Patent Application
20100009405
The invention relates to a process for the biosynthesis and production of gallidermin, a modified strain for producing an in-active pre-form of gallidermin and, to the use thereof.
Many Gram-positive bacteria produce peptide antibiotics called lantibiotics (lanthionine-containing antibiotics) which contain lanthionine, an unusual amino-acid added to the precursor peptide post-translationally (Jack et al., 1998). Gram-negative bacteria also produce similar molecules, bacteriocins, but these are larger proteins that act on target receptors and have a narrow spectrum of activity.
Lantibiotics are small (20-40 amino-acids) cationic peptides whose synthesis starts with a ribosomally synthesized pre-pro-protein, which undergoes posttranslational modification to yield the lanthionine containing but still in-active pre-form of the antibiotic, which is then exported from the cell and either during or after export activated by proteolytic cleavage by lantibiotic specific serine-protease. The producer strains possess so called immunity functions that protect to some extent against the action of the lantibiotic but which does not confer resistance (Jack et al., 1998; Hille et al., 2001). Lantibiotics are expressed during exponential and into early-stationary growth-phases meaning that they reduce competition from other flora once they themselves have become established.
Several classes of lantibiotics have been described that group the molecules according to their structure in solution (Jack et al., 1998). Type-A lantibiotics, damage the bacterial cells by disruption of anionic membranes to form wedge-like pores driven by voltage differences across membranes. This allows efflux of metabolites, ions and solutes from the bacterial cell. In addition, type-A lantibiotics bind to Lipid II, an essential precursor (carrier) for membrane building blocks. Depending on the size of the lantibiotic, two modes of killing are currently discussed (Bonelli et al. 2006, Breukink & Kruijff 2006).
Members of the typ-A lantibiotic group include cytolysin L1 and L2 from Enterococcus faecalis, epidermin, epilancins and Pep5 from Staphylococcus epidermidis, gallidermin from Staphylococcus gallinarum, lacticin 481, mutacins from Streptococcus mutans, nisin A and Z from Lactococcus lactis, salavaricin A from Streptococcus salivarius, subtilin from Bacillus subtilis, and variacin from Micrococcus varians.
Despite of being of clinical value no type-A lantibiotic have been developed into a clinical products, even though several have proven their potential to treat peptic ulcers (nisin, H. pylori, Clostridium difficile; CA2291645), MRSA protection in mice (mersacidine, B. subtilis) and others (Jack et al.,1998). Lysostaphin is being developed as a wound-anti-infective agent and in the treatment of Staphylococcus aureus infections. Mucatcins (Streptococcus mutans) are being evaluated for mouth wash and tooth paste applications. Nisin is currently the only marketed product used for food preservation (de Vuyst & Vandamme, 1993; U.S. Pat. No. 4,584,199; U.S. Pat. No. 4,597,972; U.S. Pat. No. 4,716,115).
Gallidermin is a molecule of particular interest as it possibly provides a treatment for acne or wound infection or endocarditis in human or for bovine mastitis (gallidermin, S. gallinarum; U.S. Pat. No. 5,710,124),
The major obstacle to an industrial scale production of pharmaceutical grade gallidermin or any lantibiotic is the toxicity of the lantibiotic molecules to the producing strain, resulting in the very low product titres in the fermentation broth and in the rather complex experimental processes with on-line product recovery (Kempf et al., 2001). Also feed-back inhibition by the product can prevent high level expression.
Any process to overcome this limitation in production of gallidermin would have valuable therapeutic potential enabling the economic production and further development of this compound or similar compounds in to therapeutic drugs that are urgently needed to combat the growing threat of infections by multidrug-resistant pathogenic bacteria.
According to the invention there is provided a process for the preparation of an inactive pre-forms of gallidermin comprising the steps of:
The biosynthetic gene cluster of gallidermin producing organism may be genetically Modified by any one or more of:
In one embodiment of the invention the organism is cultivated in an aqueous cultivation medium containing assimilable sources of carbon, nitrogen and inorganic substances until substantial growth and metabolic activity is detectable.
In one embodiment of the invention the inactive form of gallidermin is isolated by recovery from the cultivation medium by first separation of the biomass by centrifugation or filtration followed by a hydrophobic interaction chromatography.
In one embodiment of the invention the organism is selected from any one or more of Staphylococcus gallinarium, or any recombinant microbial organism expressing the genes for the production and secretion of pre-gallidermin or any pre-gallidermin or derivative thereof.
In another embodiment of the invention the inactive form of gallidermin is biocatalytically activated to yield a mature and active form of gallidermin.
In one embodiment of the invention the inactive form of gallidermin is activated by a gallidermin specific protease. The gallidermin specific protease may be selected from any one or more of ArgC, bromelain and trypsin.
In one embodiment of the invention the active form of gallidermin is separated from the protease and the cleaved off leader and unprocessed pregallidermin by chromatography.
In one embodiment of the invention the isolated preform comprises a truncated amino acid sequence of VNAKESNDSGAEPR (SEQ ID No. 1).
In another embodiment of the invention the isolated preform comprises a truncated amino acid sequence AKESNDSGAEPR (SEQ ID No. 2) or any other N-terminal truncated pre-forms.
The invention also provides an organism capable of producing gallidermin which has been genetically modified to eliminate and/or inactivate gallidermin specific protease(s).
The invention also provides a strain of Staphlylococcus gallinarum wherein a gallidermin specific serine protease, GdmP is inactivated.
The invention further provides modified strain of Staphlylococcus gallinarum, Staphylococcus gallinarum ΔgdmP::kan deposited with the Deutsche Sammlung von Microorganismen having a depository number of DSM 17239.
The invention also provides an isolated inactive pre-form of gallidermin and isolated active gallidermin as prepared by a process of the invention.
