CA2322241A1 - Antibiotic production - Google Patents

Abstract

Methods are provided for increasing and altering the timing of antibiotic production in Streptomyces species, particularly S.
coelicolor and S. lividans, by functionally deleting the S.
coelicolor scbA and scbR genes, respectively, or their homologues. Also provided are strains having such mutations, and methods of producing antibiotics using such strains. Also provided are methods for identifying strains in which functional deletion of the scbA and/or scbR genes or their homologues leads to the above effects.

Description

ANTIBIOTIC PRODUCTON .
FIELD OF THE INVENTION
The present invention relates to methods and materials for controlling antibiotic production in species of Streptomyces, especially increasing antibiot~ production in Streptomyces coelicolor and S. lividans.
INTRODUCTION
In addition to undergoing a complex process of morphological differentiation, streptomycetes are renowned for their ability to produce a vast array of secondary metabolites, many of which possess antibiotic or other pharmacologically useful activities. Most of these secondary metabolites are the products of complex biosynthetic pathways that are activated in a growth phase-dependent manner. While the production oz antibiotics in liquid culture is generally limited to stationary phase, in surface-grown cultures it usually coincides with the onset of morphological differentiation (Chater and Bibb, 1997).
In several streptomycetes, y-butyrolactones (GBLs) have been shown to play important, if not crucial, roles in determining the onset of antibiotic production and morphological differentiation (Horinouchi and Beppu, 1994; Yamada, 1999). The most characterised Y-butyrolactone is A-factor (2-isocaryloyl-3R-hydroxymethyl-y-butyrolactone), which is required for both streptomycin production and sporulation in Streptomyces griseus (Mori, 1983; Horinouchi and Beppu, 1994). Other well-studied Y-butyrolactones include virginiae butanolides (V8), which appears to control virginiamycin production in Streptomyces virginiae (Yamada et al., 1987; Kondo et al., 1989), and IM-2, which elicits the production of showdomycin and minimycin in Streptomyces lavendulae FRI-5 (Sato et al., 1989).
Although the details of A-factor synthesis have not been elucidated, a putative A-factor biosynthetic gene, afsA, was cloned from S. griseus and sequenced. Its predicted translation product does not resemble any protein of known function (Horinouchi et al., 1989). afsA mutants of S. griseus are deficient in A-factor synthesis, and hence in streptomycin production and sporulation.
Moreover, cloning of afsA in multiple copies leads to precocious streptomycin production in S. griseus, and to the production of a compound with A-factor activity in other streptomycetes that normally do not make it (Horinouchi et al, 1985). Culture supernatants of an Escherichia coli strain over-expressing afsA restored streptomycin production and sporulation in an A-factor deficient mutant of S. griseus (Ando et al., 1997).
A-factor is detected in culture supernatants of S.
griseus just before the onset of streptomycin production.
It diffuses freely across the cytoplasmic membrane, and binds with high affinity to a cytoplasmic A-factor-binding protein, ArpA (Onaka et al, 1995). In the absence of A-factor, ArpA acts as a negative regulator of both streptomycin production and sporulation by repressing transcription of the pleiotropic regulatory gene adpA (Ohnishi et al., 1999). Homologues of afsA
and/or arpA have been isolated from several streptomycetes, including S. virginiae (Okamoto et al., 1995; Kinoshita et al., 1997), S. lavendulae (Waki et al., 1997), S. coelicolor (Onaka et al., 1998) and S.
fradiae (Fouces et al., 1999; Bate et al., 1999).
S. coelicolor is the most genetically characterised streptomycete. It produces at least four chemically distinct antibiotics. Two of these, actinorhodin (Act) and undecylprodigiosin (Red), are pigmented. ~'he stationary phase production of Act and Red results from transcriptional activation of the pathway-specific activator genes actII-ORF4 and redD, respectively (Gramajo et al., 1993; Takano et al., 1992). Moreover, production of Act and Red in exponential phase appears to be prevented only by the absence of a threshold concentration of the pathway specific activator proteins.
Recently, four extracellular compounds were identified in culture supernatants of Streptomyces coelicolor A3(2) that elicited the precocious production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) when added to the producing strain; none of the compounds induced morphological differentiation. One of these stimulatory factors, SCB1, was purified to homogeneity and shown by structural elucidation to be a Y-butyrolactone (Takano et al., 2000).
