected phosphatase activity, growth of the host cells occurs and this growth indicates the presence of a nucleic acid molecule coding for the selected phosphatase. 7 The present invention thus provides a method with which genes that code for a certain phosphatase activity can be selected out of a number of different organisms. According to the inventive method, both the nucleic acid molecule to be investigated and a nucleic acid molecule that codes for the Kil protein of 5 phage lambda are introduced into a host cell, preferably in such a way that the two nucleic acid molecules introduced are expressed in the host cell. If the nucleic acid molecule to be analyzed codes for a protein having the selected phosphatase activity, then this is expressed in this host cell. In addition, i.e., in parallel, preferably simultaneously and/or under the same conditions, the Kil 10 protein is preferably also expressed according to this invention. If the protein expressing host cell is cultured in the inventive deficiency medium, then the growth of the host cells shows the presence of a nucleic acid molecule coding for the selected phytase activity in an especially clear manner, i.e., in an especially sensitive and selective manner. It is therefore especially advantageous that the 15 steps of the inventive method, especially steps c) and d), can be performed automatically, e.g., in an automated cell culture system that in particular also allows automated detection of the growth of the host cells. In a preferred embodiment of the inventive method, the nucleic acid molecule to be investigated and preferably also the nucleic acid molecule coding for the Kil 20 protein of phage lambda are first cloned in an expression vector (step b1). Then the host cell is transformed with the expression vector (step b2). In a particularly preferred variant, first a fragment of the genomic DNA of phage lambda carrying the kil gene is amplified and inserted into a vector (plasmid) and in particular is cloned. In another step, the nucleic acid molecule to be investigated is made 25 available, e.g., by isolating it from an organism, especially a cell that expresses the phosphatase activity, and amplifying it with suitable primers. Then the amplified nucleic acid molecule to be analyzed is inserted into a second vector (plasmid) and in particular is cloned. The nucleic acid molecules and/or gene fragments thus inserted are preferably under the control of inducible promoters in 30 the vectors. Then the fragment carrying the kil gene, especially as a complete compression cassette, of the first vector is inserted into the second vector, 8 preferably by means of restriction enzyme digestion. This yields an expression vector carrying an expressible nucleic acid molecule to the analyzed plus also preferably an expressible nucleic acid molecule coding for the Kil protein of phase lambda. 5 The inventive method can be performed especially well and solves the problem on which this invention is based especially when not only the nucleic acid molecule to be analyzed but also a nucleic acid molecule coding for the Kil protein of bacteriophage lambda is expressed, especially simultaneously, in the host cell provided according to this invention. 10 The present invention also relates to the host cells and cells originating from the host cells that have been transformed with the aforementioned inventive nucleic acids and/or vectors. According to this invention, the nucleic acid molecule to be analyzed is thus in an expression system and its expression is controlled by an inducible promoter. It is 15 also especially preferred according to the present invention for the nucleic acid molecule that codes for the Kil protein of phage lambda to be present in an expression system and for its expression to preferably be under the control of an inducible promoter. Of course instead of the inducible promoters, constitutional promoters may also be used without going beyond the teaching of the present 20 invention. In a preferred embodiment, the nucleic acid molecule that codes for the Kil protein of phage lambda, is obtained from the genomic DNA of phage lambda by amplification by means of the polymerase chain reaction (PCR) using a primer pair with SEQ ID NO: 1 and SEQ ID NO: 2. This nucleic acid molecule has a 25 length of 287 base pairs (bp). Of course a nucleic acid molecule that codes for a Kil protein can also be obtained by another method, e.g., by synthesis, cloning, preparation, etc. 9 Through the parallel expression of the kil gene in the host cell, as is preferred according to this invention, a bacterial release protein is synthesized, enabling proteins to pass from the intracellular medium, in particular the periplasm, into the extracellular space. To a certain extent, such an expression cassette with the 5 kil gene constitutes an inducible secretion of lysis system. Without being bound to the theory, expression of the kil gene especially surprisingly allows secretion of proteins having a phosphatase