The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only, with reference to the accompanying drawings, in which:
We have found a new and efficient process for the production of the type-A lantibiotic gallidermin. Due to the product toxicity, including toxicity to the producing bacterium, gallidermin currently lacks a cost efficient high yielding production process for drug grade material. The process of the invention comprises a fermentative production of the respective biologically in-active and non-toxic pre-forms of gallidermin by either genetically engineered producer strains or mutants of the natural producer strains that are deficient for the extracelluar gallidermin pathway specific serine-protease. Alternatively, fermentation conditions may be selected that specifically inhibit the serine protease. The process comprises a downstream process in which the pre-form gallidermin is isolated from the culture medium and subsequently bio-catalytically activated. Following this activation the mature and active gallidermin is isoloated.
The pre-gallidermin is isolated from the fermentation medium and a bio-catalytic step is carried out using commercially available proteases to process the pre-gallidermin into the mature and active gallidermin. The mature gallidermin may then be purified to yield a dry preparation.
The process provides a spatially separated production process for the preparation of active gallidermin. Throughout the following examples a native inactive pre-form of gallidermin is made by a genetically modified microbe, alternatively, an engineered inactive pre-form of gallidermin may be made, that is isolated and then in vitro activated by simple enzymatic cleavage.
Bacterial hosts and fermentation processes are described that predominantly yield pre-forms of gallidermin. A process for the purification of the pre-forms of gallidermin is described. Also processes are described using specific serine proteases that have the desired specificity to proteolytically cleave the pre-forms of gallidermin into their respective mature and active form. Purification of the activated gallidermin is also described.
A genetically modified strain of special interest is Staphylococcus gallinarum BC001 which has been deposited with the Deutsche Sammlung von Microorganismen, Mascheroder Weg 1B, 38124 Braunschweig, Germany under the terms of the Budapest Treaty on 13. April 2005 having a depository number of DSM 17239.
Gallidermin also binds to the membrane lipid II inhibiting the cell wall biosynthesis andinducing autolysis of the cells. Interestingly an in contrast to other, larger type-A lantibiotics, gallidermin is too short to span the membrane and to induce pores nevertheless, it acts on growing and non-growing cells very effectively (Bonelli et al., 2006; Breukink & Kruijff, 2006; Sahl & Bierbaum, 1998).
Gallidermin also inhibits cell wall biosynthesis by binding at the cytoplamic precursor, Lipid I, which would imply a membrane crossing of gallidermin. In contrast to vancomycin that recognizes the D-ala-D-alanyl residues of the peptide subunit of the peptidoglycan, gallidermin binds to the disaccharide moiety of the glycan chain that has been studied in details for nisin, a molecule that shares the respective features, a pyrophosphate cage structure, with gallidermin (Breukink & Kruijff, 2006). Gram-negative cells can also be sensitized by disrupting the outer membrane integrity (with e.g. EDTA in vitro). Furthermore, as the producing organism does not possess a lantibiotic resistance gene (and in the new process will not be forced to develop one, neither is one introduced), we can ensure that any gallidermin preparation will be free of any DNA from which infectious bacteria could acquire lantibiotic resistance.
Despite the chemical similarities among the type-A lantibiotics, distinct differences in their mode of action and their primary targets have been recognized that are also reflected in different spectrum of sensitive bacteria and by different minimal inhibitory concentrations (MIC) needed to exhibit their antibiotic activitiy (U.S. Pat. No. 5,231,013; Kellner et al., 1988).
Mammalian cells are spared because they contain cholesterol which is less anionic.
The genetics and the biosynthesis of these lantibiotics has been described in numerous publications (for review: Jack et al., 1998; Sahl & Bierbaum, 1998; McAuliffe, et al. 2001). In particular, gallidermin has been described in detail in U.S. Pat. No. 5,231,013. In general, a ribosomally produced pre-pro-peptide of the antibiotic is synthesized. This in-active pre-pro-peptide is comprised of a signal of leader peptide needed for export and the polypeptide chain that does not yet contain any dehydroamino residues and/or thioester bridges, typical for lantibiotics. The first processing of a pre-sequence polypeptide is carried out by an enzymatic complex which effects formation of dehydroamino residues and/or thioether bridges. This modified but still in-active so called pre-peptide is then exported by a dedicated lantibiotic transporter and further modified by proteolytic cleavage of the leader peptide. Both export and proteolytic functionality are encoded within the biosynthetic gene cluster. Since the active molecule exhibits toxicity to the producing organism, immunity or tolerance against these compounds is achieved by so called immunity genes found in the biosynthetic gene cluster as well. Nevertheless, immunity, the energy dependent clearance of the membrane from lantibiotics is limited to low levels, hence limiting biosynthesis and production of the lantibiotic. All genes encoding the enzymes required for these functions are encoded within the respective biosynthetic gene clusters (FIG. 1A/B). Increased tolerance to lantibiotics may also derive from changes in the membrane composition that reduce the membrane fluidity.
The major drawback for the clinical development and commercialisation of gallidermin are the high costs of production due to low fermentation titres, a result of product toxicity for the producing bacterium and in some cases negative feed-back regulation of the biosynthesis. A second problem is the low recovery during downstream processing, which is at least in part a consequence of the earlier process.
We have found a process for overcoming the low productivity of the system by eliminating the auto-toxicity of gallidermin. As a result an economic production process for synthesising gallidermin can be achieved.
The process involves fermentatively producing pre-forms of gallidermin, such as pre-gallidermin, the immediate precursor of gallidermin, rather than the mature gallidermin itself. The pre-gallidermin is essentially non-toxic to the producing strain, and since biosynthesis of the lantibiotics is growth associated, overcoming the toxicity effect leads to an increased gallidermin production. This also provides the basis for further improving strain productivity by genetic and metabolic engineering. Following the isolation and purification of the pre-gallidermin from the culture broth, the active gallidermin is obtained in an in vitro biocatalytic step. This activation is connected to a radical change in the molecular properties compared to the fermentatively produced compound that is utilized for further downstream processing (DSP) for preparing the purified galliderrnin.