SUMMARY OF THE INVENTION AND DETAILED DESCRIPTION
The present inventors have identified genes of S.
coelicolor which are involved in the regulation of Act and Red production. One gene, scbA, is a homologue of afsA (A-factor synthetase of S. griseus), and the other, scbR, encodes a Y-butyrolactone binding protein. By analogy with the S. griseus system, ScbR was expected to be a repressor of the pathway-specific activator genes actII-ORF4 and redD. Release of such repression upon binding of ScbR by the GBL SCB1 at high cell densities would lead to antibiotic expression.
S
However, it was found that ScbR binds to the transcription start sites of scbA and scbR, and is released by addition of SCB1 from S. coelicolor. An in-frame deletion mutant of scbA (a gene involved in GBL
synthesis) shows overproduction of Act and Red (when lack of antibiotic production might have been expected) and an in-frame deletion mutant of scbR shows delay in Red production and earlier Act production (scbR - see Fig.
5A). These phenotypes therefore differ from what might be expected by analogy to the S. griseus A-factor system.
Moreover, the inventors have found that S. lividans strains carrying the same in-frame deletion mutant of scbA, in place of the wild-type scbA gene usually present in S. lividans, also overproduce Act and Red.
The inventors propose, therefore, that mutations to homolgues of scbA and scbR in other Streptomyces species may have similar effects.
Accordingly, in a first aspect, the present invention provides a method of modifying an antibiotic-producing strain of a Streptomyces species to increase antibiotic production in said strain, the method comprising functionally deleting in said strain a gene which is the scbA gene of Streptomyces coelicolor or a homologue thereof.
In a second aspect, the present invention provides a method of modifying an antibiotic-producing strain of a Streptomyces species to alter the timing of antibiotic production in said strain, the method comprising functionally deleting in said strain a gene which is the scbR gene of Streptomyces coelicolor or a homologue thereof .
While it is appreciated that these effects will not be found in S. griseus and some other Streptomyces species, it is thought that the effects may not be confined to the exemplified species of S. coelicolor and S. lividans. It will be possible for the skilled person to repeat the experimental disclosure presented herein on other Streptomyces species, thereby to identify other species in which similar effects occur. In particular, it will be possible to identify in other species of Streptomyces genes which are homologues of scbR and scbA in an analogous way to the identification herein of scbR and scbA. Following identification of the genes, it will be possible to create strains in which these genes are functionally deleted, and to compare the extent and/or timing of antibiotic production in those modified strains with the extent and/or timing of production in the parent strain. Those modified strains in which similar effects are found to those presented herein are regarded also to be part of the invention.
Accordingly, in a third aspect, the present invention provides a modified strain of a Streptomyces species, the modified strain having a functional deletion of a gene which is the scbA gene of S. coelicolor or a homologue thereof, whereby production of at least one antibiotic in said modified strain is increased compared to a wild-type strain of said Streptomyces species.

Similarly, in a fourth aspect, the present invention provides a modified strain of a Streptomyces species, the modified strain having a functional deletion of a gene which the scbR gene of S. coelicolor or a homologue thereof, whereby the timing of production of at least one antibiotic in said modified strain is altered compared to a wild-type strain of said Streptomyces species.
In a fifth aspect, the present invention provides a method of producing an antibiotic, the method comprising providing a modified Streptomyces strain of any preceding aspect, and culturing said strain under conditions suitable for production of antibiotic.
The method may also comprise the additional step of purifying the antibiotic from the culture medium. It may also comprise the further step of formulating the antibiotic as a pharmaceutical.
The scbR and scbA genes are believed to be new, as is a further gene, designated scbB, which is downstream of scbR and which shows homology to the C-5 ketoreductase gene of S. avermitilis. scbB is predicted to modify the C-6 of SCB1 from keto to hydroxyl, and may therefore be important in providing specificity of SCB1 as the cognate GBL of scbR.
In a sixth aspect, the present invention provides a nucleic acid comprising a nucleotide sequence having at least about 80~ identity with a nucleic acid sequence selected from the group consisting of (1) nucleotides 3032 to 3679, (2) nucleotides 2914 to 1970, and (3) nucleotides 4529 to 3795, reading 5' to 3', of the nucleic acid deposited as EMBL AJ007731, which may alternatively be defined as (1) nucleotides 2261-2908, (2) nucleotides 2142-1199 and (3) nucleotides 3758-3024, respectively of Fig. 14.