activity, located in the intracellular space, into the extracellular medium. In an especially preferred variant, co-culturing of first cells that express phosphatase and preferably kil 10 gene and second cells, in particular other cells that are phosphatase-deficient and/or cells having no phosphatase activity is provided. Due to the extracellular phosphatase activity occurring in the case of a positive test according to this invention, the second phosphatase-deficient cells, in particular other cells, are supplied with inorganic phosphate and can also grow. 15 In an especially preferred embodiment, the phosphatase-deficient host cell is a gram-negative prokaryotic cell, especially preferably an Enterobacteria cell. The inventive host cell is especially preferably an Escherichia coli cell (E. coli). The phosphatase deficiency is preferably induced by suppressing transcription of the PHO regulon of the cell, especially E. coli. This is preferably accomplished 20 according to the present invention by deleting the phoB gene. It is especially surprising that global down-regulation of the PHO regulon is achieved in the phosphatase-deficient mutant thereby created. This advantageously results in the phosphatase-deficient mutant thereby created not have any phosphatase activity any longer and being able to grow only on a medium containing a 25 sufficient amount of inorganic orthophosphate. In the present case, this phenomenon is also subsumed under the heading of "phosphatase deficiency." In a preferred embodiment, the inventive method is used to select a certain phosphatase activity, namely the phosphatase activity of a gram-negative bacterium. The selected phosphatase is preferably an intracellular phosphatase, 30 especially a cytoplasmic or periplasmic phosphatase. Without being bound to the 10 theory, the expression plasmid codes for the signal sequence (e.g., the signal peptide of OmpA that allows translocation of phosphatase into the periplasm via the sec system). In a preferred variant, the selected phosphatase is a phytase. According to this invention, a deficiency medium containing phytic acid and/or 5 phytate as the substrate is selected in step c) of the method. In this preferred embodiment, the inventive method therefore allows especially simple and automatable selection of a nucleic acid molecule that codes for phytase activity from a group of nucleic acid molecules to be investigated, especially from a library and/or bank of nucleic acid molecules. 10 With the inventive method, it is especially surprising possible to not only directly detect a selected phosphatase activity but also it allows a quasi-quantitative determination of the selected phytase activity. Thus the observed intensity of the growth of the host cells expressing the phytase activity provides direct information about the activity of the expressed phosphatase. A high enzyme 15 activity is thus reflected in a rapid growth and/or a high rate of replication of the host cells. In step c) of the inventive method, the host cells are cultured in a deficiency medium containing the phosphatase substrate as the only phosphate source. The substrate is preferably present in the deficiency membrane in a 20 concentration of 0.1 to 0.5 mmol/L, especially preferably 0.15 to 0.4 mmol/L. The host cells are preferably cultured in the deficiency medium for a period of 2 to 4 days, especially preferably for 3 days. After this period of culturing, definite differences in the growth of the host cells to be tested can be detected. The culturing temperature is preferably from 28 to 320C, especially preferably 300C. 25 Finally, another subject matter of the present invention is the use of the nucleic acid molecule that codes for the Kil protein of the phase lambda for the purpose of discovering nucleic acid molecules that code for a certain phosphatase activity in selection processes, such that the inventive method described above is performed in particular. 11 This invention will now be explained in greater detail on the basis of the accompanying figures and examples, but these should not be understood to be restrictive. In the figures: 5 Figure 1 shows an example of a phosphatase activity: the phytase catalyzed hydrolysis of phytate to inositol and orthophosphate in the presence of divalent elements; Figure 2 shows a schematic diagram of an expression vector containing a nucleic acid molecule (gene) coding for a phosphatase activity (here: phytase activity) 10 under the control of the promoter Pte and a fragment carrying the kil gene under the control of the promoter Ptrc and an N-terminal signal peptide and a C-terminal Flag-tag; Figures 3A, 3B, 3C and 3D show analysis of the growth of E. coli cultures of the strain BL21 phoB::TnlO from a smear in Petri dishes; Figure 3A shows cells after 15 transformation of the strain with the plasma pMCS and cells derived therefrom on minimal medium containing a suitable amount of inorganic phosphate; Figure 3B shows the same organisms as in Figure 