WO2006058942A2

WO2006058942A2 - Method of producing 6-aminopenicillanic acid using the aculeacin a acylase enzyme of actinoplanes utahensis, which is purified from recombinant bacteria streptomyces lividans cect 3377

Info

Publication number: WO2006058942A2
Application number: PCT/ES2005/000643
Authority: WO
Inventors: Isabel De La Mata Riesco; Jesús TORRES BACETE; Daniel Hormigo Cisneros; Maribel STUART LEÓN; Miguel ARROYO SÁNCHEZ
Original assignee: Universidad Complutense De Madrid Rectorado
Priority date: 2004-11-26
Filing date: 2005-11-25
Publication date: 2006-06-08
Also published as: WO2006058942A3; ES2304191B2; WO2006058942B1; ES2304191A1

Abstract

The invention relates to a method of producing 6-aminopenicillanic acid using the aculeacin A acylase enzyme of Actinoplanes utahensis, which is purified from recombinant bacteria Streptomyces lividans CECT 3377. More specifically, the invention relates to a method of producing 6-aminopenicillanic acid, comprising the hydrolysis of penicillins V, K, F or dihydroF, using the aculeacin A acylase enzyme of bacteria Actinoplanes utahensis which is purified from broth cultures of cells of recombinant bacteria Streptomyces lividans CECT 3367 which carry the aac gene that encodes said enzyme.

Description

TITLE

Procedure for producing 6-aminopenicillanic acid using the enzyme acleasa aclease aclease actase actase, purified from the recombinant bacterium Streptomyces lividans CECT 3377

SECTOR OF THE TECHNIQUE

The present invention falls within the field of enzymatic biotechnology. More specifically in obtaining by enzymatic methods 6-aminopenicillanic acid, core of β-lactam antibiotics called penicillins, using as natural substrates penicillins, and more specifically from penicillins V, K, F and dihydroF.

STATE OF THE TECHNIQUE

Acylases are enzymes widely used in the industrial production of semisynthetic antibiotics and antifungal agents. In the production of semi-synthetic penicillins the starting product is acid

6-aminopenicillanic (hereinafter 6-APA) which is obtained by hydrolysis of penicillin G with penicillin G acylase (hereinafter PGA) or penicillin V with penicillin V acylase (hereinafter PVA). Penicillins G and V are produced industrially by fermentation processes in which other natural aliphatic penicillins (penicillins K, F and dihydro F) are also synthesized, which represent about 1-2% of the total penicillin in the broths. Penicillin acylases (PGAs or PVAs) used industrially are not capable of hydrolyzing these aliphatic penicillins whose presence hinders the crystallization of 6-APA, both of which imply a lower yield in obtaining 6-APA, which causes significant economic losses. Currently the only penicillin acylase described capable of hydrolyzing these aliphatic penicillins effectively is the PVA of Streptomyces lavendulae ATCC 13664 (Hamilton-Miller JMT (1966) Bacteriol. Rev. 30, 761-771; Torres R. et al. (1999) Appl. Microbiol Biotechnol 53, 81-84; Torres-Guzmán R. et al. (2002) Biochem. Biophys Res. Comm. 291, 593-597.

Kleinschmidt WJ. et al. in 1964 they described that the bacterium Actinoplanes utahensis ATCC 14539 was able to produce 6-APA by hydrolysis of penicillins produced by filamentous fungal cells of the genus Penicillium

(US Patent 3150059). However, the enzyme or enzymes responsible for hydrolysis and the genes that encode them were neither isolated nor characterized.

Subsequently, Actinoplanes utahensis ATCC 14539 has been used for the production of semisynthetic antifungal antibiotics, where cyclic lipopeptides (hereinafter echinocandins) are hydrolyzed giving rise to a cyclic hexapeptide, starting nucleus for obtaining new semi-synthetic echinocandins with improved properties (US Patent 4293482, EP0031221, US Patent 4482487, 4524135, Boeck, LD et al. (1988) J. Antibiot. 41,1085-1092; Boeck, LD et al (1989) J. Antibiot. 42, 382-388; Debono, M . (1989) J. Antibiot. 42, 389-397). In this process of hydrolysis of lipopeptides, the enzyme aquleacin A acylase (hereinafter AAC) used by A. utahensis is used. This is an extracellular enzyme that catalyzes the hydrolysis of aculeacin A and other related echinocandins. The AAC of A. utahensis has been isolated, purified and characterized (Takeshima H. et al. (1989) J. Biochem. 105, 606-610). Likewise, the aac gene that codes for the AAC of A. utahensis has been cloned, sequenced and expressed in Streptomyces spp. (Inokoshi J. et al. (1992) Gene 119, 29-35; Liokoshi J. et al. (1993) Appl. Microbiol. Biotechnol. 39, 532-536). It is a heterodimeric enzyme, with a small subunit (hereinafter subunit α) of 19 kDa and a large subunit (hereinafter subunit β) of 55 kDa. Although its structure resembles that of penicillin G acylases and that of glutaryl acylases that hydrolyze derivatives of phenylacetic and glutaric acids, respectively, the enzyme does not use these derivatives as substrates, so it seemed that the function of this enzyme was limited to the hydrolysis of the aforementioned lipopeptides and therefore their activity and utility for the hydrolysis of penicillins had not been described. DESCRIPTION OF THE INVENTION