The invention will be more clearly understood by the following examples. The examples presented are illustrative only and various changes and modifications within the scope of the invention will be apparent to those skilled in the art.
Construction of a pre-gallidermin producing strain
The natural producer of gallidermin is Staphylococcus gallinarum Tü3928. This strain carries in its chromosome all nine genes (14 kb) required for the biosynthesis and its regulation, postranslational modification, export, immunity and a gallidermin specific serine protease, designated GdmP (FIG. 1A/B; Götz & Jung, 2001; Hill; 2002). For pre-gallidermin production, said GdmP serine-type protease needs to be inactivated. Several genetic approaches for inactivating GdmP are possible to those skilled in the art, including an in frame deletion of the gene, insertional disruption and inactivation, or by preventing m-RNA translation, e.g. using antisense sequences. Alternatively, the gdmP product, the protease, can be chemically inactivated during fermentation.
Here the inactivation of the gdmP gene is carried out by insertional disruption. Care has to be taken for the expression of the translationally coupled, downstream located gdmQ gene, which is essential for regulation of gallidermin production. Preceding gdmP is an E. coli-like -10 region (5′-TATAAA) 12 by in front of the Shine-Delgamo (SD) sequence which may serve as a promoter in staphylococci. The distance between the gdmP stop codon and the ATG start codon of gdmQ is only 10 nucleotides and the gdmQ SD sequence overlaps with the gdmP termination codon as shown in
First an internal gdmP fragment from S. gallinarum was amplified by PCR, using the primer pair gdmP-PstI-F (CATATCTGCAGGGTTTGTAGCGCATCATAA TC) (SEQ ID No. 4) and gdmP-HindIII-R (CGGTCACAAGCTTAGTAAGTC CCAAGTAGAGTCC) (SEQ ID No. 5). PCR amplification was performed with an annealing temperature of 58° C., and produced a 651 bp-long amplicon. This was double digested with PstI and Hind III, and then cloned into pUC18NotI (Herrero et al., 1990). The resulting plasmid was named pUC18NotI-P (
Since gdmQ had no functional promoter, the gene was placed under control of the kanR promoter, a kanR-gdmQ fusion was created by overlap extension PCR fusing the kanamycin resistance gene without its termination sequence to the 364 by gdmQ fragment. The primer kan-R (CCTACAATATTAATAGCAATCATAT TATTTCCCTTCAAAACAATTCATCCAG) (SEQ ID No. 6) had 37 of its 52 nucleotides complementary to the gdmQ sequence, so that the amplicons could anneal upon mixing.
Amplification of the 364 by sequence of gdmQ were done by PCR with the primer pair gdmQ-EcoRI-2-F (CGGAATTCGTCTATCAATTCATCATCAA TG) (SEQ ID No. 7) and gdmQ-R (TGAAGGGAAATAATATGATTGCTAT TAATATTGTAGGTG) (SEQ ID No. 8) using S. gallinarum chromosomal DNA as template. Then the kanamycin resistance gene was amplified by PCR using the primers kan-ClaI-F (TTATCGATGCCGTATGTAAGGATTCAG) (SEQ ID No. 9) and kan-R (CCTACAATATTAATAGCAATCATATTATTTTCCTT CAAAACAATTCATCCAG) (SEQ ID No. 6) and plasmid pDG782 as a template.
Mixing the two PCR fragments, a new PCR was performed using the outer primers gdmQ-EcoRI-2-F (CGGAATTCGTCTATCAATTCATCATCAATG) (SEQ ID No. 7) and kan-ClaI-F (TTATCGATGCCGTATGTAAGGATTCAG) (SEQ ID No. 9). In order to efficiently perform overlap extension PCR, it was of fundamental importance that the melting temperature (TM) of the overlapping sequence and the outer primers were the same. The PCR product (1510 bp), was digested with EcoRI and ClaI and ligated to EcoRI, AccI digested pUC18NotI-P. The resulting plasmid was named pGV4 (
An Escherichia coli-Staphylococcus shuttle vector was constructed from plasmid pPSM1058 containing a temperature-sensitive (TS) staphylococcus-replicon, and the genes encoding resistance to ampicillin and chloramphenicol (Madsen et al., 2002). A ca.1.4 kb SacI-NheI fragment containing the nuclease gene was excised and replaced by a SacI-NotI-SpeI DNA fragment excised from pGEM5Zf(+) (Stratagene, La Jolla, Calif.) multiple cloning site. The resulting plasmid, pGV2 was used to construct the gdmP knock-out vector (
The knock-out vector pGV5 was assembled by cloning the approximately 2.2 kb Noll from pGV4, containing the internal gdmP fragment and the kanR-gdmQ construct, into pGV2, the TS Escherichia coli-Staphylococcus shuttle vector. This knock-out vector was then transformed by electroporation into Staphylococcus gallinarum (
Transformation of Staphylococcus gallinarum required a high staphylococcal DNA concentration of more than 1 μg/μl. Since pGV5 is a low copy number plasmid, its preparation in large enough quantities was achieved best by passing the plasmid through Staphylococcus aureus RN4220 as described elsewhere (Augustin & Götz, 1990). The resulting strain Staphylococcus aureus RN4220 (pGV5) was grown at 30° C. in B-Medium (1% tryptone, 0.5% yeast extract 0.5% NaCl, 0.1% glucose, and 0.1% K2HPO4*3H2O (pH 7.2)) supplemented with 20 μg/m1 chloramphenicol. A three ml over night culture was used to inoculate a 25 ml culture (1:100) that was grown for 10 hours and used to inoculate a 1-L culture (1:100). This culture was allowed to grow into late stationary phase (16hours) before cells were harvested by centrifugation at 6,000 g, 4° C. for 15 min. Plasmid was then isolated and purified with a Qiagen Plasmid purification kit (Qiagen, Hilden, Germany) following a modified protocol. The cell pellet was washed with 40 ml 0.15-mM EDTA-washing buffer, pH 8, then centrifuged at 5,000 g, 4° C. for 10 minutes, the supernatant discarded, and the cell pellet was resuspended in 60 ml NaCl-buffer (50-mM Tris, 2.5-M NaCl, EDTA, pH 7). 