3A growing on minimal medium containing phytate as the sole phosphorus source; Figure 3C shows cells after transformation with the plasmid pkil as well as cells derived therefrom, growing 20 on minimal medium containing a suitable amount of inorganic phosphate; Figure 3D shows the same organisms as in Figure 3C growing on minimal medium containing phytate as the sole phosphate source; legend: (1) control phoB strain; (2) control phoB strain containing the parent plasmid; (3) phoB strain expressing the plasmid-coded phytase 11 of E. coli; (4) phoB strain expressing the 25 plasmid-coded phytase I of E. coli; (5) phoB strain coding for the plasmid-coded phyA of Bacillus amyloliquefaciens FZB45 and in which a stop codon has been inserted between phyA and the C-terminal Flag-tag; (6) phoB strain expressing the plasmid-coded phyA of Bacillus amyloliquefaciens FZB45 in which phyA is fused to Flag-tag at the C-terminal end; 12 Figure 4 shows cloning of the kil gene in the plasmid pCrcHis2/lacZ under control of the inducible Ptc promoter; Figure 5 shows cloning of the appA gene and/or the agp gene under the control of the inducible Pac promoter; 5 Figures 6A, 6B and 6C show the insertion of Ptrckil into plasmid pMCS to obtain pkil (Figure 6A), into plasmid pappA to obtain pkilappA (Figure 6B) and into plasmid pagp to obtain pkilagp (Figure 6C); Figure 7 shows cloning of the phyA gene in the plasmid pMCS under the control of the inducible promoter Pte; 10 Figure 8 shows cloning of the phyA gene in the plasmid pkil under the control of the inducible promoter Pc; Figures 9A and 9B show Wester blot [sic; Western blot] analysis: separation of cell extract samples (Figure 9A), separation of samples from the supernatant medium (Figure 9B); legend: (1) MG1655 (wt); (2) MG 1655 induced with IPTG; 15 (3) marker; (4) MG 1655 pMCS; (5) MG1655 pMCS induced with IPTG; (6) MG1655 pkil; (7) MG1655 pkil induced with IPTG; (8) MG1655 pappA; (9) MG1655 pappA induced with IPTG; (1) MG1655 pkilappA; (11) MG1655 pkilappA induced with IPTG; (12) MG1655 pagp; (13) MG1655 pagp induced with IPTG; (14) MG1655 pkilagp; (15) MG1655 pkilagp induced with IPTG; 20 Figures 1 OA and 1 OB show Western blot analysis of the cell extract samples for pMCS and the derived cells using a primary polyclonal anti-phyA antibody and a secondary polyclonal anti-rabbit IgG AP-conjugated antibody (lanes 1 through 9) or of a monoclonal anti-Flag-tag M2 antibody (lanes 10 through 18 in Figure 1 0A, lanes 10 through 20 in Figure 1OB); legend to Figure 1 0A: (1) and (11) MG1 655 25 phoB::TnlO pphy45Flag induced with IPTG; (2) and (12) MG1655 phoB::TnlO pphy45Flag; (3) and (13) MG1655 phoB::TnlO pphy45STOP induced with IPTG; (4) and (14) MG1655 phoB::Tnl0 pphy45STOP; (5) and (10) marker; (6) and (15) MG1655 phoB::TnlO pMCS induced with IPTG; (7) and (16) MG1655 13 phoB::TnlO pMCS; (8) and (17) MG1655 phoB::Tn10 induced with IPTG; (9) and (18) MG1655 phoB::Tnl0; legend for Figure 1OB: (1) BL21 phoB pkilphy45Flag induced with IPTG; (2) BL21 phoB pkilphy45Flag induced with lactose; (3) BL21 phoB pkilphy45Flag; (4) BL21 phoB pkilphy45STOP induced with IPTG; (5) BL21 5 phoB pkilphy45STOP induced with lactose; (6) BL21 phoB:Tnl0 pkilphy45STOP; (7) marker; (8) BL21 phoB:TN10 pkil induced with IPTG; (9) BL21 phoB:TnlO pkil induced with lactose; (10) marker; (11) BL21 phoB::Tnl0 pkilphy45STOP induced with IPTG; (12) BL21 phoB::Tnl0 pkilphy45STOP; (13) BL21 phoB::TnlO pkil induced with IPTG; (14) BL21 phoB::TnlO pkil; (15) MG1655 10 phoB::Tn1O pkilphy45Flag induced with IPTG; (16) MG1655 phoB::TnlO pkilphy45Flag; (17) MG1655 phoB::TnlO pkil induced with IPTG; (18) MG1655 phoB::Tnl0 pkil; (19) MG1655 phoB:Tnl0 induced with IPTG; (20) MG1655 phoB::Tn10. Example 1: Insertion of the kil gene of bacteriophage lambda into the plasmid 15 pTrcHis2/lacZ A plasmid was constructed to obtain an inducible expression of the Kil protein. In this way a high and controllable cell lysis of a gram-negative host cell like E. coli can be achieved. From prepared genomic DNA of the bacteriophage lambda, a 287 bp DNA 20 fragment that codes for the Kil protein was amplified. The primers SEQ ID NO: 1 and SEQ ID NO: 2 (Table 1) were used. The PCR fragments were digested and cloned in the plasmid pTrcHis2/lacZ (Figure 4) as Ncol-Hindlli fragments to obtain the plasmid pTrcHis2/lacZ kil (Figure 1). The PCR conditions were (temperature in 0 Cltime in minutes): (98/10), (96/0.75; 55/0.50; 72/1.5)35, (72/10); 25 enzyme: herculase. Twelve of the resulting clones were analyzed by restriction digestion with BsaAl. The expected fragment lengths were 7532 bps for pTrcHis2/lacZ and 1894 bps and 2738 bps for pTrcHis2/lacZ kil. Three of 12 tested clones contain the correct separation profile. 