Historically, ß-lactam antibiotics constitute the most important group of antibiotic compounds, having a long and successful record of clinical use. The prominent members among this group are the penicillins and cephalosporins.

These compounds are naturally produced by filamentous fungi, such as Penicillium chrysogenum and Acremonium chrysogenum, and by filamentous bacteria.

As a result of classical strain improvement techniques, the production levels of these antibiotics in P. chrysogenum and A. chrysogenum have increased dramatically over the past decades. Increased understanding of the biosynthetic pathways leading to penicillins and cephalosporins has prompted the advent of recombinant DNA technology, providing new tools for the improvement of R-lactam production strains and for the in vivo derivatisation of R-lactam compounds.

Most enzymes involved in ß-lactam biosynthesis have been identified (Ingolia and Queener, Med. Res. Rev. 9 : 245-264, 1989), and their corresponding genes have been cloned (Aharonowitz, et al., (Ann. Rev.

Microbiol. 46 : 461-495, 1992).

The first two steps in the biosynthesis of penicillin in P. chrysogenum are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-a-aminoadipic acid) (A), L-cysteine (C) and L-valine (V) into the tripeptide LLD-ACV, followed by cyclisation of this tripeptide to form isopenicillin N. The isopenicillin N intermediate compound contains the ß-lactam structure characteristic of penicillin. These first two steps in the biosynthesis of penicillins are common in penicillin-producing, cephamycin-producing and cephalosporin- producing fungi and bacteria.

The third synthesis step involves the exchange of the hydrophilic a-

aminoadipic acid side chain derived from L-5-amino-5-carboxypentanoic acid with a hydrophobic side chain, via the action of the enzyme acyltransferase (AT). The enzymatic exchange reaction mediated by AT takes place inside a cellular organelle, the microbody, as has been described in EP-A-0448180.

Industrial fermented penicillins like-penicillin V and penicillin G, are produced by adding to the culture broth phenoxyacetic acid or phenylacetic acid, respectively, during fermentation of P. chrysogenum, thereby replacing the side chains of the natural p-iactams with phenoxyacetic acid or phenylacetic acid. In order to replace the a-aminoadipic acid side chain in the acyltransferase-catalysed reaction, the carboxylic acid group of the new side chain first must be activated, as the AT enzyme requires the side chain in an activated form. The activated form is a coenzyme A (CoA) derivative, synthesised by a CoA ligase.

Two CoA ligases have been cloned from P. chrysogenum. The gene encoding an acetyl-coenzyme A synthetase has been characterised by Van Hartingsveldt et al. (W092/07079), Gouka et al. (Appl. Microbiol. Biotechnol., 38 : 514-519 (1993)) and Martinez-Blanco et al. (Gene, 130 : 265-270 (1993) ; J.

Biol. Chem. 267 : 5474-5481 (1992)). The enzyme not only accepts acetic acid but also phenylacetic acid as a substrate in the synthesis of the corresponding acyl-coenzyme A ester, although the specificity for phenylacetic acid is very low as compared to the specificity for acetic acid. More recently, a gene encoding a homologue of the Pseudomonas putida phenylacetic acid CoA ligase has been cloned ; the pcl gene (WO 97/02349). The protein was shown to be active with both phenylacetic and phenoxyacetic acid.

In novel fermentation processes for the production of cephalosporin intermediates, P. chrysogenum strains carrying heterologous cephalosporin biosynthetic genes, such as the expandase gene of S. clavuligerus or the expandase/hydroxylase gene of A. chrysogenum, are used for the production of the corresponding adipoyl-cephalosporins by feeding adipic acid to these recombinant strains. Cephalosporin intermediates produced in this fashion include 7-aminodeacetoxycephalosporanic acid (7-ADCA), 7-

aminodeacetylcephalosporanic acid (7-ADAC) and 7-aminocephalosporanic acid (7-ACA). Subsequent removal of the adipoyl side chain from the adipoyl- cephalosporins can be achieved by using an amidase (EP 532341 and Crawford et al., Bio/Technology 13 : 58-62 (1995)).

During the latter stages of industrial penicillin fermentation processes extremely high p- ! actam titers are found, using Penicillium chrysogenum as the production host (estimated to be 120 mM by Hillenga (1999) PhD thesis, University of Groningen, The Netherlands). From literature it is known that the ergosterol content of P. chrysogenum plasma membranes is around 50% (Hillenga, 1999). At this concentration the membrane is almost impermeable for passive diffusion of penicillins. Therefore, it remains unclear how the fungus can export such an amount of ß-lactam antibiotics. Especially at the end of fermentation this must be an efficient system to export against a high external concentration.

Recently, organic cation/peptide transporters were suggested to be involved in the transport of p- ! actam antibiotics in humans (Ganapathy et al., J. Biol. Chem (2000) 275 : 1699-1707). Until now no such proteins are reported in filamentous fungi.

Industrial strains of the filamentous fungus Penicillium chrysogenum have been selected for their capacity to produce and secrete large quantities of a variety of p- ! actam compounds into culture medium. Various lines of evidence indicate that ß-lactam secretion is an active process, possibly via transport proteins. During the latter stages of such fermentation processes extremely high p- ! actam titers are found in fermentations on an industrial scale. However, despite traditional genetic selection of fungal strains for ß-lactam output and evidence of how ß-lactam compounds are secreted, micro-organisms have yet to be optimised for maximal secretion of p-iactam compounds. Moreover, the transport process might be optimised for each organism and each antibiotic.

For example, the production rate of heterologous cephalosporins produced in P. chrysogenum will be limited due to the fact that the penicillin transporter will

have a low affinity for the product. This could be overcome either by cloning one or more homologue ß-lactam transporters from natural cephalosporin producers like A. chrysogenum with the aid of the transporter subject of this invention or, alternatively, by mutagenesing the penicillin transporter according to the present invention into a derivative more apt to transporting cephalosporins.