As will be evident from Fig. 1, both strands of DNA in this region encode polypeptides. Nucleotide.~umbering is given in relation to the strand which runs from 5' to 3' from right to left in Fig. 1. The sequence of part of this strand is given in Fig. 14. However, the coding sequences of scbA and scbB are on the complementary strand. References to the nucleotide sequences in EMBL
AJ007731 and Fig. 14 which relate to these genes (i.e.
where the nucleotide numbering is shown as [higher number)-[lower number]) should therefore be interpreted as references, to the strand complementary to that shown.
Preferably the nucleic acid sequence identity is at least 85%, 90%, 95%, 98% or 99% or is 100%.
In a seventh aspect, the present invention provides a nucleic acid comprising a nucleotide sequence which encodes a polypeptide having at least about 70% amino acid sequence identity with an amino acid sequence selected from the group consisting of (1) the amino acid sequence of ScbR, as shown in Fig. 9 , (2) the amino acid sequence of ScbA, as shown in Fig. 10, and (3) the amino acid sequence of ScbB, as shown in Fig. 11.
In further aspects, the present invention provides:
polypeptides encoded by the nucleic acid molecules of the sixth and seventh aspects; vectors including the nucleic acids of those aspects, optionally in operative association with control sequences, e.g. promoter and/or enhancer sequences; host cells transfected with said vectors; and methods of producing said polypeptides, comprising culturing said host cells under conditions suitable for polypeptide production and extracting said polypeptides from the culture medium.
As used herein, the term "functional deletion" of a gene may mean any alteration of the nucleic acid in a cell or cells of the strain containing the functional deletion, which alteration has the effect of preventing normal expression of that gene. For example, the gene may comprise a deletion in the coding sequence, leading to a shortened transcript which is translated into a protein lacking the normal function of the expression product of the gene; or the transcriptional and/or translational regulatory sites (e. g. promoter and/or enhancer sequences) may be altered to prevent normal transcription and/or translation of the gene; or the coding sequence may contain an insertion or mutation (e. g. to introduce or produce a stop codon or to cause a shift in reading frame), leading to a non-functional expression product.
Alterations of the coding sequence may be in frame or may cause a shift in reading frame. As a further alternative, the cells) may be modified to produce antisense mRNA, which prevents correct translation, preventing gene expression even if the gene itself is unmodified.
In using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense"
strand of the target gene. See, for example, Rothstein et al, 1987; Smith et a1,(1988) Nature 334, 724-726;
Zhang et a1,(1992) The Plant Cell 4, 1575-1588, English et al., (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995), Plant S Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.
An alternative is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression. See, for example, van der Krol et al., (1990) The Plant Cell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992) The Plant Cell 4, 1575-1588, and US-A-5,231,020.
The complete sequence corresponding to the coding sequence (in reverse orientation for anti-sense) need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene. Total complementarity or similarity of sequence is not essential. The sequence need not include an open reading frame or specify an RNA
that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene.
Preferred Streptomyces species for the practice of the invention are species which possess adjacent and divergent scbA and scbR genes of S. coelicolor or adjacent and divergent homologues thereof, since it is thought that this arrangement of genes may correlate with the effects on amount and timing of antibiotic production seen in S. coelicolor and S. lividans.
The closely related species S. coelicolor, S.
violaceoruber, S. lividans and S. parvulus are particularly preferred. Strains of such species (i.e.
wild-type strains) are commonly available, e.g. from the ATCC, for example under ATCC deposit numbers 12434 for S.
parvulus and 19832 for S. violaceoruber. S. coelicolor A3(2) and S. lividans 66 are particularly preferred wild-type strains and are available from the John Innes Culture Collection (Norwich, UK) under JICC deposit numbers 1147 and 1326, respectively. However, the invention is not limited to such particular strains.
The present invention may exclude the modification of barX and/or farX, the afsA homologues in S. virginiae and S. fradiae, respectively.