For the realization of this invention, the Gram positive bacterium Actinoplanes utahensis NRRL 12052 has been used, which corresponds to Actinoplanes utahensis ATCC 14539 as a donor of the deoxyribonucleic acid (hereinafter referred to as DNA) producing the AAC enzyme.

Based on the known sequence of the aac gene encoding the AAC enzyme from A. utahensis, synthetic oligonucleotides were designed to amplify the aac gene by the amplification process known as the polymerase chain reaction (hereinafter referred to as PCR) using the genomic DNA of A. utahensis NRRL 12052 as a template. Escherichia ribosome binding sequences were included in these synthetic oligonucleotides. coli (RBS), ATG as the translation initiation codon, since GTG is described as an A. utahensis aac gene initiation codon, and a translation termination codon, as well as various restriction sites useful for subsequent cloning . A DNA fragment with the gene sequence encoding the CAA was thus obtained including the sequence encoding the signal peptide, which was cloned into a bifunctional plasmid vector of Escherichia coli and Streptomyces lividans carrying two ampicillin resistance markers, for its selection in E. coli, and to thioestreptone, for its selection in S. lividans. Said construction was verified by sequencing and then the transformation of the Streptomyces lividans 1326 strain was carried out. Recombinant clones were obtained constitutively expressing the aac gene under the control of the erythromycin gene promoter. One such clone of S. lividans that contained the plasmid vector carrying the aac gene and that produced the AAC of A. utahensis was deposited in the Spanish Type Culture Collection (CECT), located at the University of Valencia, Research Building, Campus de Burjassot, 46100 Burjassot, Valencia, Spain, with n ° 3377. For the production of the CAA using the previously selected CECT 3377 recombinant clone, the organism was cultured in TSB (Soy triptone, sodium chloride, glucose and bipotassium phosphate) at 30 ⁰ C. The recombinant microorganism was stabilized by the addition of thioestreptone to the culture medium, antibiotic for which the vector containing the aac gene confers resistance. The presence of the antibiotic, in addition to conferring production stability, prevents contamination of the culture medium by other unwanted microorganisms. The CAA produced by S. lividans CECT 3377 is extracellular, obtained from the culture broth by separation of the cells by centrifugation. The cell-free extract has an AAC with a high degree of purity, and the AAC enzyme can be purified to homogeneity by a simple procedure using conventional chromatographic techniques.

Once the pure CAA was obtained, its activity against penicillins V, K, F and dihydro F was determined, finding that it hydrolyzed said compounds and determining the kinetic parameters of the enzyme with each of them.

The novelty of this invention lies in the fact that it is the first time that the penicillin acylase activity of the acyleain A acylase enzyme of Actinoplanes utahensis NRRL 12052 (ATCC 14539), described as hexacyclylpolipopeptidyl acylase, is described, which provides added value to the use of this enzyme on an industrial scale for its application in the production of 6- aminopenicillanic acid from the hydrolysis of penicillins V, K, F or dihydroF, which has a great industrial value.

BRIEF DESCRIPTION OF THE FIGURES

The invention is illustrated by the following figure:

Figure 1. Obtaining 6- APA from penicillins V, K, dihydroF and F using the CAA produced by S. lividans CECT 3377, represented in μmoles of 6-APA per minute and milligram of enzyme. EMBODIMENT OF THE INVENTION

The present invention will be illustrated in more detail in the following examples:

Example 1: Determination of penicillin V acylase activity of fermentation broths of Actinoplanes utahensis NRRL 12052 (ATCC 14539)

Actinoplanes utahensis NRRL 12052, which corresponds to Actinoplanes utahensis ATCC 14539 was cultured as described (Takeshima H. et al. (1989) J.