14 Table 1: Primer[SEQ ID NO.] Sequence 1 kilNcolhin CATGCCATGGCATGCCATTGCAGGGTGGCCTGT 5 2 kilHindllher CCCAAGCTTGTGAATGCTTTTGCTTGATCTCAG 3 appaXholhin CCGCTCGAGCGAAAGCGATCTTAATCCCATTT 4 appaBglllher GAAGATCTTCCCAAACTGCACGCCGGTATGCGT 5 agpXholhin 10 CCGCTCGAGCGAACAAAACGCTAATCGCCGCAG 6 agpBgillher GAAGATCTTCCTTTCACCGCTTCATTCAACACG 7 45hinXhol CCGCTCGAGCGAAGCATAAGCTGTCTGATCCTTAT 8 45herBglllSTOP 15 GGAAGATCTTTATTTTCCGCTTCTGTCGGTCAG 9 45herBglllFlag GGAAGATCTTTTTCCGCTTCTGTCGGTCAGTTT Example 2: Cloning the appA gene and the aqp gene of E. coli Plasmids were constructed to obtain a high and controlled expression of AppA 20 (phytase 1) and Agp (glucose-1-phosphatase and/or phytase 11). a) Cloning appA A 1316 bp DNA fragment, the appA gene that codes for an acid phosphatase (phytase 1) was amplified from prepared genomic DNA from the E. coli strain MG1655. The primers SEQ ID NO: 3 and SEQ ID NO: 4 (Table 1) were used for 25 this. The PCR fragments were digested and cloned as Xhol-Bglll fragments to obtain the plasmid pappA (Figure 5). 15 Two of the resulting clones were analyzed by restriction digestion with Sall. The expected fragment lengths were 5403 bps for pMCS and 1081 bps and 5546 bps for pagp. One of the two clones tested had the correct separation profile. b) Cloning agp 5 A 1258 bp DNA fragment, the agp gene that codes for an acid phosphatase (phytase 11), was amplified from repaired genomic DNA from the E. coli strain MG1655. The primers SEQ ID NO: NO: 5 and SEQ ID NO: NO: 6 (Table 1) were used for this. The PCR fragments were digested and cloned as Xhol-Bglll fragments to obtain the plasmid pagp (Figure 5). 10 Eleven of the resulting clones were analyzed by restriction digestion with Sall. The expected fragment lengths were 5403 bps for pMCS and 975 bps and 5713 bps for pagp. Three of 11 of the tested clones had the correct separation profile. Example 3: Insertion of PI-kil into the plasmids pMCS, pappA and Paqp 15 a) Insertion of Ptrckil into plasmid pMCS Plasmid pTrcHis2/lacz kil (see Example 1) was digested with the restriction enzymes Pmel and BssHl, and a DNA fragment with 1126 bps was isolated. Plasmid pMCS was digested with the restriction enzymes BssHll and Bstl 1071, and a DNA fragment with 4477 bps was isolated. These PCR fragments were 20 cloned to obtain the plasmid pkil (Figure 6A). Twelve of the resulting clones were analyzed by restriction digestion with Ncol. The expected fragment lengths were 4632 bps for pTrcHis2/lacz kil, 5408 bps for pMCS and 1606 bps and 3993 bps for pkil. All of the 12 tested clones had the correct separation profile. 25 b) Insertion of Ptrckil into plasmid pappA 16 Plasmid pTrcHis2/lacz kil (see Example 1) was digested with the restriction enzymes Pmel and Mlul and a DNA fragment with 1537 bps was isolated. Plasmid pappA was digested with restriction enzymes Mlul and Bstl 1071 and a DNA fragment with 5519 bps was isolated. These PCR fragments were cloned to 5 obtain the plasmid pkilappA (Figure 6B). Twelve of the resulting clones were analyzed by restriction digestion with Ncol. The expected fragment lengths were 4632 bps for pTrcHis2/lacz kil, 6688 bps for pappA and 1606 bps and 5278 bps for pkilagp. Seven of the 13 tested clones had the correct separation profile. 10 c) Insertion of Ptrekil into plasmid pagp The plasmid pTrcHis2/lacz kil (see Example 1) was digested with the restriction enzymes Pmel and Mlul and a 1537 bps DNA fragment was isolated. Plasmid pagp was digested with restriction enzymes Mlul and Bstl 1071 and a DNA fragment with 5351 bps was isolated. The PCR fragments were cloned to obtain 15 the plasmid pkilagp (Figure 6C). Twelve of the resulting clones were analyzed by restriction digestion with Ncol. The expected fragment lengths were 4632 bps for pTrcHis2/lacz kil, 6630 bps for pagp and 1606 bps and 5220 bps for pkilagp. Eight of the 12 tested clones had the correct separation profile. 20 Example 4: Insertion of the phyA gene from Bacillus amyloliquefaciens FZB45 into the plasmid pMCS The plasmid pMCS allows strong and controllable expression via the promoter Ptac. The C-terminal Flag-tag occurring therein facilitates purification and detection of the expressed target protein by means of anti-Flag antibody. 25 However in the present selected example, the C-terminus is directed at the center of the phytase protein PhyA which has a p-propeller structure with highly complex folding. To rule out the possible influence of fusion of the phyA gene with the plasmid-coded Flag-tag on the correct folding of the protein or on 17 inhibition of enzyme activity, two cloning strategies were developed. First, phyA gene was cloned in the expression frame of Flag-tag; in a second batch, a stop codon was inserted between the phyA gene and Flag-tag. Accordingly, plasmids having a high and controllable expression of phytase from 5 the Bacillus amyloliquefaciens FZB45 (PhyA) in a host cell such as E. coli were constructed. a) Batch PhyA in the Flag-tag expression frame A 1082 bp DNA fragment that codes for PhyA was amplified from the prepared genomic DNA from Bacillus amyloliquefaciens FZB45. The primers SEQ ID 10 NO: 7 and SEQ ID NO: 8 (Table 1) were used for this purpose. PCR fragments were digested and cloned in an Xhol-Bglll fragment into the plasmid pMCS to obtain the plasmid pphy45STOP (Figure 7). The PCR conditions were (temperature in *C/time in minutes): (95/5) (95/0.5; 58/0.5; 72/1.5)35, (72/10); enzyme: herculase. 