50 μl lysostaphin (10 mg/ml, AMBI, USA) were added to the suspension, which was then incubated at 37° C. until viscosity increased (40 minutes). Thereafter, 60 ml lysis buffer (50-mM Tris, 300-mM EDTA, 0.5% Brij 58, 0.04% Na-deoxycholat, pH 8) at 4° C. were added. The cell suspension was mixed and incubated on ice for 1 hour. Cell debris was removed by centrifugation with 19,000 g, 4° C. for 30 min. The supernatant was filtered before 0.4 vol. of 50% PEG 6,000 were added. After mixing, the solution was incubated 2.5 hours on ice for DNA precipitation to proceed. The precipitated DNA was collected by centrifugation with 5,500 g, 4° C. for 20 minutes, discarding the supernatant. The DNA pellet was resuspended in 4 ml buffer P1 (10-mM EDTA, 0.01% RNase, 50-mM Tris-HCl, pH8), and 4 ml buffer P2 (200-mM NaOH, 1% SDS) were added. After careful mixing the solution was incubated 5 minutes at room temperature. Thereafter, 4 ml buffer P3 (3-M potassium acetate, pH 5.5) at 4° C. were added and the tubes immediately inverted 4-6 times. After 10 minutes incubation on ice followed by centrifugation with 15,000 g, 4° C. for 30 minutes the cleared lysate was applied onto an anion exchange column (Qiagen Midi Plasmid Kit), which was then washed twice with QC washing buffer (1-M NaCl, 50-mM MOPS, pH 7, 15% isopropanol). The DNA was eluted with 2 ml buffer QF (1.25-M NaCl, 50-mM Tris-HCl, pH 8.5, 15% isopropanol). The DNA solution was precipitated by adding 0.7× vol. isopropanol vortexing, and centrifugation with 14,000 g, 4° C. for 15 min. The DNA pellet was washed twice with 70% ethanol, then dried, and dissolved in 20 μl 1×TE, pH 8 (Sambrook et al. 1989). This isolation method yielded about 2 μg/μl of pGV5 that was used for electroporation of Staphylococcus gallinarum.
Electrocompetent cells of Staphylococcus gallinarum were obtained by preparing a ten ml culture B-Medium (1% tryptone, 0.5% yeast extract 0.5% NaCl, 0.1% glucose, 0.1% K2HPO4*3H2O, pH 7.2) incubated overnight, shaking with 250 rpm at 37° C. Five ml of this culture were used to inoculate 500 ml B-medium. The culture was grown until an OD600 of between 0.45 and 0.5 was reached. The cells were centrifuged at RT, then washed with 500 ml washing buffer (0.75-M sucrose, 10-mM EDTA, pH 7.5). This washing step was repeated twice using 250 ml of washing buffer. The pellet was resuspended in a volume of washing buffer that corresponds in ml to the OD600 value at which cells were harvested. Aliquots of 50 μl were distributed into cryo vials and stored at −80° C.
For electroporation, the cells were thawed on ice and incubated for 20 min with 4 μl plasmid DNA (ca. 4-8 μg). Then cells were transferred into an ice-cold 0.2 corn cuvette, and electroporated with the BioRad Genepulser at 2.5 kV, 1000Ω and 1 μF. Resulting time constants were 0.6 ms. Then 1 ml SMMP70 medium (SMMP 70 consisted of 7.5 parts of 2× filter-sterilised SMM (1-M sucrose, 40-mM maleic acid, 40-mM MgCl2, pH adjusted with NaOH to 6.8); 2 parts autoclaved Pennassay broth; and 0.5 parts of filter-sterilised 10% bovine serum albumin) was added, and the cells were incubated for 2 hours at 30° C., shaking. with 225 rpm. Thereafter, cells were plated on selective B-agar plates, B-medium containing 1.2% agar, supplemented with 20 μg/ml chloramphenicol and incubated at 30° C. First colonies were visible after 24 h. Plates were totally incubated for 48 h.
The electroporation conditions balance ideally the conditions for DNA uptake by Staphylococcus gallinarum while preventing excessive cell death.
Following transformation and selection, gene replacement was induced in the S. gallinarum (pGV5) isolates grown at 30° C. in B-medium under various selective conditions following the general outline described by Bruckner (1997) (
A first S. gallinarum (pGV5) culture was started inoculating 10 ml kanamycin-supplemented (25 μg/μl) B-medium with a single colony of a transformed cell. Culture was incubated shaking with 225 rpm at 30° C., a temperature that allowed pGV5 to replicate as free plasmid (permissive temperature), until late stationary phase (16h). With this culture, new cultures were inoculated (1:100) and incubated shaking with 225 rpm at 30° C. into the late stationary phase (16 h). Of these, 100 ml cultures in B-medium containing 7.5 μg/μl kanamycin (MIC value) were inoculated (1:100) and grown for 24 hours at the non-permissive temperature of 40° C. This step was repeated once, before diluting the cultures 1:100 into 100 ml B-medium containing no antibiotic. Growth at 40° C. was extended for another 24 hours. The selection for plasmid integration resulted in 253 colonies with CmRKanR phenotype.
A 1:10,000 dilution of the last culture was spread onto B-agar plates supplemented with 7.5 μg/μl kanamycin. After over night incubation at 40° C., colonies were replicated onto B-medium plates supplemented with either chloramphenicol (20 μg/μl) or kananmycin (7.5 μg/μl). All colonies, which were found to be sensitive to chloramphenicol but resistant to kanamycin, were strains where double homologous recombination took place. Three colonies, designated Staphyloccus gallinarum ΔgdmP::kan, with CmS KanR phenotype were isolated and further analyzed.