EP-A-0908516 discloses a Staphylococcus ATP-binding cassette (ABC) transporter polypeptide, an ABC transporter polynucleotide and materials and methods for their production. The transporter polypeptide of EP-A-0908516 may be used to determine the presence of drug resistant bacteria in a mammalian host. The polypeptide of EP-A-0908516 may be obtained from other organisms, but has no bearing on transport of p- ! actam compounds.

Certain transporters have been described in eukaryotic yeast and moulds, but none appear related to improving p- ! actam output from industrial strains of fungi. For example, a histidine transporter (Tanaka and Fink, Gene 38 : 205-214 (1985)) and a proline transporter (Vandenbol et al., Gene 83 : 153- 159 (1989)) have been identified in Saccharomyces cerevisiae. Over- expression of the yeast multi-drug-resistance gene CDR1 in Candida albicans can confer resistance to cycloheximide, chloramphenicol, and miconazole (Prasad et al., Curr. Genet., 27 (4) : 320-329 (1995)). An ATP-binding cassette transporter p-glycoprotein encoded by a Penicillium multi-drug resistance gene is over-expressed in strains of Penicillium digitatum resistant to fungicide, suggesting the toxicant efflux system of the fungus is directly involved with fungicide resistance (Nakaune et al., Appl. Environ. Microbiol. 64 : 3983-3988 (1998)). However, such protein carriers are not associated with p- ! actam secretion from fungi.

Thus, a need exists for improving the secreted amounts of ß-lactam compounds recoverable from industrial strains of micro-organisms. The invention solves the above-mentioned problem by manipulation of the ABC- transport protein system, providing a means for enhancing p- ! actam secretion

from the micro-organisms in which these compounds are or can be synthesised.

Present invention therefore provides a method for producing p- ! actam compounds comprising the step of enhancing ABC-transporter activity of a micro-organism and subsequently culturing said micro-organism under conditions allowing the production of said p- ! actam compounds. In another embodiment of the present invention a method is provided for enhancing the secretion of a ß-lactam compound from a micro-organism comprising the step of enhancing an ABC transporter activity of said micro-organism.

The present invention further provides an isolated polynucleotide encoding a ABC transporter polypeptide selected from the group consisting of SEQ ID NO. 7, 8, 9, 11, 12, 14, 15, 17, 18, 19, 21, 22, 24, 25, 26, 29, 30, 32, 33, 34, 35, 37, 38, 40, 41, 43, 44, 46 and 47 as incorporated in the present application, or a sequence substantially homologous thereto.

The present invention also provides for isolated ABC transporters encoded by an ABC transporter gene comprising at least part of the nucleotide sequence according to SEQ ID NO selected from the group consisting of SEQ ID NO. 7, 8, 9, 11, 12, 14, 15, 17, 18, 19, 21, 22, 24, 25, 26, 29, 30, 32, 33, 34, 35, 37, 38, 40, 41, 43, 44, 46 and 47 or functional equivalents thereof.

An ABC-transport protein utilises ATP to drive transport processes of a diverse range of substrates including various antibiotics, oligopeptides, polysaccharides and unrelated toxic drugs (reviewed by Schneider et a/ (Schneider, E. and Hunke, S. FEMS Microbiol. Rev. 22 : 1-20, 1998) ; Mondez et al (Mendez, C. and Salas, J. A., FEMS Microbiol. Lett. 158 : 1-8, 1998) ; van Veen et al (Veen, H. W. van and Konings, W. N. Biochim. Biophys. Acta 1365 : 31-36, 1998).

An embodiment of the invention provides a method for enhancing the secretion of a ß-lactam compound from a micro-organism which method

comprises the step of enhancing an ABC transporter activity of said micro- organism. Secretion according to the invention comprises any step involved in transport of ß-lactam compounds or intermediates thereof occuring inside the micro-organism and from inside to outside the micro-organism. p- ! actam compounds according to the invention comprise Penicillins, Cephalosporins, Amoxycillins, Calvulanic acid and intermediates thereof such as PenV, PenG, 6-APA, 7-ADCA, 7-ACA, 7-ACDA.

In a preferred embodiment of the invention, the step of enhancing the ABC transporter activity comprises the step of transforming said micro-organism with at least one vector comprising a polynucleotide encoding a homologous or heterologous ABC transporter protein. According to another embodiment of the invention the method comprises the use of polynucleotides encoding an ABC transporter polypeptide selected from the group consisting of SEQ ID NO. 7, 8, 9, 11, 12, 14, 15, 17, 18, 19, 21, 22, 24, 25, 26, 29, 30, 32, 33, 34, 35, 37, 38, 40, 41, 43, 44, 46 and 47 or substantially homologous peptides. The micro- organisms useful as a source for a polynucleotide according to the present invention, may be micro-organisms capable of producing ß-lactam compounds either naturally or upon induction or mutation according to methods known in the art. In a preferred embodiment of the invention, micro-organisms are ß- lactam-producing fungi, filamentous fungi or filamentous bacteria, more preferably Penicillium, Acremonium, Saccharomyces, Hansenula, Pichia and Aspergillus strains. Most preferably Penicillium chrysogenum, Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Acremonium chrysogenum or Aspergillus nidulans, or Actinomycetes like Streptomyces clavuligerus, Nocardia lactamdurans, Flavobacterium sp.

The isolated polynucleotide according to the invention comprises a nucleotide sequence encoding an ABC transporter polypeptide may suitably be of genomic or of cDNA origin, for instance obtained by preparing a genomic or a cDNA library and screening for DNA sequences coding for all or part of the

polypeptide by hybridisation using at least part of the novel nucleotide sequence as disclosed herein as a probe applying standard techniques (cf.

Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd. Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 1989). The isolated polynucleotides of the invention comprising the gene or cDNA encoding a ABC transporter polypeptide may also be prepared by polymerase chain reaction using specific primers advantageously primer derived from the nucleotide sequence as disclosed herein. To this end use can be made, for instance of the method described in US 4, 683, 202 or by Saiki et a/. (Science, 239 (1988) : 487- 491).