15 Thirty of the resulting clones were analyzed by PCR using the primers SEQ ID NO: 7 and SEQ ID NO: 8. Six positive clones were further analyzed by restriction digestion with Eco47111. The expected fragment lengths were 5403 bps for pMCS and 5388 bps and 1077 bps for pphy45STOP. Four of the six tested clones had the correct separation profile. 20 b) Batch with a stop codon A 1079 bp DNA fragment that codes for PhyA was amplified from the prepared genomic DNA from Bacillus amyloliquefaciens FZB45. The primers SEQ ID NO: 7 and SEQ ID NO: 9 (Table 1) were used for this purpose. The PCR fragments were digested and cloned in the form of an Xhol-Bglll fragment into the plasmid 25 pMCS to obtain the plasmid pphy45Flag (Figure 7). The PCR conditions were (temperature in *C/time in minutes): (95/5) (95/0.5; 58/0.5; 72/1.5)35, (72/10); enzyme: herculase. 18 Thirty-eight of the resulting clones were analyzed by PCR using the primers SEQ ID NO: 7 and SEQ ID NO: 9. Six positive clones were further analyzed by restriction digestion with Eco47111. The expected fragment lengths were 5403 bps for pMCS and 5385 bps and 1077 bps for pphy45Flag. All six tested clones had 5 the correct separation profile. Example 5: Insertion of the phyA gene from Bacillus amyloliquefaciens FZB45 into the plasmid pkil These plasmids were constructed to achieve high and controlled expression of phytase from Bacillus amyloliquefaciens FZB45 (PhyA) in host cells such as 10 E. coli. These plasmids allow controlled release of the expressed enzyme into the extracellular space. To rule out the influence of Flag-tag (see Example 4), two alternative cloning strategies were developed. a) Batch PhyA in the Flag-tag expression frame A 1082 bp DNA fragment that codes for PhyA was amplified from the prepared 15 genomic DNA from Bacillus amyloliquefaciens FZB45. The primers SEQ ID NO: 7 and SEQ ID NO: 8 (Table 1) were used for this purpose. The PCR fragments were digested and cloned in the form of an Xhol-Bglll fragment into the plasmid pMCS to obtain the plasmid pkilphy45STOP (Figure 8). The PCR conditions were (temperature in *C/time in minutes): (95/5) (95/0.5; 58/0.5; 20 72/1.5)35, (72/10); enzyme: herculase. Eight of the resulting clones were analyzed by PCR using the primers SEQ ID NO: 7 and SEQ ID NO: 8. Five positive clones were further analyzed by restriction digestion with Eco47111. The expected fragment lengths were 5599 bps for pkil and 5584 bps and 1077 bps for pkilphy45STOP. All five tested clones had 25 the correct separation profile. b) Batch PhyA with a stop codon 19 A 1079 bp DNA fragment that codes for PhyA was amplified from the prepared genomic DNA from Bacillus amyloliquefaciens FZB45. The primers SEQ ID NO: 7 and SEQ ID NO: 9 (Table 1) were used for this purpose. PCR fragments were digested and cloned in the form of an Xhol-Bglll fragment into the plasmid pMCS 5 to obtain the plasmid pkilphy45Flag (Figure 8). The PCR conditions were (temperature in OC/time in minutes): (95/5) (95/0.5; 58/0.5; 72/1.5)35, (72/10); enzyme: herculase. Five of the resulting clones were analyzed by PCR using the primers SEQ ID NO: 7 and SEQ ID NO: 9. One positive clone was further analyzed by restriction 10 digestion with Eco47l1l. The expected fragment lengths were 5599 bps for pkil and 5581 bps and 1077 bps for pkilphy45Flag. The tested clone had the correct separation profile. Example 6: Translation of aqp and apDA in host cells a) Culturing 15 10 pL portions of overnight cultures of the bacterial strains MG1655, MG1655 pMCS, MG1655 pkil, MG1655 pappA, MG1655 pkilappA, MG1655 pagp and MG1655 pagpkil (see Table 2) in 10 mL LB medium were inoculated with 100 pg/mL ampicillin at 370C. During the mean log phase (OD 600 : 0.4-0.7), IPTG (isopropyl-p-D-thiogalactopyranoside) was added in a final concentration of 20 1 mmol/L. This induced expression of AppA and Agp by the promoter Ptac and expression of Kil by the promoter Pt,,. The cells were then shaken for 2.5 hours at 37 0 C. Next the cell cultures were centrifuged and the cell pellets and supernatants (medium) stored at -200C. b) SDS-PAGE 25 For cell lysis, the sample pellets were resuspended in 200 pL of a 1 x SDS PAGE sample buffer. Next (for extraction) 20 pL CHCl 3 was added. For extraction, the samples were vortexed and centrifuged and the supernatants of the aqueous phase were mixed with sample buffer. The supernatants (medium) 20 of the cell cultures were also mixed with sample buffer. The extract samples and samples of the cell supernatant were separated in one run in SDS-PAGE (12%). c) Western blot analysis The proteins were transferred to nitrocellulose membrane using a transfer buffer 5 with 25 mmol/L Tris at pH 8.3, 192 mmol glycine and 20 vol% methanol (1 hour to 1.5 hours at 100 V and 40C). The membrane was removed and blocked with 5% milk powder for 20 