The site of integration was analyzed by PCR using primers designed to generate characteristic amplicons for Staphyloccus gallinarum ΔgdmP::kan and the Staphyloccus gallinarum wild-type chromosomal DNA, which was isolated from 8 ml Luria Broth grown cultures (14 hours) using the Genomic tips (Qiagen, Hilden, Germany) with a modified protocol, omitting the proteinase K treatment for lysis, but including addition of 7 μl lysostaphin (10 mg/ml) in addition to lysozyme. Incubation at 37° C. was extended to one hour. The second incubation was performed at 55° C. instead of 50° C. In the final step, DNA was eluted with 3 ml hot (50° C.) buffer QF (1.25-M NaCl, 50-mM Tris-HCl, pH 8.5, 15% isopropanol). DNA was removed with a glass rod and dissolved in 1×TE, pH 8.
The PCR reactions were performed with 1:10 diluted DNA of which five microliter where used as DNA template. PCR reactions were performed with primer pairs PROOF-K (ACGAACTCCAATTCACTGTTCCTTG) (SEQ ID No. 10) and PROOF-P (GGTGAGGGGTGCTATATGAAGAAATT) (SEQ ID No. 11), and with PROOF-Q (CTTTGCACACCCTTAAATTATCTCTTAATC) (SEQ ID No. 12) and PROOF-P.
PCR reactions were analysed in a 0.8% agarose gel. Amplification with KP primer pair yielded a 1.14 kb fragment indicating the proper integration of the kan gene in Staphyloccus gallinarum ΔgdmP::kan; no KP primer product was identified in the wild-type. Also the QP primer pair resulted in the expected amplicon length of 2.5 kb and 2 kb for Staphyloccus gallinarum ΔgdmP::kan and the wild-type, respectively (
Following the successful genetic examination Staphyloccus gallinarum ΔgdmP::kan culture supernatant was analyzed by HPLC for the presence of gallidermin and pre-gallidermin. Cultures were grown in YE4 medium (5% yeast extract (Ohly Cat, Deutsche Hefe Werke, Germany), 4.5% CaCl2, and 0.5% maltose, pH 7.2). A three ml preculture was incubated shaking with 190 rpm for 24 hours at 37° C. and then used to inoculate (1:100) a 100 ml YE4 culture (in 250 ml Erlenmeyer flasks with one baffle) that was grown for 18 hours under the same conditions. Filtered culture supernatants were subjected to reversed-phase liquid chromatography (C18 Nucleosil column). The mobile phase was composed of 0.1% aqueous H3PO4 (v/v) (solvent A) and 100% acetonitrile (solvent B). Gallidermin and pre-gallidermin were separated using a linear gradient from 90% A/10% B to 45% A/55% B in 14 minutes at a flow rate of 1 ml/min. Regeneration of the column and equilibrium to start conditions was achieved in five more minutes. Analysis revealed that the Staphyloccus gallinarum wild-type supernatant contained gallidermin, calculated molecular mass of 2165.6 Da (peak at ca 9.7 minutes). Pre-gallidermins (peaks at ca. 9.1 and 9.5 minutes) was not detected (
It is possible that these truncated forms are produced during translocation through the membrane. Furthermore, this information may be used to alter the GdmA gene such as to shorten the native leader sequence or to modify the leader, with e.g. a His-tags, labels or other derivations to facilitate certain downstream processing steps, such as capture chromatography and a like.
Further evidence for the lack of gallidermin production and the precursor accumulation in strain Staphyloccus gallinarum ΔgdmP::kan came from protein analysis of the culture supernatants by Tricine-SDS-PAGE, blotting and silver staining of the proteins. Tricine-SDS-PAGE (16% acrylamide) that allows separation of proteins from 1 to 100 kDa was done following the protocol described elsewhere (Schägger & von Jagow 1987) Samples, 15 μl of a 1:5 dilution of supernatants were adjusted to pH 8. Purified gallidermin (Alexis Corp) dissolved in water was used as standard. To allow for detection of very small peptides like gallidermin and pre-gallidermin, gels are transferred to a nitrocellulose membrane and then stained with silver staining (Kovarik et al. 1987). Blotting was done using a BIO RAD blotting apparatus. Prior to blotting, the transfer pads, filter pads and 0.45 μm nitrocellulose membranes (PROTRAN, Schleicher and Schuell) had to be soaked for at least 15 min in 4° C. transfer buffer (3 g Tris, 1.4 g glycine, 0.1% SDS, and 200 ml MeOH, ad 1 L). Blotting sandwich was prepared, and blotting was performed in 4° C. transfer buffer at 64 mA for 40 minutes. A protocol described elsewhere was used for silver staining the membrane (Kovarik et al. 1987). As shown in
To further substantiate this, supernatants from both wild-type and mutant strain was subjected to a bioassay following the protocol of Kies and coworker (Kies, et al., 2003). Briefly, Kocuria rhizophila (formerly Micrococcus luteus) was grown in LB-medium at 30° C. for 24 hours. The culture was diluted to an OD600 of one. Then 0.3 ml of this dilution were mixed into 150 ml cooled molten B-agar, to which 0.3 ml 50% glucose were added. Plates containing 10 ml medium were prepared. 20 μl supernatant of test cultures were pipetted onto 6 mm sterile paper disks. A negative control, 20 μl of medium YE4, was applied as well. Dried filters were placed onto the K. rhizophila containing plates, which were then incubated at 30° C. for 24 hours. The bioassay (
These results show that the GdmP protease mutant strain Staphylococcus gallinarum ΔgdmP::kan indeed lacked the expression of a functional extracellular protease that is required to proteolytically process pre-gallidermin precursor molecules into mature and active gallidermin. Moreover, it was shown that strain Staphylococcus gallinarum ΔgdmP::kan accumulated novel truncated pre-gallidermin in the culture supernatant.
While the generation of a GdmP deletion strain is the most critical step during the strain development program, other genetic modifications to increase the production of pre-gallidermin are obvious to those skilled in the art and include increasing the gene dosage by adding an additional copy of the gdmP deleted gallidermin cluster, removal of the no longer required genes lanI and lanEFG encoding the immunity functions, changing promoter strength and regulation including and alike, known to those skilled in the art can be employed to further improve the strain's productivity.