The isolated polynucleotide according to a preferred embodiment of the invention may be selected from the group consisting of polynucleotides as depicted in SEQ ID NO 7, 8, 9, 11, 12, 14, 15, 17, 18, 19, 21, 22, 24, 25, 26, 29, 30, 32, 33, 34, 35, 37, 38, 40, 41, 43, 44, 46 and 47 or sequences substantially homologous thereto.

A homologous ABC transporter polynucleotide according to the invention comprises a polynucleotide that is obtained from a micro-organisms belonging to the same species as the micro-organism to which said polynucleotide is transformed, whereas a heterologous ABC transporter comprises a polynucleotide that is obtained from a micro-organisms belonging to another species as the micro-organism to which said polynucleotide is transformed.

Polynucleotide and polypeptide sequences that are substantially homologous to the polynucleotide or polypeptide sequences as referred to in present invention can be determined using the method described in experiment 2. In this experiment it is described how the percentage of sequence identity and percentage of similarity can be determined. Basically, a comparison of the sequences of two polynucleotides (or more than two) may be restricted to a "comparison window"in order to determine sequence similarity in a local region of the two polynucleotides. A"comparison window"is defined to be a continuous series of at least 20 nucleotides in a first polynucleotide sequence,

which when aligned with a reference sequence of at least 20 contiguous nucleotides, enables a comparison of the first polynucleotide and reference sequences. The segment of contiguous nucleotides in the"comparison window" of the first polynucleotide may comprise additions and/or deletions such as, for example, gaps, of up to 20% of the reference sequence. Local homology or sequence identity within the"comparison window"of the first polynucleotide and reference polynucleotide may be achieved via alignment of the sequences according to the algorithm of Smith and Waterman (Appl. Math. 2 : 482 (1981), or by a another suitable alignment method or algorithm (Needleman and Wunsch, J. Mol. Bio. 48 : 443 (1970) ; Pearson and Lipman, Proc. Natl. Acad.

Sci. (USA) 85 : 2444 (1988)).

Sequence identity is established when the first polynucleotide and reference polynucleotide exhibit complete sequence similarity on a nucleotide by nucleotide basis over the span of the"comparison window". However, the invention contemplates polynucleotides having substantial identity with a reference polynucleotide, wherein a polynucleotide having substantial identity with the reference polynucleotide exhibits the same function as the reference polynucleotide. By"substantial identity"is meant that a polynucleotide has nearly identical nucleic acid bases in a"comparison window"located in positions corresponding to those of the reference polynucleotide. The number of matched base positions divided by the number of total positions compared, multiplied by 100 percent, yields what is known as the percentage sequence identity (also known as percentage sequence homology) between compared polynucleotides. The invention contemplates a reference sequence having a "comparison window"of at least 20 nucleotides (preferably 20-50 nucleotides).

The reference sequence of this invention is obtained via a PCR reaction on a homologous boxes in the so-called ATP Binding Cassette (ABC-) proteins : the Walker A and B domains. Although the similarities of these sequences to known ABC-transporters can be quite high, this is concentrated in these conserved regions. The variability in sequence identity within the known sequences is due to the existence of two sequence subclasses, determined via

clustal sequence alignment. The above outlined procedure is applicable to all the sequences which are part of this invention.

For the purposes of this invention, the BlastP method and Blast N method (Version 2. 0. 6, release date September 1998, running under the software of Biomax Bioinformatics GmbH, Martinsried, GE) is preferred for determining the degree of identity between two polypeptide sequences or polynucleotide sequences, using the following parameters : Expectation value (E) : 10 Cost to open a gap. 11 Cost to extend a gap. 1 Matrix Blosum62 Polynucleotide and polypeptide sequences which are substantially homologous according to present invention may show a percentage of similarity of at least 60%, preferably at least 80 %, more preferably 90 %, and more preferred at least 95%, 96%, 97%, 98%, 99% with the polynucleotide or polypeptide sequence represented by SEQ ID NO. 7-48 as incorporated in the present. application.

Functional equivalents of the ABC transporter protein according to the present invention include nucleotide sequences encoding a ABC transporter polypeptide which may have one or more amino acid substitutions, deletions or additions as compared to their natural counterparts and still resulting in a functional ABC transporter polypeptide.

Fragments of the ABC transporter protein of present invention may include biologically active fragments of the polypeptide comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the ABC transporter protein, which include fewer amino acids than the full length protein. Typically, biologically active fragments comprise a domain or motif with at least one activity of the ABC transporter protein. Amino acid substitutions may encompass changes made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic

nature of the residues involved. These changes are preferably of a minor nature (that is conservative amino acid substitutions that do not adversely affect the folding or activity of the protein), small deletions (typically of one to about 30 amino acids), small amino-or carboxyl-terminal extensions (such as an amino- terminal methionine residue), a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (such as a poly- histidine tract, an antigenic epitope or a binding domain). See in general Ford et al. (Protein Expression and Purification, 2 (1991), 95-107,).

Examples of conservative substitutions are substitutions within the respective groups of + basic amino acids (such as arginine, lysine, histidine), + acidic amino acids (such as glutamic acid and aspartic acid), + polar amino acids (such as glutamine and asparagine), + hydrophobic amino acids (such as leucine, isoleucine, valine), + aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and * small amino acids (such as glycine, alanine, serine, threonine, methionine).

It will be apparent to persons skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still have to result in an active polypeptide. A test which can be applied to determine the ABC transporter activity of a polypeptide is described in example12.

Amino acids essential to the activity of the ABC transporter polypeptide of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science, 244 (1989) : 1081-1085). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity to identify amino acid residues that are critical to the activity of the molecule. Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photo-affinity labelling. See, for

example, de Vos et al. (Science, 255 (1992) : 306-312), Smith et al. (J. Mol.

Biol., 224 (1992) : 899-904) and Wlodaver et al. (FEBS Lett., 309 (1992) : 59- 64).