Biochem 105, 606-610). In the cell-free fermentation broths after centrifugation the determination of penicillin V acylase activity was carried out using penicillin V (30 mM) as a substrate, according to a previously described protocol (Torres et al. (1998) Progess in Biotechnology 15: Stability and Stabilization of Biocatalysts, A. Ballesteros, FJ Plou, JL Iborra and PI Halling Eds.,

Elservier, Amsterdam, pp 719-724). The incubation is carried out in 100 mM potassium phosphate buffer, pH 8, for 20 minutes at 40 ⁰ C. The reaction is stopped by adding 400 μl of 50 mM sodium acetate, pH 4.5 and, after centrifugation, the release of 6 -APA is spectrophotometrically assessed by reaction with p-dimethylaminobenzaldehyde (PDAB) according to the specifications of Balasingham K. et al. (1972) (Balasingham K. et α /. (1972) Biochim. Biophys. Acta 276, 250-256).

Example 2: PCR cloning of the aac gene

1. Preparation of Actinoplanes utahensis NRJRL 12052 DNA (ATCC 14539)

The strain of A. utahensis NRRL 12052 (ATCC 14539) was grown in medium

YEME (yeast extract (0.3%), bactopeptone (0.5%), malt extract (0.3%), glucose (1%), MgCl ₂ 5mM), supplemented with sucrose (34%) and glycine (0.5%). The incubation was carried out for 18 hours at 300 rpm and 30 ⁰ C. Next, the mycelium was collected by centrifugation at 3000 xg for 15 minutes, washed with sucrose at 10% and then cell lysis was carried out by treatment with lysozyme. Finally, the DNA was purified following a previously described procedure (Hopwood DA et al. (1985) Genetic Manipulation of Streptomyces. A laboratory Manual. The John Innes Foundation, Norwich, England). To achieve greater DNA purity, it was treated with RNase, extracted several times with neutral phenol and chloroform-isoamyl alcohol 24: 1 (CIA) and subsequently precipitated with isopropanol. After washing with 70% ethanol and 100% ethanol, the DNA was dissolved in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA).

2. Preparation of the bifunctional vector pEM4 and competent E. coli cells for the cloning of the acin gene of Actinoplanes utahensis

The plasmid vector pEM4, bifunctional with origin of replication in E. coli and S. lividans, contains resistance markers for ampicillin and thioestreptone, for selection in E. coli and S. lividans respectively (Quirós LM et al. (1998) Mol. Microbiol. 28,1177-1186). Said vector was prepared according to the conditions described: E. coli strains, which individually possessed the aforementioned plasmid, were incubated for 16 hours with stirring on an orbital shaker at 250 rpm and at 37 ⁰ C in 0.05 liters of LB medium (Sambrook et al. (1989) Molecular Cloning: A laboratory Manual, CoId Spring Harbor, New York, USA). After the time the cells settled and the plasmids were extracted and purified by the High Puré Plasmid Isolation Kit (Roche) procedure following the protocol provided by the supplier. The strain used for cloning was E. coli DH5α. The RbCl procedure was used to obtain the competent cells (Sambrook et al. (1989) Molecular Cloning: A laboratory Manual, CoId Spring Harbor, New York, USA).

3. Collection by PCR of the DNA fragment containing the aac gene of A. utahensis From the known amino terminal sequences (üiokoshi J. et al. (1992) Gene 119, 29-35) and taking into account the use of codons in Streptomyces the synthetic oligonucleotides described in SEQ ID NO: 1 and 2 were designed.

To carry out the amplification reaction by PCR, 16 μl (5 μM) of each of the oligonucleotides synthesized with 0.5 μg of the genomic DNA of A. utahensis, obtained as described in section 1 of example 2, were mixed. 2.5 units of the Pfu DNA polymerase enzyme (Promega) were added to the mixture together with the appropriate buffer recommended by the suppliers. The final volume of each reaction was 0.1 ml containing 5% dimethylsulfoxide (DMSO). The preparation was subjected to an amplification process in an Eppendorf Mastercycler Gradient thermal cycler under the following conditions: 96 ⁰ C (2 minutes) then 5 cycles, each cycle being 96 ⁰ C (1 minute), 70 ⁰ C (2 minutes) and 72 ⁰ C (8 minutes); then another 5 cycles, each cycle being 96 ⁰ C (1 minute), 68 ⁰ C (2 minutes) and 72 ⁰ C (8 minutes); 5 cycles, each cycle being 96 ⁰ C (1 minute), 63 ⁰ C (2 minutes) and 72 ⁰ C (8 minutes); and finally 20 cycles, each cycle being 96 ⁰ C (1 minute), 60 ⁰ C (2 minutes) and 72 ⁰ C (8 minutes). The result of the amplification was visualized by staining ethidium bromide after an electrophoresis in a 1% agarose gel.