Gallidermin is produced by Staphylococcus gallinarum Tü3928. Several protocols have been described in the recent literature that detailed the production and yields of gallidermin and its partial purification (Fiedler et al., 1988; Kellner et al., 1988; Kempf et al., 2001; EP0342486). These protocols include the direct adsorption of gallidermin from the fermentation broth with Amberlite XAD-1180. The water washed resin was eluted with acidified methanol, and the gallidermin was recovered by drying the eluate in vacuo yielding some 2.7 g crude material from a 20-L fermentation. The crude gallidermin was further purified by ion exchange chromatography on Amberlite IRC-50 resin followed by a desalting step again on XAD-1180 yielding about 1 g of the crude antibiotic. Final purification was achieved on a reversed-phase chromatography with a 10C18 column, reducing the yield to 100 mg purified gallidermin obtained from a 30-L fermentation. Due to the toxicity of gallidermin to the producing organism, all the efforts to increase yields focussed in fermentation regimes that require on-line product removal or repeated partial harvests (Kempf et al., 2001).
We found a process using an engineered strain of Staphylococcus gallinarum that is deficient in the final step of proteolytic gallidermin activation (example 1). Said strain only makes the non-toxic and non-active gallidermin precursor molecules, exported into the culture supernatant. Said pre-gallidermin was then partially purified for its biocatalytic activation (example 4).
Production of these truncated pre-forms of gallidermin allows for all fermentation regimes known to those skilled in the art, which include high cell density, batch-, fed-batch or continuous cultivation with either complete or repeated partial harvests; all with or without in situ product removal strategies.
To evaluate and prove that the gallidermin precursor molecules are indeed non-toxic to the producing organism, dilutions of a partially purified pre-gallidermin stock solution were prepared of which 20 μl aliquots were applied to filter discs that were then placed onto GM plate freshly inoculated with wild type S. gallinarum. As shown in
The following cultivations show the potential of this approach to reach production titres well above the current limit of 300-400 mg/l, at which the gallidermin toxicity prevents further improvement of fermentation titres.
The analytical procedure for quantification and determination of the purity of pre-gallidermin and gallidermin, respectively, were as follows.
A La Chrom system (Merck, Germany) equipped with an L-7100 pump, an L-7200 autosampler with a 100 μl injection loop, an L-7455 diode array detector (DAD) and an L-7490 refractive index detector (RI) was used. Data collection was done with a Merck-Hitachi model D-7000 chromatography station software. A Prontosil Eurobond C18 5.0 μm, (125×4.6 mm I.D.) with guard column (10×4.6 mm I.D.) of the same material (Bischoff, Leonberg, Germany) was used.
The injection volume was 10 μl; column temperature was maintained at 25° C. The mobile phase was made up from 0.1% H3PO4 (mobile phase A) and acetonitrile (Mobile phase B). Separation was achieved at a flow rate of 1 ml/min and a linear gradient that is detailed in the table below:
Further improvements in the fermentation protocol will evidently result in higher titers. In particular, since gallidermin precursor production is growth associated and that extending growth by feeding an utilisable source of carbon such as glycerol with or without supplementation with utilisable sources of nitrogen, sulphur, and or other micro nutrients such as vitamins or selected amino acids such as prolin, cystine, serine or lysins or any other amino acid or mixes thereof, will improve the fermentation outcome. Also replacement of the yeast extract by e.g soya meal or protein or cotton seed flower that can contain amino acids and micro elements more amenable for good growth of S. gallinarum, will further improve growth.
The fermentation regime and data are provided as an example only and does not preclude further improvement in productivity by employing techniques commonly used by those skilled in the art to optimise the fermentation regime including media composition, feed composition and feed rates, as well as other commonly controlled parameter that will alter the growth kinetic and production dynamics of a microbial cultivation.
Following the fermentation, the initial steps of the isolation of the gallidermin precursor molecules involve the removal of the biomass using filtration technologies or centrifugation. For demonstration purposes, centrifugation with 10,000×g for 10 minutes was employed. The clear, cell-free supernatant containing the gallidermin precursor molecules was than applied to a hydrophobic interaction chromatography using commercially available resins such as Amberlite XAD1600, XAD1180, XAD16, XAD7, XAD7HP and)(AD-4 Amberlite resins and all chemicals were provided from Fluka (Buchs, Switzerland), Rohm & Haas (Philadelphia, Pa., U.S.A.) or Roth (Reinach, Switzerland) unless mentioned otherwise. It was critical for that resins were washed prior use. Packed into a column (20×1.5 cm I.D.) resins were washed with 8 bed volumes methanol. The alcohol was then displaced with 8 bed volumes of double distilled water prior to adsorption. Adsorption was tested at pH 6, 7 and 8 by adjusting the pH of the cleared with 5 mM H2SO4 to pH 6, 7, or 8 and prior to loading of the aforementioned resins. 1800 μl cell free fermentation supernatant and 200 μl 1-M Na-phosphate buffer were added into a 2m1 Eppendorf tube. Eighty mg (dry weight basis) washed resin were added to the solution and incubated at 30° C., shaking at 220 rpm. After 90 min incubation, liquid phase and resin phase were separated by short centrifugation; pre-gallidermin concentration in the supernatant was determined by HPLC. Desorption was carried out with an elution buffer composed of (9:1, v/v) methanol/5 mM H2SO4 (pH 4). Elution buffer, 600 was added to the resin and incubated at 30° C., shaking with 220 rpm. After 30 min incubation liquid phase and resin phase were separated by short centrifugation and the pre-gallidermin concentration in the supernatant was determined. After removal of the liquid phase, a second 600 μl elution buffer were added to the resin phase and the same procedure was repeated. The results of these screens are summarized in
The methanol from the eluate was evaporated in vacuo at up to 40° C. to obtain a methanol free preparation of pregallidermin for further analysis.