Another embodiment of the invention provides for a micro-organism transformed with at least one vector comprising at least one polynucleotide encoding a microbial ABC-transporter. Said polynucleotide might be operably linked to a regulatory sequence, such as a heterologous or homologous promoter, or a constitutive or regulatable promoter. The term"operably linked" indicates that the sequence encoding at least part of a microbial ABC transporter and the regulatory sequence are arranged so that they function in concert for their intended purposes, e. g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide.

An overview of fungal promoters can be found in, for instance, Applied Molecular Genetics of filamentous fungi (Kinghorn, Turner (eds.), Blackie, Glasgow, UK, 1992). Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et a/., EMBO J., 4 (1985) : 2093-2099,) or the tpiA promoter. Examples of other useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral, B-amylase, A. niger acid stable a- amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase, P. chrysogenum ACV synthetase, P. chrysogenum isopenicillin N synthase, P. chrysogenum acyltransferase, P. chrysogenum phosphoglycerate kinase, P. chrysogenum gene Y. Preferred are the A. niger glucoamylase or P. chrysogenum promoters.

The DNA sequence of the invention encoding the ABC-transporter polypeptide according to the invention may also, if necessary, be operably connected to a suitable terminator.

The vector may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously

replicating vector, i. e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e. g. a plasmid.

Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.

The recombinant vector may also comprise a selectable marker, e. g. a gene the product of which complements a defect in the host cell, or one which confers resistance to a drug. Examples of the latter are phleomycin or hygromycin. For filamentous fungi, additional selectable markers include amdS, pyrG, argB, niaD, facA, and sC (Applied Molecular Genetics of Filamentous fungi (ibid.), Biotechnology of Filamentous fungi, Finkelstein, Ball (eds.), Butterworth-Heinemann, Boston, 1992).

It is also possible to introduce the vector comprising the DNA sequence of the invention encoding a ABC transporter polypeptide into a host cell on a DNA fragment separate from the vector comprising a selectable marker, by a so-called co-transformation process.

In another embodiment the invention relates to the transformed host cell comprising the vector that contain the polynucleotide according to SEQ ID NO selected from the group consisting of : 7, 8, 9, 11, 12, 14, 15, 17, 18, 19, 21, 22, 24, 25, 26, 29, 30, 32, 33, 34, 35, 37, 38, 40, 41, 43, 44, 46 and 47 or a sequence substantially homologous thereto.

A preferred embodiment of the invention is a process for enhancing ABC transporter activity comprising expression from the host cell under conditions sufficient for the production of the polypeptide according to SEQ ID.

10, 13, 16, 20, 23, 28, 31, 36, 39, 42, 45 or 48 or a fragment or functional equivalent thereof.

Another embodiment is the use of an isolated polynucleotide according to the invention or a vector comprising said polynucleotide in a method for the production of-lactam compounds in a micro-organism.

Another embodiment of the invention provides a method for producing beta-lactam compounds in a miro-organism comprising the step of enhancing

ABC-transporter activity of said micro-organism and subsequently culturing said micro-organism under conditions allowing the production of said p-lactam compounds.

The invention also provides an isolated ABC transporter encoded by an ABC transporter gene comprising at least part of the nucleotide sequence according to SEQ ID NO. 7, 8, 9, 11, 12, 14, 15, 17, 18, 19, 21, 22, 24, 25, 26, 29, 30, 32, 33, 34, 35, 37, 38, 40, 41, 43, 44, 46 and 47 or a functional equivalent thereof.

The invention also provides a method for producing a ß-lactam compound or intermediate from a cultured micro-organism comprising transforming a ß-lactam-producing micro-organism with an isolated polynucleotide encoding the microbial ABC-transporter according to the invention under the control of a suitable promoter, and subsequent culturing the R-lactam-producing micro-organism under conditions which permit (i) synthesis of a ß-lactam compound, (ii) expression of the isolated polynucleotide encoding the microbial ABC- transporter under the control of a suitable promoter, and (iii) ABC-transporter-mediated secretion of the ß-lactam compound by the micro-organism into culture media, and thereafter, recovery of the secreted ß-lactam compound from the culture media.

Another embodiment of the invention is the use of an isolated polynucleotide encoding the microbial ABC-transport protein according to the invention under the control of a suitable promoter in the production of a secreted microbial ß-lactam compound.

EXAMPLE 1 INTRACELLULAR ACCUMULATION OF ß-LACTAM ANTIBIOTICS AFTER INHIBITION OF ABC-TRANSPORTERS P. chrysogenum was cultivated in 100 ml shake flasks with 25 ml of defined medium containing lactose as the carbon source to ensure non- repressing conditions for penicillin production (280 rpm at 25 degrees Celsius).

Table l : Intracellular accumulation ß-lactam antibiotics as a result of the inhibition of ABC transporters. Inhibitor1 Intracellular Intracellular penicillinG isopenicillinN conc. 2 conc. 2 Control without phenyl acetic acid - 0 5.2 Control with phenyl acetic acid 0. 4 0. 1 Addition of inhibitor at the start of BeF 0. 4 0. 1 penicillin production Verapamil 1. 2 0 Cyclosporin 0. 6 0 A Control with 100mM penicillinG 1. 8 0. 1 incubation Addition of inhibitor at the end of the BeF 3. 1 0. 1 fermentation, after 1 hour followed Verapamil 5. 3 0 by the 100mM penicillinG incubation Cyclosporin 6. 1 0 A

1Berilliumfluoride was added in the a final concentration of 400pM BeSO4 and 2mM NaF ; together with 400µM ATP, 4mM MgSO4 and 0.1mM EDTA.

Verapamil and cyclosporinA were added in a final concentration of 20µM and 2, uM, respectively ; together with 40pM vanadate, 4 mM MgCI2 and 4mM ATP.

2 In arbritary units

The precursor for penicillinG, phenyl acetic acid, was added as potassium salt 48 hours after inoculation. The ABC transporter inhibitors were added either at the start of penicillin production (48 hours) or at the end of the fermentation (144 hours). In the latter case 100 mM penicillinG was added after the cultures were incubated for 1 hour with the inhibitors. Exactly 1 hour later all cultures were harvested by filtration and washed three times with cold physiological salt (0. 9%).