A gene of a Streptomyces species or strain, which gene is a "homologue" of or is "homologous" to the scbA gene of S. coelicolor, may be the gene which shows greatest deduced amino acid sequence identity to scbA of all genes of said species or strain; alternatively or additionally, it may be a gene which is capable of specific hybridisation with the amplification product obtained using the primers oligol c5'-GACCACGT(CG)CC(CG)GGCATG) (SEQ.ID.NO.:1) and O 1 i go2 (5'-GTCCTG(CG)TGGCC(CG)GT(CG)AC(CG)CG(CG)AC) (SEQ.ID.N0.:2) to amplify total DNA of said species or strain (bracketed nucleotides indicate positions of degeneracy);
alternatively or additionally, it may be a gene encoding a polypeptide having at least about 35% sequence identity with the deduced amino acid sequence of scbA as shown in Fig. 10, preferably at least about 40% (which,~.s the homology found between scbA and other homologues of the afsA gene of S. griseus) more preferably about 50%, 60%, 65% (which is the homology found between scbA and afsA of S. griseus), 70%, 80%, 90%, or 95%.
A gene of a Streptomyces species or strain, which gene is a "homologue" of or is "homologous" to the scbR gene of S. coelicolor, may be the gene which shows greatest deduced amino~acid sequence identity to scbR of all genes of said species or strain; alternatively or additionally, it may be a gene which is adjacent to and divergent from a gene which is capable of specific hybridisation with the amplification product obtained using the primers oligol (5'-GACCACGT(CG)CC(CG)GGCATG) and oligo2 (5'-GTCCTG (CG) TGGCC (CG) GT (CG) AC (CG) CG (CG) AC) to amplify total DNA of said species or strain (bracketed nucleotides indicate positions of degeneracy); alternatively or additionally, it may be a gene encoding a polypeptide having at least about 35% sequence identity with the deduced amino acid sequence of scbR as shown in Fig. 9, preferably at least about 40%, more preferably about 45%
(which is the homology found between scbR and arpA of S.
griseus), 50%, 55% (which is the homology found between scbR and the FarA gene of S. lavendulae) 60%, 65%, 70%, 80%, 90%, or 95%.
"Percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the sequence with which it is being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The identity values used herein are generated by WU-BLAST-2 which was obtained from Altschul et al. (1996);
http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span =1, overlap fraction = 0.125, word threshold (T) - 11. The HSPS and HSPS2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. A ~ amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer"
sequence in the aligned region, multiplied by 100. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-BLAST-2 to maximize the alignment score are ignored).
"Percent (%) nucleic acid sequence identity" is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the sequence under comparison. The identity values used herein were generated by the BLASTN module of WU
BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 Restriction map of 7.5kb DNA fragment isolated S from S. coelicolor which includes scbA and scbR. The positions of scbA, scbR, orfX (also referred to herein as scbB) are indicated by shaded,~.oxes and the other ORFs with no apparent homology to other known streptomyces antibiotic regulatory genes (as assessed by the BLAST program) are indicated by open boxes. The ORFs are deduced from the FRAME program (Bibb et al., 1983). pIJ6111 and pIJ6114 were used for sequencing analysis of the 7.5kb fragment.
Restriction maps of the in-frame deletion mutant constructs are also shown. Dotted lines indicate the in-frame deletion of scbA for pIJ6120 or scbR
for pIJ6124. The PstI site in pIJ6124 was generated by using a designed primer for PCR to allow ligation with the corresponding PstI site. The BamHI site in pIJ6140 was end filled and ligated with PvuII.
Single arrows (PrimersR) and double arrows (PrimersA) denote the primers used to determine the scbR and scbA mutation, respectively, after the second crossover event. pIJ 6135 and pIJ6143 (insert cloned into pSET152) were used to complement the scbR and scbA mutant, respectively. pIJ6120 (insert in pIJ2925) was used to express ScbR for gel retardation and Dnase I footprinting experiments.
Fig. 2a S1 nuclease mapping of the transcriptional start site of scbA and scbR. Asterisks indicate the probable start points of the transcription; the sequences given are those of the template strand.
Lanes T, G, C, and A are sequence ladders derived from the same primers as the probe generated by PCR
and using the Taqtrack kit along with the these primers.
Fig. 2b S1 nuclease mapping of scbA, scbR, and the major sigma factor hrdB, using RNA isolated from a liquid time course of S. coelicolor M145 at the time (hours) indicated. The EXP, TRANSITION and STAT
indicated the exponential, transition and stationary phases of growth, respectively, and the shaded box labelled RED denote the presence of undecylprodigiosin in the mycelium; SM, end-labelled HpaII-digested pBR322 size marker.