Proteolytic in vitro activation of pre-gallidermin into gallidermin The serine-type protease GdmP cleaves the pre-gallidermin molecule recognizing the amino acid sequence Ala-Glu-Pro-Arg-1-Ile-gallidermin of the N-terminal leader peptide, and cleaving off the leader peptide between arginie and isoleucine as indicated by the arrow. The protease cleavage site has been described earlier (Ottenwälder et al., 1995; Bierbaum et al., 1996; Furmanek et al, 1999). Nevertheless, reports on the in-vitro proteolytic digestion of type-A lantibiotics are controversial. Gallidermin and epidermin, both are digested by trypsin under standard conditions to yield several inactive fragments (Allgaier et al., 1986; Kellner et al., 1988) that were used for structural analysis. This result is not surprising, as both molecules possess several potential trypsin cleavage sites. Hence it is not surprising, that an activation of pre-gallidermin using proteases other then GdmP or EpiP has never been reported. Other lantibiotics, such as nisin that do not possess additional sites for proteolysis may be derived by in-vitro proteolysis under standard conditions (US2004/0009550 (A1)).
Trypsin has an aspartate residue (189) at the bottom of the pocket of the active site, and this Asp forms a salt bridge with the positively charged group at the end of the substrate Lysine and Arginine side chains, on which this enzyme acts. The two relevant sites in gallidermin are the C-terminal arginine residue of the leader peptide and a lysine at position 13. This lysine, however, is located next to a 2,3-dihydrobutyrin (Dhb) residue hat was thought to possibly be protected under when tryptic digestion is done under carefully selected non-standard conditions. In order to identify proteases and conditions that would only cleave off specifically the N-terminal leader peptide, pre-gallidermin isolated from cultivations (see example 2 and 3) was incubated in the presence of several serine proteases, including clostripain (endonuclease ArgC, Roche Diagnostics), papain, bromelain (Ananas comosus) and trypsin using choosing conditions that was not obvious to those skilled in the art.
Endonuclease ArgC (Roche Diagnostics), a proteinase isolated from Clostridium histolyticum cleaving peptide bonds C-terminal from arginine as found in pre-gallidermin, was examined for converting gallidermin precursor molecules into gallidermin. A crude pre-gallidermin powder obtained by foam separation from a fermentation of Staphylococcus gallinarum Tü3928 that contained mostly pre-gallidermin with only traces of gallidermin. Resuspended in 100 μl incubation buffer (100-mM Tris-HCl, 10-mM CaCl2, pH 7.6) Of these 85 μl, 10 μl activation buffer (50-mM DTT, 5-mM EDTA) and 5 μl endoproteinase ArgC were mixed. Following incubation for 12 hours at 37° C., a sample was taken and the gallidermin/pre-gallidermin ratio was determined by HPLC and compared to the initial ratio. After 12 h incubation with ArgC, the gallidermin/pre-gallidermin ratio increased from 1:9.8 to 1:1.54 and the amount of gallidermin increased as expected (
Papain (American Laboratories Inc, 2000 USP (IU) ca 30%) and stem bromelain (American Laboratories Inc, 600 GDU ca 25%) were evaluated using an aqueous solution of partially purified pre-gallidermin (250 mg/l) was adjusted to pH 6.5 and incubated with Bromelain 600 (0.05 g/l; American Laboratories) at room temperature (˜20° C.) for 12 h. (
In contrast, papain Papain (160 mg/l) in 15 mM potassium phosphate buffer did not convert the gallidermin precursor (˜500 mg/l) molecules (not shown).
Surprisingly trypsin that previously had been reported to digest and to inactivate gallidermin was the most suitable enzyme, albeit under conditions not commonly used. Gallidermin precursor molecules isolated as outlined in example 2 were subjected to proteolysis with trypsin.
The selective protease digestion conditions, as defined by the selective processing of pre-lantibiotics, can be further optimized in a variety of ways. This can include, for example, low protease to antibiotic ratio, decreased temperature, altered pH, etc. The various relevant parameters can be altered independently or in combination.
Of all proteases examined, only trypsin appeared to be suitable for a commercial and industrialised process. In order to demonstrate that the tryptic digest indeed produced active and mature gallidermin, samples of trypsin-treated pre-gallidermin were used in a bioassay, alongside with gallidermin isolated from Staphylococcus gallinarum Tü3928 cultivations. ESI-MS analysis (Water Q-TOT Ultima API in cationic mode) of the tryptic digest (
After identification of trypsin as a suitable protease, the conditions were analyzed in more details. In particular the use of an industrially and pharmaceutically amenable enzyme, gamma-irradiated trypsin (porcine 1:250, powder, SAFC Biosciences, USA) was compared to the trypsin from American Laboratories Inc. used in example 4. Methanol free and pre-gallidermin solutions containing 20, 40, and 60% methanol were evaluated under the following conditions: trypsin was used at 200 mg/1, pre-gallidermin concentration was ca. 700 mg/l in 25 mM Na-phosphate buffer pH 6; reaction volume was 1000 μl. The activities were calculated during the first 7 minutes of the reaction and the reaction yields are based on the maximum gallidermin obtained during the reaction. The results obtained with the gamma-irradiated trypsin are summarized in the Table below and in
Max[Gdm]/[P/gdm]t0 * 100
Interestingly there was no difference in catalytic activity and gallidermin yield in the presence of 0 or 20% methanol in the reaction (
This example describes one possible way to isolate maturated gallidermin after its proteolytic activation. The initial tryptic digest was performed with 3 g/l pre-gallidermin in solution. After trypric digest was completed (
Further purification steps employing hydrophobic interaction chromatography with e.g. Amberlite XAD1180 or equivalent, ion exchange chromatography and gelfiltration to yield pure and dried gallidermin have been published elsewhere (Fiedler et al., 1988).