These washing steps are very critical ; they must be done thoroughly. The mycelial pellets were frozen in liquid nitrogen, freeze-dried and the internal penicillinG concentration was determined by NMR.

There was a clear increase in intracellular penicillinG when the organism was incubated with ABC transporter inhibitors (see Table I). The best results were obtained with verapamil and cyclosporinA, where a two-to-three fold increase in intracellular penicillinG was found.

This is a clear indication that ABC transporters are directly involved in the export of p- ! actam antibiotics as penicillinG.

EXAMPLE 2 DESCRIPTION OF THE METHODS USED IN CLONING ABC TRANSPORTER FRAGMENTS PCR oligonucleotides Degenerate oligonucleotides complementary to the rather conserved C-terminal WalkerA and WalkerB motifs (present in the nucleotide binding domain of ABC- transporters) were designed (see Figure 1). As a result, the forward primers (complementary to the WalkerA motif ; SGA/CGKT/ST), 5'-TCI GGI G/TG/CI GGI AAA/G A/TCI AC-3' (SEQ ID NO. 3) and 5'-TCI GGI G/TG/CI GGI AAA/G AGC/T AC-3' (SEQ ID NO. 4) and the reverse primers (complementary to the WalkerB motif ; DEA/PTSA/GLD), 5'-TCI AA/GI G/CCI GAI GTI GG/CC/T TCA/G TC-3' (SEQ ID NO. 5) and 5'-TCI AA/GI G/CCA/G CTI GTI GG/CC/T

TCA/G TC-3' (SEQ ID NO. 6) were applied.

RT-PCR Total RNA was isolated from a high penicillin producing strain of P. chrysogenum (e. g. Panlabs P2) under penicillin producing conditions to enlarge the presence of putative penicillin transporter transcripts in the total RNA pool. RT-PCR was performed on these total RNA isolates using the above mentioned degenerate primers. cDNA was synthesized with AMV reverse transcriptase (Boehringer Mannheim) using the reverse (WalkerB) primers (incubation for 1 hr at 50°C). This cDNA material was subsequently used in a PCR procedure using the following program. As the standard PCR programs (with one standard cycle) gave no satisfactory results, we applied a special"staircase"program to obtain good PCR products A'hot start'was made for 3 min at 95°C after which 10 cycles of 0. 15 min at 95°C, 0. 30 min at 55°C and 0. 40 min at 72°C were done. This was followed by 20 cycles in which the last step at 72°C was extended each cycle with 20 sec., followed by 5 min at 72°C.

Cloning of PCR fragments PCR reaction products were analyzed by electrophoresis on agarose gels containing ethidium bromide. PCR products of proper size (about 400 bp) were isolated from gel, purified and cloned in the pGEM-T Easy vector (Promega).

This vector contains 3'protruding thymidine residues which makes ligation with PCR-fragments that contain 3'protruding adenosine residues (when generated with Taq-polymerase, for example), possible.

AFLP analysis PCR techniques as described above restrict ones choice to one set of genes.

As another approach to obtain genes involved in the export of-lactams a DNA fingerprinting technique called AFLP (selective restriction fragment amplification) is employed.

The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA.

The technique involves three steps : (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplifie fragments.

PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of their nucleotide sequence. The method allows the specific co- amplification of high numbers of restriction fragments.

The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplifie and detected on denaturing polyacrylamide gels.

The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity. It can also be used in combination with RNA and thereby applied to pinpoint differences in mRNA levels between two strains or two conditions. This is called : Differential Display.

We applied this technique to identify genes which are expressed higher with increased penicillin titers.

Sequence analysis After determining the sequences of the PCR-fragments, homology searches and sequence comparisons were executed as following. The terms used herein to describe the sequence relationships between two or more polynucleotides are well known in the art, as exemplified in U. S. Patent Nos. 5, 912, 120 and 5, 914, 393.

Polynucleotide sequences discussed herein refer to single or double- stranded polymers of deoxyribonucleotide or ribonucleotide bases as read from their respective 5'to 3'ends. A reference sequence in this regard is defined as a polynucleotide sequence, at least 20 nucleotides in length, used as a basis for a comparison between sequences. The reference sequence comprise a full- length cDNA or gene sequence in a sequence listing, or the reference sequence comprise a portion of a full-length cDNA or gene sequence.

A comparison of the sequences of two polynucleotides (or more than two) may be restricted to a"comparison window"in order to determine sequence similarity in a local region of the two polynucleotides. A"comparison window"is defined to be a continuous series of at least 20 nucleotides in a first polynucleotide sequence, which when aligned with a reference sequence of at least 20 contiguous nucleotides, enables a comparison of the first polynucleotide and reference sequences. The segment of contiguous nucleotides in the"comparison window"of the first polynucleotide may comprise additions and/or deletions such as, for example, gaps, of up to 20% of the reference sequence. Local homology or sequence identity within the "comparison window"of the first polynucleotide and reference polynucleotide may be achieved via alignment of the sequences according to the algorithm of Smith and Waterman (Appl. Math. 2 : 482 (1981), or by a another suitable alignment method or algorithm (Needleman and Wunsch, J. Mol. Bio. 48 : 443 (1970) ; Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85 : 2444 (1988)).

Sequence identity is established when the first polynucleotide and reference polynucleotide exhibit complete sequence similarity on a nucleotide by nucleotide basis over the span of the"comparison window". However, the invention contemplates polynucleotides having substantial identity with a reference polynucleotide, wherein a polynucleotide having substantial identity with the reference polynucleotide exhibits the same function as the reference polynucleotide. By"substantial identity"is meant that a polynucleotide has nearly identical nucleic acid bases in a"comparison window"located in positions corresponding to those of the reference polynucleotide. The number

of matched base positions divided by the number of total positions compared, multiplie by 100 percent, yields what is known as the percentage sequence identity (also known as percentage sequence homology) between compared polynucleotides.