minutes in phosphate-buffered saline (PBS), rinsed with water and then incubated overnight at room temperature with a monoclonal anti Flag AP-conjugated antibody (dilution 1:2000; Sigma) in PBS mixed with 3% milk 10 powder. The membrane was then washed three times for 15 minutes with PBS containing 0.1% Tween 20. Then the membrane was washed again once for 10 minutes with PBS and treated with the Alkaline Phosphatase Conjugated Substrate Kit from BIO-RAD (see Figures 9A and 9B). d) Result 15 In both cases, strong expression of the target protein was observed. In addition, an effective secretion of both proteins into the extracellular space was achieved (Figures 9A and 9B). Example 7: Translation of phyA in host cells a) Culturing 20 10 pL portions of overnight cultures of the bacterial strains BL21 phoB::TnlO, BL21 phoB::TnlO pMCS, BL21 phoB::TnlO pphy45STOP, BL21 phoB::TnlO pphy45Flag, BL21 phoB::TnlO pkil, BL21 phoB::TnlO pkilphy45STOP and BL21 phoB::Tn10 pkilphy45Flag (see Table 2) were inoculated with 12.5 pg/mL tetracycline and 100 pg/mL ampicillin at 370C in 10 mL LB medium. During the 25 mean log phase (OD 600 : 0.7) IPTG was added in a final concentration of 1 mmol/L. Expression of PhyA by the Ptac promoter and the expression of Kil by the Prc promoter were thereby induced. The cells were then shaken for 2 hours 21 at 370C. Next the cell cultures were centrifuged and the cell pellets and supernatants (medium) were stored at -201C. b) SDS-PAGE For cell lysis, the sample pellets were resuspended in 200 pL of a 1 x SDS 5 PAGE sample buffer. Next (for extraction) 20 pL CHC1 3 was added. For extraction, the samples were vortexed and centrifuged and the supernatants of the aqueous phase were mixed with sample buffer. The supernatants (medium) of the cell cultures were also mixed with sample buffer. The extract samples and the samples of the cell supernatant were separated in SDS-PAGE (12%). 10 c) Western blot analysis The proteins were transferred to nitrocellulose membrane using a transfer buffer with 25 mmol/L Tris at pH 8.3, 192 mmol glycine and 20 vol% methanol (1 hour at 100 V and 40C). The membrane was removed and blocked with 5% milk powder for 1 hour in Tris-buffered saline (TBS), rinsed with water and then 15 incubated with a primary polyclonal anti-PhyA antibody (dilution 1:3000) or a monoclonal AP-conjugated anti-Flag antibody (dilution 1:2000; Sigma) in TBS mixed with 1% bovine serum albumin (BSA) and then incubated overnight at room temperature. The membrane was washed three times for 5 minutes in TBS containing 1% BSA. If a secondary antibody was necessary the membrane was 20 incubated with anti-rabbit IgG AP-conjugated antibody (dilution 1:1000; cell signaling) in TBS mixed with 1% BSA. Then the membrane was washed three times for 15 minutes in TBS containing 0.1% Tween 20, then rinsed with TBS and treated with the Alkaline Phosphatase Conjugated Substrate Kit from B10 RAD (see Figures 10A and 1OB). 25 d) Results The plasmids investigated expressed the protein PhyA from Bacillus amyloliquefaciens FZB45. Both the anti-PhyA antibody and the anti-Flag antibody are capable of recognizing the fusion protein PhyA Flag (see 22 Figure 10A, lanes 1, 2, 11 and 12). Expression of the PhyA protein has been detected with the anti-PhyA antibody (see Figure 10A, lanes 3 and 4). In addition, a high expression of proteins was detected in the presence of IPTG. The plasmids investigated, derived from pKIL-derived plasmids express the PhyA 5 protein from Bacillus amyloliquefaciens FZB45. Both the anti-PhyA antibody and the anti-Flag antibody are capable of recognizing the fusion protein PhyA Flag (see Figure 10B, lanes 1, 2, 3, 15 and 16). Recognition of the PhyA protein not furnished with a tag peptide by means of the anti-PhyA antibody is less definite (see Figure 10B, lanes 4, 5 and 6). 10 Example 8: Construction of the DhoB deletion mutants a) Preparation of the P1,ir lysate To prepare a P1ir lysate from the bacterial strain HA15, 10 pL of an overnight culture of the strain was inoculated in 10 mL LB medium containing 5 mmol/L CaCl2 at 370C. During the exponential growth phase, the bacterial cells were 15 infected with the pretreated P1vir lysate of wild type MG1655. The infected culture was incubated for 4 hours more at 37*C until cell lysis was observed. For extraction, CHCl 3 was added, the lysate was centrifuged, the supernatant was transferred to a new test tube with CHCla and stored at 4 0 C. b) P1 transduction 20 50 pL of an overnight culture of the recipient strains BL21, TG1, IS568, ES1 568, JM110, MG1655 mutL::Tn5 was inoculated in LB medium at 370C until reaching the exponential growth phase. 