The DNA fragment obtained by PCR with an approximate size of 2.3 kb was purified by extraction of the agarose gel by GeneClean from BiolOl (Inc.) following the recommended protocol.

4. Cloning and sequencing of the fragment obtained by PCR containing the aac gene

1 μg of the purified DNA fragment resulting from the PCR procedure was digested simultaneously with the restriction enzymes Xbal and EcoRI (Roche), since the oligonucleotides were designed with Xbal targets for the 5 'end and EcoRI for the 3' end. The digestions were carried out in a buffer recommended by the suppliers and compatible for both enzymes, at 37 ⁰ C for three hours, after which the reaction was stopped by heating the mixture for 15 minutes at 85 ⁰ C. The digested fragment was incubated with a sample of 0.5 ug DNA plasmid pEM4 bifunctional digested with XbaI and EcoRI enzymes in same conditions as the fragment. The DNA fragment was ligated to the plasmid DNA by the enzyme T4 DNA ligase (USB) in the presence of ATP using a buffer recommended by the suppliers.

The resulting ligation mixture was used to transform competent E. coli DH5α cells. The transformants were selected in solid LB medium to which ampicillin had been added (100 μg / ml). Through this procedure several clones were obtained which were analyzed by electrophoresis in 1% agarose gels of the purified plasmids. For this, the colonies of E. coli DH5α transformed in liquid LB medium were grown at 37 ⁰ C for 16 hours and with stirring at 250 rpm, then the plasmids were extracted and purified by the High Puré Plasmid Isolation Kit (Roche) procedure. following the protocol provided by the supplier. One of these clones that contained the recombinant plasmid carrying the aac gene fragment was identified as pAACAU.

The DNA fragment contained in plasmid pAACAU was sequenced at both ends of the fragment by automatic sequencing in an ABI PRISM 3700 unit in the Automatic DNA Sequencing Service (SSAD) of the Biological Research Center (CIB) of the Higher Council for Scientific Research (CSIC). Plasmid sequence analysis found that the cloned DNA fragment contained the aac gene from the gene sequence encoding the signal peptide to the translation termination codon, the gene sequence encoding the RBS of E. coli and ATG as initiation codon.

Example 3: Production of the AAC of A. utahensis in S. lividans.

1. Preparation of competent S. lividans cells for expression of the aac gene The strain used for cloning and expression of the DNA fragment that carries the aac gene was S. lividans 1326. Obtaining recombinant cells was carried out by transforming protoplasts, which were obtained following the procedure described (Hopwood DA. et al. (1985) Genetic Manipulation of S .. A laboratory Manual. The John Innes Foundation, Norwich, England)

2. Obtaining a recombinant strain of S. lividans producing the CAA of A. utahensis

Plasmid pAACAU (2 μg), purified from a pure culture of a recombinant strain of E. coli DH5α, as previously described, was used to transform S. lividans 1326 protoplasts following the method described (Hopwood DA. et al. (1985) Genetic Manipulation of S .. A laboratory Manual. The John Innes Foundation, Norwich, England). Transformants were plated on R2YE solid medium and incubated at 30 ⁰ C for 24 hours, then thiostrepton (5 ug / ml) was added in order to select cells carrying the recombinant plasmid pAACAU. Numerous clones were obtained that were analyzed by electrophoresis in 1% agarose gels of the purified plasmids, selecting one of them named S. lividans pAACAU and deposited in the Spanish Type Culture Collection (CECT), with the No. 3377.

3. Production of the AAC of A. utahensis in S. lividans CECT 3377

The S. lividans CECT 3377 strain was fermented in TSB medium for 140 hours at

30 ⁰ C and 250 rpm Aliquots of the culture broths were taken every 24 hours and, after centrifugation at 3000 xg for 30 minutes at 4 ⁰ C ₅ , the AAC activity was determined both in the supernatant (cell-free extract ) as in the sediment (cell extract). In the latter case, a previous rupture of the cells was performed by sonication. The activity test was carried out using penicillin V as a substrate, as described in example 1. AAC activity was only detected in the supernatant, where the maximum occurs at 96 hours of culture.