The reference sequence of this invention is obtained via a PCR reaction on a homologous boxes in the so-called ATP Binding Cassette (ABC-) proteins : the Walker A and B domains. Although the similarities of these sequences to known ABC-transporters can be quite high, this is concentrated in these conserved regions. The variability in sequence identity within the known sequences is due to the existence of two sequence subclasses, determined via clustal sequence alignment. Yeast ABC-transporters have been analyzed for sequence and function (substrate) relationship to other known ABC- transporters, and classified as MDR-like ABC-transporters and ABC- transporters involved in the secretion of substances (Decottignies and Goffeau, Nature Gen. 15 : 137-145 (1998)). The substrate specificity of these proteins is in part conferred to the regions outside the conserved domains.

The above outlined procedure is applicable to all the sequences which are part of this invention.

For the purposes of this invention, the BlastP or BlastN method (Version 2. 0. 6, release date September 1998, running under the software of Biomax Bioinformatics GmbH, Martinsried, GE) is preferred for determining the degree of identity between two amino acid sequences, using the following parameters : Expectation value (E) 10 Cost to open a gap : 11 Cost to extend a gap : 1 Matrix : Blosum62

EXAMPLE 3 PCR AMPLIFICATION OF ABC TRANSPORTER FRAGMENTS Degenerate oligonucleotides complementary to the rather conserved WalkerA and WalkerB motifs, present in the nucleotide binding domain (NBD) of ABC-transporters, were used in RT-PCR reactions (Reverse Transcriptase Polymerase Chain Reaction) on total RNA isolated from P. chrysogenum strains (e. g. Wisconsin 54-1255 and Panlabs P2). To enlarge the presence of putative penicillin transporter transcripts in the total RNA pools, the above mentioned strains were grown under penicillin producing conditions. The RT- PCR resulted in the formation of a DNA fragment of about 400 bp, which is the expected size. The fragment was cloned in the vector pGEM-T Easy (Promega) and 32 individual clones were analyzed. Ten different sequences were obtained (due two multiple clones containing identical sequences). The sequence of these fragments (see SEQ ID NO. 7, 11, 14, 17, 21, 24, 29, 32, 37 and 40) showed high sequence identity to several ABC transporter genes from other organisms.

EXAMPLE 4 AFLP ANALYSIS IDENTIFYING ABC TRANSPORTER FRAGMENTS Samples of a p-tactam producing fed-batch culture of Penicillium chrysogenum were used to isolate RNA. An AFLP analysis as described above between the samples A and B (see Figure 2) was performed. Two transcripts were found which were approximately 8-10 times increased during the fermentation. Surprisingly, these two polynucleotide sequences (see SEQ ID NO. 43 and 46) showed a high sequence identity to several ABC transporter genes from other organisms.

This indicates that there is need for high ABC transporter activity when the penicillinG concentration is high, which confirms the direct role for these transporters in the export of penicillinG.

EXAMPLE 5 NORTHERN ANALYSIS : TRANSCRIPTION OF THE ISOLATED PENICILLIUM RT-PCR FRAGMENTS ENCODING ABC-TRANSPORTERS The isolated Penicillium chrysogenum ABC transporter clones were subjected to an expression study to determine the conditions under which the transporter genes are regulated. Northern analysis was conducted on RNA samples obtained from the organism subjected to two different growth conditions. Total RNA was isolated from a high yielding strain grown on either glucose or under conditions optimal for penicillinV production. RNA electrophoresis, blotting and hybridizations were performed as described by Schuurs et al., 1997 (Genetics 147 : 589-596) Northern analysis showed a remarkable elevated expression of the 5 tested ABC transporter genes (aa1, aa5, aa6, aa7 and aa10) during penicillinV fermentation conditions (see Figure 3). Thus, during penicillin fermentation, P. chrysogenum specifically expresses ABC-transporter genes whose protein product is shown to be a transporter of p-lactam.

EXAMPLE 6 DNA ARRAY HYBRIDISATION : TRANSCRIPTION OF THE ISOLATED PENICILLIUM RT-PCR FRAGMENTS ENCODING ABC-TRANSPORTERS All PCR-isolated Penicillium chrysogenum ABC transporter clones were subjected to an expression study using a DNA array. Opposite to Northern analysis here the DNA is linked to a filter and hybridized with labeled mRNA.

Moreover, we applied the technique in such a way that it is possible to omit the RT-PCR step normally applied in DNA experiments and directly labeled the mRNA.

The filter was prepared by spotting 12) je of denatured pGEM-T Easy vector containing the PCR amplified fragment (as described in example 3) on a Hybond-N+ filter (Amersham Pharmacia Biotech) ; this means that approximately 1 ug of each ABC transporter fragment is on the filter.

The RNA samples were obtained from the strain Wisconsin54-1255 subjected to two different growth conditions. Total RNA was isolated from the fungus grown on either glucose or under conditions optimal for penicillinG production. RNA was prepared by mortaring the washed and frozen fungal pellet with trizol. The continuing steps were essentially as described in laboratory manuals (e. g. Sambrook et al (1989) Molecular Cloning : a laboratory manual (2"d ed), Cold Spring Harbor Press, CSH, NY). mRNA was isolated from 3, ug of total RNA with the Oligotex mRNA Midi Kit (Qiagen).

Prior to hybridization 1ug of mRNA was labeled according to the ECL kit (Pharmacia Amersham Biotech) : 1, ug/20 pI mRNA was denatured by boiling for 5 minutes in a water bath 'cafter 5 minutes the mRNA was directly placed on ice for 5 minutes 20 uI labeling reagent was added and gently mixed 20 ul glutaraldehyde solution was added and mixed the mixture was incubated for 15 minutes at 37 °C The blot was first pre-hybridized for 30-45 minutes with hybridizationbuffer (500 ml ECL hybridizationbuffer+ 25 mg ECL blockingreagent + 14 g NaCL) at 42 °C in a tube in a rotisserie oven. The labeled probe was added to 50 ml hybridizationbuffer after pre-hybridization. Hybridization was carried out overnight at 42 °C in a tube in a rotisserie oven. Afterwards, the blot was washed 2 times for 20 minutes at 42 °C with primary washbuffer (6 M ureum, 0. 4% SDS, 0. 2 x SSC) in a tube in a rotisserie oven and washed 2 times for 5 minutes at roomtemperature with secondary washbuffer (2 x SSC) in a container with gentle agitation. Detection was started by mixing 10 ml ECL detection reagent 1 with 10 ml ECL detection reagent 2. The washed blots were placed in the detection mixture for one minute, wrapped in plastic bags and

placed in a cassette with a photographic film.