900 pL portions of these cells were mixed with 2.5 pL CaC1 2 (1 mol/L) and 100 pL P1vir lysate of bacterial strain HA15 (phoB::Tn10). Phage absorption, which was continued for 38 minutes at 370C, 25 was stopped by adding 5 mmol/mL sodium citrate in LB. 23 The cells were incubated for 1 hour at 370C without agitation for phenotypic expression. The bacterial culture was centrifuged out and plated out on selection plates mixed with 12.5 pg/mL tetracycline. The resulting individual colonies were purified and incubated overnight at 370C. 5 Example 9: Transformation of competent cells Competent cells of the strains MG1655, MG1655 phoB::Tnl0, MG1655 mutL::Tn5, MG1655 mutL::Tn10 phoB::Tnl0, BL21, BL21 phoB::Tnl0, TG1, TG1 phoB::Tn10, ES568, ES568 phoB::Tnl 0, ES1 578, ES1 578 phoB::Tnl 0, JM 110, JM110 phoB::Tnl0 and DH5a (phoA-) were prepared according to the rubidium 10 chloride protocol (Promega). Finally, 200 pL aliquots were allocated per test tube and stored at -800C. Each strain was transformed with one of the following plasmids: pMCS, pkil, pappA, pkilappA, pagp and pkilagp. In addition, the strains MG1655 phoB::Tn10, TG1 phoB::Tnl0 and BL21 phoB::Tnl0 were each transformed with the plasmids 15 pphy45STOP, pkylphy45Flag [sic; pkilphy45Flag] and pkilphySTOP. The resulting individual colonies were purified and incubated overnight at 370C on LB agar plates or selection plates containing 100 pg/mL ampicillin. The bacterial strains thereby produced and the plasmid-carrying derivatives derived therefrom were mixed with glycerol and stored at -800C. Table 2 lists the 20 bacterial strains. The strain Escherichia coli BL21 phoB::Tnl0 pappA was deposited with the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig (DSMZ) on June 20, 2005 under the deposition number DSM 17406. 25 The strain Escherichia coli BL21 phoB::Tnl0 pagp was deposited with the DSMZ on June 20, 2005 under the deposition number DSM 17407. 24 The strain Escherichia coli BL21 phoB::Tn10 pphy45FLAG was deposited with the DSMZ on June 20, 2005 under the deposition number DSM 17408. The strain Escherichia coli BL21 phoB::Tnl0 pphy45STOP was deposited with the DSMZ on June 20, 2005 under the deposition number DSM 17409. 5 The strain Escherichia coli BL21 phoB::Tn10 pKiIappA was deposited with the DSMZ on June 20, 2005 under the deposition number DSM 17410. The strain Escherichia coli BL21 phoB::Tnl0 pKilagp was deposited with the DSMZ on June 20, 2005 under the deposition number DSM 17411. The strain Escherichia coli BL21 phoB::Tnl0 pKilphy45FLAG was deposited with 10 the DSMZ on June 20, 2005 under the deposition number DSM 17412. The strain Escherichia coli BL21 phoB::Tn10 pKilphy45STOP was deposited with the DSMZ on June 20, 2005 under the deposition number DSM 17413. Table 2 ES568 pMCS ES568 phoB::Tn10 pMCS ESS68 pkil ES568 phoR:Tn1o pkil ES568 pappA ESS68 phoB:Tn10 pappA ES568 pkilappA ES568 pho&:Tn10 pkilappA ES568 pagp ES568 pho&:TnlO pagp 15 ES568 pkilagp ES568 phoa:Tn10 pkilagp BL21 BL21 phoB:Tn1o BL21 pMCS BL21 phoB::Tn10 pMCS BL21 pkil BL21 phoBR:Tn1O pkil BL21 pappA BL21 phoa.:Tn10 pappA BL21 pkilappA BL21 phoB::Tn1OpkilappA BL21 pagp BL21 phoB:Tn1O pagp BL21 pkilagp BL21 phoB:Tn1O pkilagp BL21 pho&R:Tn10 pphy45STOP BL21 phoa.:Tn1O pphy45FLAG 8L21 phog:Tn10 pkilphy45STOP BL21 phoR:Tn10 pkilphy45FLAG MG1655 MG1655 phoR:Tn10 20 MG1655pMCS MG1655 pho&:Tn10 pMCS MG1655 pkil MG1655 phoB.:Tn10 pkil MG1655 pappA MG1655 phoB::Tn10 pappA MG 1655 pkilappA MG1655 pho&R:Tn1O pkilappA MG1655 pagp MG1655 phoa:Tn1O pagp MG 1655 pkilagp MG1655 phoB:Tn1O pkilagp MG16S5pho&:Tn1O pphy45STOP 25 MG I 655phoa.:Tn 10 pphy45FLAG MG1 655pho8:Tn 10 pklIphy45STOP MG I 655pho:Tn 10 pkilphy45FLAG ES1578 ES1578 phoB.:Tn10 ES1578 pMCS ES1578 phoB:Tn10 pMCS ES1576 pkil ES1578 phoB.:Tn10 pkil ES1578 pappA ES1578 pho&:Tn1o pappA ES 1578 pkilappA ES1578 pho&:Tn1O pkilappA ES1578 pagp ES1578 phoa:Tn10 pagp ES1578 pkilagp ES1578 phoa:Tn1O pkllagp TG1 TG1 phoa:Tn1O TG1 pMCS TG1 pho&:Tn10 pMCS TG1 pkil TG1 phoa:Tn10 pki TGI pappA TGI phoB::Tn10 pappA TG1 pkilappA TG1 pho&:Tn10 pkilappA TGI pagp TG1 phoB::Tn10 pagp 5 TG1 pkilagp TG1 pho&:TnlO0 pkiagp TG1 phoft:Tn10 pphy45STOP TG1 phoB::Tn10 pphy45FLAG TG1 phoa:Tnl0 pkilphy45STOP TG1 pho&:Tn10 pkilphy45FLAG MG1655 mutL ::Kn MG1655 mutL ::Kn pho&:Tnl0 MG1655 mutL ::Kn pMCS M01655 mulL::Kn phoa:TnlO pMCS MG1655 mutt ::Kn pkil MG1655 mutL::Kn pho&t:TnlO pkil MG1655 mutL ::Kn pappA MG1655 mulL::Kn phoa:Tn10 pappA MG1655 mutL ::Kn pkilappA MG1655 mulL::Kn phoa:Tnl0 pkiappA MG1655 mutL ::Kn pagp MG1655 mutL ::Kn phoB::TnIO pagp MG1655 mutL::Kn pkilagp MG1655 mutL ::Kn phoa:TnO pkilagp 1 0 DH5a (phoA.) DHSa5pMCS DH5M pmil DH6 mpappA DH5a pkilappA DH5a pagp DH5a pkilagp Example 10: Selection of phytase activity 15 a) Growth conditions The bacterial strains listed in Table 2 (see Example 9) were plated out on agar plates of different minimal media. The media compositions are based on MOPS buffer (Table 4). The MOPS buffer was prepared according to Neidhardt, Block and Smith, 1974, Culture medium for Enterobacteria (J. Bacteriol. 119:736-47). 20 In general, 22 mmol/L glucose and 0.3 mmol/L thiamine were added to the media compositions. In addition, so-called micronutrients were added in the concentrations listed in Table 5. Depending on the composition of the minimal 26 medium, inorganic phosphate (K 2

