Title of Invention

MUTATED PENICILLIN EXPANDASE ENZYME AND ITS USE

Abstract

The present invention relates to a mutant pencillin expandase which comprises an amino acid substitution at one or more residue positions corresponding to those of a wild-type expandase selected from the group consisting of threonine at position 42, isoleucine at position 50, histidine at position 57, threonine at position 67, valine at position 133, threonine at position 143, proline at position 145, glycine at position 148, phenyl alanine at position 152, proline at position 196, alanine at position 240.

Full Text

Field of the invention The present invention relates to a modified expandase enzyme and in particular penicillin N expandase having increased specificity for a substrate such as penicillin G. P-lactam antibiotics occupy major portion of anti-infective segment due to low toxicity, high specificity and clinical efficacy against wide variety of pathogenic organisms. Numerous organisms of both bacterial and fungal species produce classical (3-lactam antibiotics such as penicillins, cephalosporins, cephamycins and non-classical antibiotics such as clavulanic acid and thienamycin. Penicillium chrysogenum and Cephalosporium acremonium, both of fungal family, produce Penicillin G and Cephalosporin C respectively. Streptomyces clavuligerus, a bacterium, produce classical antibiotic cephamycin and non-classical antibiotic clavulanic acid, which is widely known for its P-lactamase inhibition. Reviews such as (Jensen, S. E. & Demain, A. L. (1993) in Biochemistry and Genetics of Antibiotics/Biosynthesis, eds Vining, L. C & Stuttard, C, Butterworth - Heinemann, Boston) can be consulted for biosynthesis, genetic regulation and biochemical characterization of various enzymes involved in the synthesis of these antibiotics. Although penicillin antibiotics are used for treating infections, many of the infectious organisms become resistant to naturally occurring penicillin antibiotics and the resistance mechanisms operate through degradation by p-lactamase, thereby requiring novel and more potent antibiotics. Cephalosporins have been remarkable in their effectiveness against resistant bacteria and hence, significant research is devoted to develop novel semisynthetic derivatives. Cephalosporin derivatives such as Cephalexin, cephadoxyl and Cefradine are produced by coupling 7-Amino deacetoxy cephalosporinic acid (7-ADC A) with appropriate side chains. 7-ADC A is produced from Cephalosporin G, which in turn gets synthesized by expensive and polluting chemical processes from penicillin G. Biotransformation, a process per se environmentally friendly and that could be cheaper than the chemical process is required. Penicillin N expandase also known as desacetoxy cephalosporin C synthase (DAOCS), an enzyme found in species such as Streptomyces clavuligerus, Streptomyces lactamdurans, Xanthomonas lactamgenus, Flavobacterium sp, Streptomyces organanensis, Streptomyces lactamgens, Streptomyces fradiae, Streptomyces griseus and Streptomyces ofivaceus catalysing the conversion of five membered thiazolidine ring nucleus of penicillin into six membered dihydrothiazine nucleus of cephalosporins has become an obvious choice for an alternative route for the synthesis of cephalosporin derivatives. Penicillin N expandase isolated from Streptomyces clavuligerus has been extensively characterized and described in Kovacevic S, et aL, J. Bacteriol. 171(2): 754-760, 1989, Dotzlaf, J. E. et al, J, Biol. Chem 264:10219-10226, 1989 and Valegard, K. et a/., Nature. 394: 805-809, 1998. Penicillin N, although a natural substrate of expandase, is not readily available and cleaving the adipoyl side chain is highly inefficient. On the other hand, penicillin G is readily available and phenyl acetyl side chain group can be cleaved with penicillin G amidase at high efficiency. However, penicillin G is a poor substrate for Penicillin N expandase. Hence, commercial capitalization requires engineering the expandase and such modified expandases and their uses are described in WOOl/85951 and US 6,699,69982. US publication No. 20030186354 discloses a mutated penicillin expandase comprising an amino acid substitution at one or more residue positions corresponding to those in a wild-type expandase selected from the group consisting of methionine 73, glycine 79, valine 275, leucine 277, cysteine 281, glycine 300, asparagine 304, isoleucine 305, threonine 91, alanine 106, cysteine 155, tyrosine 184, methionine 188 and histidine 244, provided that the amino acid substitution at the residue position of asparagine 304 is not N304L and the amino acid substitution at the residue position ot cysteine li!) is C155Y. The main objective of the present invention is to provide mutated expandase having expansion