Example 4: Purification of the AAC from A. utahensis expressed in S. lividans CECT 3377

Strain CECT 3377 lividans in TSB medium was fermented for 72 hours at 30 ⁰ C and 250 rpm agitation. The culture broths were then centrifuged at 3000 xg for 30 minutes at 4 ⁰ C. With the cell-free supernatant obtained, the AAC was purified. To that end, the cell-free production broths were adjusted to pH 6 and subjected to cation exchange chromatography by application on a Pharmacia S-Sepharose Fast Flow column (2.6 x 28 cm), buffer-balanced. 10 mM sodium phosphate, pH 6 and connected to a Pharmacia FPLC system. Under these conditions the AAC was retained in the column and eluted by applying a discontinuous gradient of 0 to 1.5 M NaCl in the same buffer. The set of fractions with AAC activity were concentrated in a bed of 35,000 polyethylene glycol (Fluka) and subjected to a penetrability chromatography in Superóse 12 Fast Flow (I x 30 cm) from Pharmacia, equilibrated in 50 mM potassium phosphate buffer, pH 7 and connected to a Pharmacia FPLC system. After this last chromatographic step, the CAA was obtained pure to homogeneity.

Example 5: Determination and characterization of the penicillin acylase activity of the AAC of A. utahensis produced by S. lividans CECT 3377 on penicillins V, K, F and dihydro F.

The pure CAA was functionally characterized by determining the stability and activity of the enzyme against pH, temperature and ionic strength, using penicillin

V (30 mM) as substrate. The release of 6-APA after the reaction was assessed spectrophotometrically by reaction with fluorescamine (fluorescamine method) according to the specifications of Udenfriend et al. (1972) modified by Reyes et al. (1989) (Udenfiiend S. et al. (1972) Science, 178, 871-872; Reyes F. et al. (1989) J. Pharma. Pharmacol., 41, 136-137).

Regarding the temperature, the enzyme is stable up to 50 ° C, presenting a maximum activity at 75 ⁰ C. Regarding the pH, the enzyme is stable between pH 6 and 10, 8.2 being its optimum pH. The optimal reaction conditions have been established at pH 8.2 and 45 ° C temperature.

After determining the optimal activity conditions of the CAA produced by S. lividans CECT 3377, the ability of the enzyme to produce 6- APA from penicillins V, K, F and dihydro F was characterized, determining the kinetic parameters of the enzyme for each of the penicillins. For this purpose, activity tests were carried out using as a substrate penicillin V, penicillin K, penicillin F or penicillin dihydro F at different concentrations in 100 mM potassium phosphate buffer pH 8.2 for 20 minutes at 45 ° C, according to the protocol described in example 1. The release of 6- APA was spectrophotometrically assessed by the fluorescamine method described in this example. As described in Figure 1, the AAC cloned and expressed in S. lividans is capable of generating 6- APA from penicillins V, K, F and dihydro F, being collected in table 1 the kinetic parameters of the enzyme for Each of the penicillins. As can be seen in this table, the AAC of A. utahensis produced by S. lividans CECT 3377, presents greater catalytic efficacy against penicillin K.

Table 1: Kinetic parameters of the A. utahensis CAA produced by S. lividans CECT 3377 against different penicillins

Claims

1. Procedure for producing 6-aminopenicillanic acid from penicillin using the acyleacin A acylase enzyme of Actinoplanes utahensis purified from the recombinant bacterium Streptomyces lividans CECT 3377, characterized by the following operations:

a) culturing cells of Streptomyces lividans recombinant bacteria CECT 3377 aculeacin A acylase producers in defined medium TSB during at the least 96 hours at 30 ⁰ C and 250 rpm agitation. b) Obtain cell-free production broths by centrifugation at 3000 xg for 30 minutes at 4 ⁰ C and adjust them to pH 6. c) Subject the adjusted broths to a cation exchange chromatography by application on a balanced S-Sepharose column in 10 mM sodium phosphate buffer, pH 6 and elute the retained enzyme by a discontinuous gradient of 0 to 1.5 M NaCl in the same buffer. d) Concentrate the set of fractions with aqualeacin A acylase activity in a polyethylene glycol bed 35,000. e) Submit the set of fractions with concentrated acylase A acylase activity to a penetrability chromatography by applying it on a Superóse 12 column and isolating the most active fractions, f) Producing 6-aminopenicillanic acid hydrolyzing penicillin with the active fractions of the CAA

2. Process for producing 6-aminopenicillanic acid, according to claim 1, from penicillin V.

3. Process for producing 6-aminopenicillanic acid, according to claim 1, from penicillin K.

4. Process for producing 6-aminopenicillanic acid, according to claim 1, from penicillin F.

5. Process for producing 6-aminopenicillanic acid, according to claim 1, from penicillin dihydro F.