As depicted in Figure 4 all 10 ABC transporter genes clearly show elevated expression during penicillinG fermentation conditions.

EXAMPLE 7 INDUCTION OF TRANSCRIPTION OF THE ISOLATED PENICILLIUM RT-PCR FRAGMENTS, ENCODING ABC-TRANSPORTERS, BY P-LACTAM ANTIBIOTICS Penicillium chrysogenum was pre-grown overnight on a rich medium (e. g. YEPD) and then diluted into a defined medium containing lactose as the sole carbon source to ensure derepressing conditions. After 24 hours the culture was washed and resuspended in fresh medium containing 100mM penicillinV, 10mM 7-amino deacetoxycephalosporanic acid (7-ADCA) and 100mM adipoyl-7-ADCA, respectively. At several time-points samples were taken for RNA isolation with trizol. This material was used as a template for a semi-quantitative RT-PCR, using RT-PCR beads from Amersham/Pharmacia.

The PCR program was as follows : 30 minutes at 42°C (the RT-step) ; 5 minutes at 95°C ; followed by 20 cycles of 30 seconds at 95°C, 30 seconds at 59°C and 45 seconds at 72°C ; followed by a final step of 7 minutes at 72°C and cooling at 4°C. The primers used were : aa7 forward primer (SEQ ID. 49) : 5'-ATT GGC TGA GAC AGC AAA TCT CGC-3'. aa7 reverse primer (SEQ ID. 50) 5'-AGG ATC GGA AAT GAG GGC GC-3'. aa10 forward primer (SEQ ID. 51) : 5'-AGC GGT TCT ATC TCC CAG TCG GAG G-3'. aa10 reverse primer (SEQ ID. 52) : 5'-CAC GAG CGA TGG CAA TAC GTT GC-3'. dd062 forward primer (SEQ ID.

53) 5'-TGG AGT GCC CGA TGA TGT ATG-3'. dd062 reverseprimer (SEQ ID.

54) : 5'-CGC GAG GTT GCT ACC TGG AGA G-3'.

Of the three ABC transporters tested (aa7, aa10and DD062) all responded positively to the addition of p-tactams (see respectively Figure 5, 6 and 7).

EXAMPLE 8 INDUCTION OF TRANSCRIPTION OF AA5, ENCODING AN ABC-TRANSPORTER, BY PENICILLING Penicillium chrysogenum was pre-grown to mid-logarithmic phase on a rich medium (e. g. YEPD) when 100mM penicillinG was added to the culture. At several time-points samples were taken for RNA isolation with trizol. This material was analyzed with a Northern blot (as described in example 5) using the PCR-fragment cloned in the pGEM-T easy vector (as described in example 3) as a probe.

Theinduction of penicillinG on the transcription of the ABC transporter gene takes place in the presence of high glucose concentrations and subsequently the absence of penicillin production (see Figure 8).

EXAMPLE 9 CLONING OF THE COMPLETE GENES Aa7 and aa10 A phage lambda genomic P. chrysogenum DNA library was successfully screened using the 400 bp fragments from the pGEM-T Easy vector (see example 3) as probes. Hybridization was carried out overnight in : 1 mM EDTA ; 0. 5 M Na2HP04, pH 7. 2 ; 7% SDS ; 1% Blocking agent (Cat. No. 1096176, Roche Molecular Biochemicals). Washing was done 2X 30 minutes in : 1 mM EDTA ; 40 mM Na2HP04, pH 7. 2 ; 5% SDS, followed by 2X 30 minutes in : 1 mM EDTA ; 40 mM Na2HP04, pH 7. 2 ; 1% SDS. All steps were performed at the stringent temperature of 65°C. The probes were labeled with 32P via PCR (25 microCi 32P dCTP, standard protocol Oligolabeling Kit (27-9250-01, Pharmacia Biotech.). DNA from positive clones was excised in vivo by picking the core phage of the plaque of interest ; transfer it into 500psi buffer and 20, u1 chloroform ; the subsequent incubation of 2 hours at room temperature releases the phage particles. 200u1 of XL1-Blue cells (grown in LB-medium to an OD600=1) were mixed with 200p1 of the phage particles and 10p1 R408 helper phage

(Predigested Lambda ZAP II/EcoRl Cloning Kit ; Catalog&num 236211, Stratagene).

This was incubated for 15 minutes at 37°C, and afterwards 5 ml 2XTrypton medium was added for another incubation of 3 hours at 37°C. The temperature was increased to 70 °C for 20 min, the mixture was spun down for 5 minutes at 4000 g and transformed to XL1-Blue host cells.

These clones were sequenced (see SEQ ID NO. 25 and 34). The gene aa7 contains one putative intron, while aa10 contains no introns (see SEQ ID NO. 26 and 35).

Aa1 and aa5 These clones were isolated directly from the phage lambda library. The methods employed were the same as described for aa7 and aa10. The genomic sequences of aa1 and aa5 are depicted in SEQ ID NO. 8 and 18. As presented in SEQ ID NO. 9 and 19 these genes contain a lot more introns : 8 and 4, respectively.

Aa3. aa4. aa6. aa8. aa20. aa32. dd034 and dd062 Chromosomal DNA of a high-production strain of Pencillium chrysogenum (e. g.