activities multiple folds higher on penicillin G or penicillin V than wild-type expandase or disclosed in prior art. Description of the Figure Figure 1: SEQ ID NO: 1 describes the nucleotide and amino acid sequence for penicillin N expandase of Streptomyces clavuligerus. Summary of the Invention Accordingly, the present invention provides a mutant penicillin expandase having modified or improved ring-expanding activity. Preferably, the expandase is a penicillin N expandase, which is modified to increase the activity on penicillin G or penicillin V as a substrate. In another embodiment of the present invention, there is provided a modified expandase gene encoding the expandase mutation. In another embodiment of the present invention, there is provided a protein having modified expandase activity. In another embodiment of the present invention, there is provided an expression vector, which comprises a modified expandase gene. In another embodiment of the present invention, there is provided a host strain transformed with an expression vector. In another aspect, the invention provides a mutant pencillin expandase which comprises an amino acid substitution at one or more residue positions corresponding to those of a wild-type expandase selected from the group consisting of threonine at position 42, isoleucine at position 50, histidine at position 57, threonine at position 67, valine at position 133, threonine at position 143, proline at position 145, glycine at position 148, phenyl alanine at position 152, proline at position 196, alanine at position 240, cysteine at position 281, Serine at position 309, provided that the amino acid substitution at the residue position of cysteine at position 281 is not tyrosine. In particular, the invention provides a mutated penicillin expandase which comprises one or more specific amino acid substitutions selected from the group consisting of T42A, I50V, H57R, T67A, V133I, T143S, P145L, G148E, F152L, P196S, A240T, C281R, S309P, V133I and P196S; F152L and C281R; T42A, F152L and S309P; V133I, T143S, P196S and S309P wherein the residue positions of the amino acid substitution correspond to those of a wild-type expandase. In another aspect, the invention provides a variant of SEQ ID No. 1 defined above and seen Streptomyces lactamdurans, Xanthomonas lactamgenus, Flavobacterium sp., Flavobacterium chitinovoruna, Streptomyces organanensis, Nocardia lactamdurans, Streptomyces lipmanii, Streptomyces jumonjinensis, Streptomyces wadayamensi, Streptomyces cattleya, Streptomyces lactamgens, Streptomyces fradiae, Streptomyces griseus, Streptomyces oHvaceus, Streptomyces sp and Acremonium chrysogenum. The variation can be identified by a person skilled in the art by aligning a variant with the sequence of SEQ ID No:l. For example the equivalent amino acid to asparagine at position 304 of SEQ ID No: 1 can be identified by a person skilled in the art by aligning a variant with the sequence of SEQ ID No:l and thus identify the equivalent residue for position 304 of SEQ ID No:l. The modified strains of Escherichia coli DH5a containing the moditiecl expanaase genes were designated with the following accession numbers; MTCC 5133, MTCC 5134, MTCC 5135, MTCC 5136, MTCC 5137, MTCC 5138, MTCC 5139, MTCC 5140, MTCC 5141, MTCC 5142 and MTCC 5143. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a mutant penicillin expandase which shows increased or modified ring-expanding activity preferably on penicillin G or penciUin V as a substrate. A mutant penicillin expandase according to the invention comprise an expandase derived from Streptomyces clavuligerus or an expandase derived from other organisms. The nucleotide and amino acid sequence of penicillin N expandase from Streptomyces clavuligerus is set out in SEQ ID NO : 1 The primary aspect of the present invention is to provide a mutated penicillin expandase having a better substrate specificity to penicillin G or penicillin V, wherein the mutated penicillin expandase comprises an amino acid substitution at one or more residual positions corresponding to those in a wild-type expandase selected from the group consisting of threonine at position 42, isoleucine at position 50, histidine at position 57, threonine at position 67, valine at position 133, threonine at position 143, proline at position 145, glycine at position 148, phenyl alanine at position 152, proline at position 196, alanine at position 240, cysteine at position 281, Serine at position 309, provided that the amino acid substitution at the residue position of cysteine at position 281 is not tyrosine. The variations in the sequence of SEQ ID NO. 1 are as given here: isoleucine at position 50 is substituted by valine. histidine at position 57 is substituted by arginine; threonine at position 67 is substituted