The invention is not limited to the embodiments hereinbefore described, with reference to the accompanying drawings, which may be varied in construction and detail.
Allgaier, H., G. Jung, R. G. Werner, U. Schneider and H. Zähner, 1986. Epidermin: Sequencing of a heterodetic tetracyclic 21-peptide amide antibiotic. Eur. J. Biochem. 160(1):9-22.
Augustin, J., and F. Götz, 1990. Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation. FEMS Microbiol Lett 54:203-7.
Bierbaum, G., F. Götz, A. Peschel, T. Kupke, M. van der Kamp, and H. G. Sahl. 1996. The biosynthesis of the lantibiotics epidermin, gallidermin, Pep5 and epilancin K7. Antonie Van Leeuwenhoek International Journal of Gen Mol Microbiol 69:119-127.
Bonelli, R. R., T. Schneider, H. G. Sahl, and I., Wiedemann, 2006. Insights into in vivo activities of lantibiotics from gallidermin and epidermin mode-of-action studies. Antimicrob. Agents Chemother. 50(4): 1449-57.
Brükner, R. 1997. Gene replacement in Staphylococcus camosus and Staphylococcus xylosus. FEMS Microbiol Lett 151:1-8.
Breukink E. and de Kruijff B., 2006. Lipid II as a target for antibiotics. Nat. Rev. Drug Discov. 5(4): 321-32.
de Vuyst, L. and E. Vandamme, 1993. Nisin a lantibiotic produced by Lactococcus lactis supsp. lactis: properties, biosynthesis, fermentation and applications. In: de Vuyst, L. and E. Vandamme (eds.) “Bactiocins of lactic acid bacteria: Microbiology, genetics and applications”. Elsevier Applied Biotech. Series, pp151-221.
Fiedler, H-P., T. Homer and H. Decker, 1988. Purification of the hydrophilic antibiotics epidermin, gallidermin, and nikkomycin Z by preparative reversed-phase HPLC. Chromatographia 26:215-220.
Furmanek, B., T. Kaczorowski, R. Bugalski, K. Bielawski, J. Bohdanowicz, A. J. Podhajska, and J. Bogdanowicz. 1999. Identification, characterization and purification of the lantibiotic staphylococcin T, a natural gallidermin variant. J Appl Microbiol 87:856-66.
Götz, F. and G. Jung, 2001. Biosynthesis of the lantibiotics epidermin and gallidermin. In: Braun, V. and F. Götz (ed.) “Microbial fundamentals of biotechnology”. WILEY-VCH Verlag GmbH, Weinheim, pp: 52-92.
Guerot-Fleury, A.-M., N. Shazand, N. Frandsen and P. Stagier. 1995. Antibiotic resistance cassettes for Bacillus subtilis. Gene 167:335-336.
Herrero, M., V. de Lorenzo and K. N. Timinis, 1990. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J. Bacteriol. 172:6557-67.
Hille, M. 2002. Untersuchungen zur Biosynthese der Lantibiotika Gallidermin and Epidermin. Dissertation University of Tubingen, Germany.
Hille, M., S. Kies, F. Götz and A. Peschel, 2001. Dual role of GdmH in producer immunity and secretion of the staphylococcal lantibiotics gallidermin and epidermin. Appl. Environ. Microbiol. 67:1380-1383.
Jack, R. W., G. Bierbaum and H.-G. Sahl (eds.) 1998. Lantibiotics and related peptides, Springer-Verlag Berlin, pp. 224.
Kellner, R., G. Jung, T. Hörner, H. Zähner, N. Schnell, K.-D. Entian and F. Götz, 1988. Gallidermin: a new lanthionine-containing polypeptide antibiotic. Eur. J. Biochem. 177:53-59.
Kempf, M., U. Theobald and H.-P. Fiedler, 2001. The antibiotic gallidermin—evolution of a production process. In: Van Broekhoven, A., F. Shapiro and J. Anne (eds.) “Novel frontiers in the production of compounds for biomedical use”. Vol. 1, Kluwer Academic Publishers, Dordrecht, pp 35-55.
Kies, S., C. Vuong, M. Hille, A. Peschel, C. Meyer, F. Götz and M. Otto, 2003. Control of antimicrobial peptide synthesis by the agr quorum sensing system in Staphylococcus epidermidis: activity of the lantibiotic epidermin is regulated at the level of precursor processing. Peptides 24:329-338.
Kovarik, A., K. Hlubinova, A. Vrbenska and J. Prachar, 1987. An improved colloidal silver staining method of protein blots on nitrocellulose membranes. Folia Biol (Praha) 33:253-7.
Madsen, S. M., H. C. Beck, P. Ravn, A. Vrang, A. M. Hansen, and H. Israelsen. 2002. Cloning and inactivation of a branched-chain-amino-acid aminotransferase gene from Staphylococcus carnosus and characterization of the enzyme. Appl Environ Microbiol 68:4007-14.
McAuliffe, 0., R. P. Ross and C. Hill, 2001. Lantibiotics: structure, biosynthesis and mode of action. FEMS Microbiol. Rev. 25:285-308.
Ottenwälder, B., T. Kupke, S. Brecht, V. Gnau, J. Metzger, G. Jung, and F. Götz. 1995. Isolation and characterization of genetically engineered gallidermin and epidermin analogs. Appl Environ Microbiol 61:3894-903.
Sahl, H. G. and G. Bierbaum, 1998. Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria. Annu. Rev. Microbiol. 52:41-79.
Sambrook, J., E. F. Fritsch and T. Maniatis, 1989. Molecular Cloning, a laboratory manual, Second Edition, C old Spring Harbor Laboratory Press, NY.
Schägger, H. and G. von Jagow (1987). “Tricine sodium dodecyl-sulfate polyacrylamide-gel electrophoresis for the separation of proteins in the range from 1-Kda to 100-Kda.” Anal. Biochem. 166: 368-379.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05394019.3 | Jun 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/006240 | 6/28/2006 | WO | 00 | 12/19/2007 |