Panlabs P2) was digested with 5 different restriction enzymes ; EcoRl, Hindlll, Sall, BamHl and Pstl. 3 ug of the digested chromosomal DNA was separated on an 0. 4 % agarose gel with ethidium bromide and the DNA was transferred on a hybond-N+ filter (Pharmacia Amersham Biotech) by vacuum blotting and afterwards cross-linked by UV light.

As probes we amplifie the approximately 400 bp fragments of the ABC- transporter genes by PCR from the pGEM-T easy vectors (see example 3). The PCR products were purified from gel with the Gel Extraction Kit (Qiagen). The hybridization was essentially the same as described in example 6. Hybridization signals were obtained for all probes.

EXAMPLE 10 SIMILARITY/IDENTITY SEARCHES USING BLASTP The longest protein sequence derived from each ABC transporter gene described in this invention were analyzed using the blastP software with settings as described in example 2. DNA Protein Similari Identity Closest homologue ABC translation sequence ty score trans depicted in depicted score (%) porter SEQ ID inSEQ ID (%) gene NO : NO : aa1 9 10 71 54 TREMBL AF071410 1 gene: "atrC"; product :"ATP-binding cassette multidrug transport protein ATRC" ; Emericella nidulans aa3@ 12 13 67 46 TREMBL AC004411 16 gene: "At2g47000" ; product :"putative ABC transporter" ; Arabidopsis thaliana aa4 15 16 89 83 PIRIT30566 ATP-binding cassette multidrug transport protein-Emericella nidulans aa5 19 20 79 60 TREMBL ANPGP2_1 product :"ATP- binding cassette multidrug transporter"; A. nidulans aa6 22 23 92 87 PIRIT30567 ATP-binding cassette multidrug transport protein-Emericella nidulans aa7 26 28 66 47 TREMBL AF173826_1 gene: "abcD"; product: "ABC-transporter"; Emericella nidulans aa8 30 31 90 86 TREMBLIMGR243113 1 gene :"atr2"; product :"putative ABC transporter"; Mycosphaerella graminicola aal0 35 36 85 75 TREMBLIU62934_1 gene :"mdrl" ; product :"multidrug resistance protein 1", Aspergillus fumigatus aa20 38 39 85 77 PIR T43022 ATP-binding multidrug cassette transport protein-Botryotinia fuckeliana aa32 41 42 78 64 PATENTPROT | Y21815 A. nidulans atrC polypeptide ddO34 44 45 89 69 TREMBL BFU6217_1 gene :"atbB"; product :"ABC transporter" ; Botryotinia fuckeliana dd062 47 48 56 38 PIR T43551 multidrug resistance protein fnx1 fission yeast (Schizosaccharomyces pombe)

EXAMPLE 11 TRANSFECTION OF THE ABC TRANSPORT PROTEIN GENE INTO A P-LACTAM-PRODUCING MICRO-ORGANISM The above described full genomic gene was used as template DNA for a PCR reaction with two homologous primers, which insert a Ndel restriction site just 5'of the start codon and a Nsil restriction site just 3'of the stop codon. The resulting fragment was cloned into an expression vector. For this the vector pISEWA, as described in PCT/EP99/03455, was digested with Ndel and Nsil.

The pcbC promoter was replace by one of the suitable promoters mentioned in the text. The resulting plasmids were transformed to Penicillium chrysogenum (using co-transformation with the A. nidulans amdS gene under control of the A. nidulans gpdA promoter) and the penicillin production was tested as described above and found to be enhanced.

EXAMPLE 12 DISRUPTION OF THE ABC TRANSPORT GENE.

The above described full genomic genes of aa7 and aa 10 were disrupted by inserting a heterologous selectionmarker like the Aspergillus nidulans amdS gene under control of the A. nidulans gpdA promoter.

Transformants growing on agar plates with acetamide as the sole nitrogen source were selected. Colony PCR was employed to select the homologous integrants from the random transformants. Southern analysis confirmed the disruption of the aa7 and aa10 gene described in this invention.

DESCRIPTIONS OF FIGURES Figure 1.

Schematic representation of the C-terminal nucleotide binding domain (NBD) found in ABC-transporters. Examples of some WalkerA and WalkerB sequences found in a variety of ABC-tranporters are given. The boxed amino acid sequences were used to develop degenerate primers. (AtrA and AtrB of A. nidulans ; MDR1 of human ; CDR1 of C. albicans ; ATM1 and STE6 of S. cerevisiae ; MaIK and HisP of E. colt).

Figure 2.

Schematic representation of a typical penicillin production curve with Penicillium chrysogenum. Samples at indicated timepoints A and B were used for mRNA isolation and subsequent AFLP analysis.

Figure 3.

Northern analyses showing expression of several isolated ABC-transporter gene fragments, from a high yielding strain (e. g. Panlabs P2 or Wisconsin54- 1255) grown on glucose-containing medium or under conditions optimal for penicillin V production. Expression of the pcbC gene (encoding the penicillin biosynthetic enzyme isopenicillin N synthase) was used as a positive control ; specific expression under penicillin producing conditions. 18S rRNA and the actA gene (actin gene, isolated from a cDNA library by PCR) were used to check for equal gel loading.

Figure 4.

DNA filter analysis showing expression of several isolated ABC-transporter gene fragments. mRNA was isolated from Wisconsin54-1255 grown on glucose-containing medium or under conditions optimal for penicillinG production. All genes show elevated expression when producing penicillinG.

Top-panel : Wisconsin54-1255 producing penicillinG

Bottom-panel : Wisconsin54-1255 grown on glucose (not producing penicillinG).

Figure 5.

Time-course experiments after addition of p- ! actam antibiotics to growing cultures of Penicillium chrysogenum aa7 mutants. In the experiment the actin gene was used a a control for equal amounts of starting material. The time is indicated in minutes.

Figure 6.

As in figure 5, but now for Penicillium chrysogenum aa 10 mutants.

Figure 7.

As in figure 5, but now for Penicillium chrysogenum DD062 mutants.

Figure 8.

Time-course analysis of aa5 transcription after addition of penicillinG to a growing culture of Penicillium chrysogenum.