HPO

4 ; Pi) and/or phytate (Na, 2

C

6

P

6 0 24 ) were present (Table 3). Table 3 Medium (mmol/L) K 2

HPO

4 Na 12

C

6

P

6

O

24 5 A MOPS 1.32 - B MOPS P-limited 0.066 - C MOPS phytate -- 1.32 D MOPS phytate limited -- 0.246 E MOPS phytate, Pi-limited 0.066 1.32 10 F MOPS phytate, P-hyperlimited 0.0132 1.32 G MOPS phytate-limited, P-limited 0.066 0.246 H MOPS phytate-limited, P-hyperlimited 0.0132 0.246 Table 4 MOPS buffer (mmol/L) 15 NH 4 CI 9.52 MgCl 2 0.532

K

2

SO

4 0.276 FeSO 4

-H

2 0 0.010 CaCl 2

.H

2 0 5 x 104 20 NaCl 50 MOPS 40 Tricine 4 Micronutrients + (see Table 5) Table 5 25 Micronutrients (mmol/L)

(NH

4

)

6

(MO

7

)

24 3 x 10-6

H

3 B0 3 4 x 10-4 CoC1 2 3 x 10-5 CuSO 4 10-5 30 MnC1 2 8 x 10-5 27 ZnSO 4 10-1 The growth studies were conducted at temperatures of 25 0 C to 370C. The agar plates were expediently incubated for 7 days at 250C or 300C. The cell growth was checked each day. 5 b) Results Availability of results Clear-cut results were obtained after three or max. 4 days, depending on the strain used. No further incubation would then be necessary. Expression of AppA in E. coli 10 It was found that E. coliphoB strains that expressed the plasmid-coded AppA protein (phytase 1) or glucose-1-phosphtase (phytase 11) could grow on minimal media (medium D) containing phytate as the sole phosphate source. Selection for phytase activity was additionally performed with other phytase-limited media containing 0.1 to 0.4 mmol/L phytate. The phytate concentration ideally 15 amounted to 0.15 to 0.4 mmol/L. Expression of PhyA from Bacillus amyloliquefaciens FZB45 E. coli phoB strain (BL21 phoB::Tn10) that express the plasmid-coded PhyA protein from Bacillus amyloliquefaciens FZB45 were also able to grow on minimal media (medium D) containing phytate as the sole phosphate source (see Figures 20 3A, 3B, 3C, 3D). Selection for phytase activity could also be performed with additional phytate-limited media containing 0.1 to 0.4 mmol/L phytate. The phytate concentration ideally amounted to 0.15 to 0.4 mmol/L. Control Under the same conditions (medium D), no growth was found at all (see 25 Figures 3A, 38, 3C, 3D) in untransformed host cells and/or host cells 28 transformed with the plasmid pMCS or pkil by comparison. These host cells express only the naturally occurring chromosomally coded AppA protein. Quantification of activity In addition, it was found that the observed growth rate correlates directly with the 5 activity of the expressed phosphatase. This has been verified by parallel quantitative Western blot analyses for quantification of the expression intensity and by quantitative experiments in the liquid phase to determine the enzyme activity. Transferability 10 These results can also be confirmed with the other strains produced (Table 2) and/or with the other expression vectors. 29

Claims (12)

1. Method for selection of a nucleic acid molecule coding for a selected phosphatase activity from a group of nucleic acid molecules to be analyzed, comprising the steps: 5 a) providing the nucleic acid molecule to be analyzed, b) inserting the nucleic acid molecule to be analyzed into a phosphatase deficiency host cell, c) culturing the host cell in deficiency medium for inorganic phosphate containing the substrate of the selected phosphatase 10 activity as the sole phosphate source and in sufficient amount and d) detecting the growth of the host cells in the medium, where growth indicates the presence of the nucleic acid molecule coding for the selected phosphatase activity.

2. Method according to Claim 1, wherein step b) comprises the following 15 substeps: b1) cloning the nucleic acid molecule in an expression vector, b2) transforming the host cell with the expression vector.

3. Method according to Claim 1 or 2, wherein in step b) a nucleic acid molecule coding for the Kil protein of phage lambda is additionally inserted into 20 the phosphatase-deficient host cell.

4. Method according to Claim 3, wherein the nucleic acid molecule coding for the Kil protein of phage lambda has a length of 287 bp and is accessible by PCR amplification of genomic DNA of phage lambda with the primer pair having SEQ ID NO: 1 and SEQ ID NO: 2. 30

5. Method according to any one of the preceding claims, wherein the phosphatase-deficient host cell is an E. coli cell, in particular a phoB strain of E. coli.

6. Method according to any one of the preceding claims, wherein the 5 deficiency medium contains the substrate in a concentration of 0.1 to 0.5 mmol/L, preferably 0.15 to 0.4 mmol/L.

7. Method according to any one of the preceding claims, wherein the host cell is cultured in the deficiency medium for 2 to 4 days, preferably 3 days.

8. Method according to any one of the preceding claims, wherein the host 10 cells are cultured in the deficiency medium at 28 to 320C, preferably 300C.

9. Method according to any one of the preceding claims, wherein the selected phosphatase is the phosphatase of a gram-negative bacterium.

10. Method according to any one of the preceding claims, wherein the selected phosphatase is an intracellular phosphatase, in particular a periplasmic 15 phosphatase.

11. Method according to any one of the preceding claims, wherein the selected phosphatase is a phytase and the substrate of the selected phosphatase is phytic acid and/or phytate.

12. Use of a nucleic acid molecule coding for the Kil protein of phage lambda 20 for selection of nucleic acid molecules coding for phosphatase activity, in particular in a method according to any one of the preceding claims. 31