by alanine ; proHne at position 145 is substituted by leucine ; glycine at position 148 is substituted by glutamic acid ; phenyl alanine at position 152 is substituted by leucine ; alanine at position 240 is substituted by threonine ; valine at position 133 is substituted by isoleucine; and proline at position 196 is substituted by serine; phenyl alanine at position 152 is substituted by leucine and cysteine at position 281 is substituted by arginine not tyrosine; threonine at position 42 is substituted by alanine ; phenyl alanine at position 152 is substituted by leucine, and Serine at position 309 is substituted by proline ; valine at position 133 is substituted by isoleucine; threonine at position 143 is substituted by serine; proline at position 196 is substituted by serine, and serine at position 309 is substituted by proline. A modified peptide in accordance with the present invention may incorporate the modifications described for example modification of leucine at position 50. As described above, a variant polypeptide having an amino acid sequence which varies from that of SEQ ID NO: 1 may be modified in accordance with the present invention. A variant for use in accordance with the invention is one having expandase activity. A modified variant in accordance with the invention is one which demonstrates an improved ability to ring expand a substrate such as penicillin G or penicillin V when compared to a variant sequence not so modified. Amino acid substitutions may be made to the amino acid sequence. One or more amino acid residues of the amino acid sequence of SEQ ID NO: 1 may alternatively or additionally be deleted. Polypeptides of the invention also include fragments of the above-mentioned sequences. Such fragments retain expandase activity. Such fragments may be used to produce chimeric enzymes using portions of enzyme derived from other expandase polypeptides. Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e. g. 95%, 98% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention. The polypeptides of the invention may be introduced into a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. Such cell culture systems in which polypeptides of the invention are expressed may be used in production of phenylacetyl 7-ADC A. All biochemicals, reagents and oligonucleotides were obtained either from from Sigma-Aldrich Chemicals Pvt. Ltd or USB, USA. Restriction enzymes and strains were purchased from New England Biolabs Inc, USA. pET24a vector and bugbuster reagent were purchased from Novagen, USA. Streptomyces clavuligerus and Penicillium chrysogenum strains were obtained from ATCC. Cloning of expandase gene Streptomyces clavuligerus (ATCC 27064) culture was grown in 50 ml of YMG medium (Yeast Extract 4g, Malt Extract lOg, Glucose 4g per litre (pH7.0) in a 250 ml conical flask at 26°C in a rotary shaker at 180 rpm until the O.D at 600 nm reached 3.00. The culture was centrifuged at 13000 rpm for 10 minutes at 20°C to harvest the cell pellet. The cell pellet was resuspended in TE buffer pH 8.0 (1/10* of the culture volume) containing 10 mg lysozyme (200 [il of 50 mg/ml stock) and the mixture was incubated at 30°C for 30 min followed by the addition of 1 ml of 10% SDS, 5 ml of phenol saturated with Tris-HCl (pH 8.0) and 750 |il of 5M NaCl. The tubes containing the mixture were inverted gently for few times and kept at room temperature for 20 minutes. The suspension was centrifuged at 16,000 rpm for 10 minutes at 20°C to separate the phases. After transferring the aqueous layer to a fresh centrifuge tube, two volumes of Isopropyl alcohol was added and the tubes were inverted gently for few times and the resulting mixture was left at room temperature for 10 minutes. It was centrifuged again at 16,000 rpm for 10 minutes at 20°C and the pellet was resuspended in TE buffer pH 8,0. After dissolving the DNA, RnaseA was added to a final concentration of 20 |ig/ml and the suspension was incubated at 50°C for 1 hr. Subsequently, Proteinase K was added to a final concentration of 200 |ig/ml followed by 100 mM NaCl and 0.4% SDS and the mixture was incubated at 37°C for 1 hr. The suspension was again extracted with equal volume of phenol, centrifuged at 16000 rpm for 10 minutes at 20°C and the aqueous layer was transferred to a fresh tube. The mixture was extracted similarly with chloroform and the aqueous layer was treated with Isopropanol and left at -20*^C for one hour. The DNA was precipitated by centrifuging at 13,000 rpm for 10 minutes at 20°C and the pellet was resuspended in 50 |al TE (pH 8). Expandase gene was amplified from Streptomyces clavuligerus (ATCC 27064) genomic DNA isolated as described above using the oligonuecotides (5' G AGC AT ATGG AC ACGACGGTGCCC3', 5'GATTGCTGCTGTGACCATGACGGT3') in a reaction volume of 100 \i\ containing IX Vent DNA polymerae buffer, 10% DMSO, 1 mM MgS04, 2.5 units of Deep Vent DNA polymerase with an oil overlay of 50 |J,1. The amplification process consisted a cycle of 5 minutes incubation at 95^C, 25 cycles of 40 seconds incubation at 95^C for denaturation, 30 seconds incubation at 60^C for annealing and 5 minutes incubation for extension at ITC followed by a final cycle of extension at 72^C for 15 minutes. The fragment resulting from amplification was purified and cloned into Smal restricted pUC 19 vector. The sequence of the gene was further verified by sequencing. Generation of mutants Expandase gene was mutagenised using error prone polymerase chain reaction by biasing the nucleotide concentration using oligonuecotides 5' ATCGGTGCGGGCCTCTTCGCTATT3', 5'CTCACTCATTAGGCACCCCAGGCT3' in a reaction volume of 100 |il containing IX Taq DNA polymerase buffer, 10%DMSO, \0 ng of template and 3 units ofTaq DNA polymerase. The amplification process was carried out as described for cloning of expandase gene. The fragment was further purified, digested with Ndel and BamHi and cloned into similarly digested pET24a by standard molecular biology procedures. In some cases, mutant templates were used to generate additional mutants. Expression of expandase mutants Single colony of BL21(DE3) harbouring putative mutant constructs were inoculated in 96 well culture plates containing LB supplemented with antibiotics and grown at 37°C and 220 rpm. When the optical density reached between 0.6-0.8, IPTG was added to induce the expression and cultured further for 3 hours at 25°C, Subsequently, the plates were centrifuged at 4000 rpm in a microplate centrifuge and the pellet was resuspended in buffer containing 50 mM Tris.HCL (pH7.5), 1 mM Dithiothreitol, 0.01 mM EDTA, 10% Glycerol and 50 mM Glucose and stored at -SO'^C. Assay of expandase mutants Expression isolates were thawed in ice and treated with 100 \i\ of Bugbuster reagent at 25'^C for 10 minutes to promote lysis of the bacteria. The expandase assay was started by adding 30 |il of freshly prepared lOX mix and 30 |il of 100 mM Penicillin G substrate to the wells, mixed nicely, covered with breathseal and incubated at 25°C for 30 minutes in a shaker. The final concentrations of the constituents in the mix were 50 mM ammonium sulphate, 1 mM a-ketoglutaric acid, 50 |iM ascorbate, 2 mM dithiothreitol, 2 mM FeS04 and 10 mM Penicillin G. The reaction was quenched by adding 150 [d of CH3OHandl50ialofH2O. Primary screening of expandase mutants 25 |il of the assay mix was loaded into blank paper discs, allowed to dry and placed on LB plates containing penicillinase spread with E. coli ESS (kindly provided by Professor S. E. Jensen, University of Alberta, Canada). The plates were incubated at 37*^C for ovemight and the clones with larger zone of inhibition than native expandase was short-listed for further quantification and confirmation by sequencing. Quantification of expandase activity using HPLC Assay samples were centrifuged for 30 minutes at 4*^C and 20 |il of it was injected and the elution profile was monitored in a CI 8 column using a mixture of methanol and phosphate buffer by HPLC. The conversion of Penicillin G into Cephalosporin G was quantified using Cephalosporin G as a standard and the relative activity levels for few of the expandase mutants are indicated in Table 1. We claim : 1. A mutated penicillin expandase comprising an amino acid substitution at one or more residue positions corresponding to those in a wild-type expandase selected from the group consisting of threonine at position 42, isoleucine at position 50, histidine at position 57, threonine at position 67, valine at position 133, threonine at position 143, proline at position 145, glycine at position 148, phenyl alanine at position 152, proline at position 196, alanine at position 240, cysteine at position 281, Serine at position 309, provided that the amino acid substitution at the residue position of cysteine at position 281 is not tyrosine 2. The mutated penicillin expandase of claim 1, wherein the wild-type expandase is obtained from Streptomyces clavuligerus. 3. The mutated penicillin expandase of claim 1, comprising an amino acid substitution at one or more residue positions selected from the group consisting of T42A, I50V, H57R, T67A, V133I, T143S, P145L, G148E, F152L, P196S, A240T, C281R and S309P. 4. The mutated penicillin expandase of claim 3, comprising the amino acid substitutions of V133I and P196S; F152L and C281R; T42A, F152L and S309P; V133I, T143S, P196S and S309P. 5. A modified expandase gene encoding the expandase mutation as claimed in claim 1. Dated this twenty first (21'^) day of April 2004 for Orchid Chemicals & Pharmaceuticals Ltd.,

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