Fig. 3a Gel retardation experiment shown with crude extract of E. coli JM101 harboring scbR. Various crude extracts or unlabelled DNA fragments that were used in the experiment are indicated as +.
Fig. 3b Gel retardation experiments using E. coli JM101 crude extract harboring scbR and various Y-butyrolactones are indicated. CD denotes Circular Dichroism positive (+) or negative (-).
Fig. 4a Dnase I footprinting experiment of ScbR.
Protection of the scbA and scbR promoter region by ScbR from cleavage by Dnase I is shown by vertical lines. No.l denotes ScbR binding site No.l and No.2 binding site No.2. Both DNA strands were tested for protection by Dnase I by using two different 3zP
labelled oligonucleotides. The A and G sequence ladder were used as size standards. Asterisk indicates the oligonucleotide which has been labelled. The presence or absence of crude extracts from E. coli JM101 harboring scbR is indicated by +
or -. The numbers underneath the symbol > denote the concentration gradient of the crude extract added to the reaction.
Fig. 4b (sEQ.zD.rros.:l2-15) ScbR binding sites No.1 and No.2 from Dnase I footprinting experiments. The protected sequences are indicated by lines and the numbering is with respect to the transcriptional start site of scbA
for binding site No.l and scbR for binding site No.2. The arrows and pscbA, pscbR indicate the transcriptional start site and direction of scbA and scbR, respectively. [ScbA] and [ScbR] indicate the coding sequence for scbA and scbR, respectively.
Fig. 5a Effect of deletion of scbA or scbR on antibiotic production in solid grown S. coelicolor M145. Confluent lawn of M145, M751 and M752 were grown on SMMS at 30°C for 20h (top plates) or 40h (bottom plates).
Fig. 5b Effect of deletion of scbA or scbR on response to SCB1, a y-butyrolactone. Bioassay using spare suspension of M145(top), M751(left bottom) and M752(right bottom) as indicator strain and spotted with 1 ug of SCB1. The plates were incubated at 30°C
for 20h (left panel) or 40h (right panel).
Fig. 6 Effect of deletion of scbA or scbR on the production of Y-butyrolactones with antibiotic stimulatory activity. Bioassay of ethyl acetate extracts from SMMS solid cultures of M145 (pset152) (top), M751 (pset152) (left side) and M751 complemented with scbA (right side), M752 (pset152) (right side) and M752 complemented with scbR (left side). In each case the indicator lawn is M145 and grown on SMMS at 30°C for 30h.
Fig. 7a S1 nuclease mapping of scbA, scbR, and the major sigma factor hrdB, using RNA isolated from a liquid time course of S. coelicolor M145, M751 and M752 at the numbers indicated. The E, TRAM and S
indicates the exponential, transition and stationary phases of growth, respectively, and the shaded box labelled RED and A denote the presence of undecylprodigiosin and actinorhodin in the mycelium.
The numbers in the box denotes the measurement of antibiotic production, ACT for actinorhodin, and RED
for undecylprodigiosin, respectively. Numbers in bold refer to the time points when the antibiotics were measured, which corresponds to the time of RNA
isolation.
Fig. 7b Bioassay of supernatants isolated at the time of RNA isolation from M145. The numbers denotes the different time points indicated in Figure 7a.
Fig. 8 S1 nuclease mapping of scbA, scbR, and the major sigma factor hrdB, using RNA isolated from M571 grown on liquid media SMM, with 0 or 31.25ng final concentration addition of SCB1.
Fig. 9 cssQ ID.NO.:ls) Deduced amino acid sequence of ScbR.
Fig. 10 csEQ iD.NO.:m~ Deduced amino acid sequence of ScbA.
Fig. 11 ~sEQ zD.NO.:se) Deduced amino acid sequence of ScbB.

Fig. 12 csEQ zD.NO.:i9> Production of Act by S. lividans strains carrying pIJ68.
Fig. 13 Production of Red by S. lividans strains carrying pIJ6014.
Fig. 14 Nucleic acid sequence of region containing scbA, scbR and scbB. M751 (OSCbA) is deleted from nt position 1320 to 2021; M752 (~scbR) is deleted from nt position 2359 to 2796 with five bases added;
pIJ6134 runs from nt position 2021 to 4346; and pIJ6140 runs from nt position 1 to 3430.
The work on which the present invention is based will now be described, by way of example only, with reference to these figures,.
EXAMPLE 1 scbA - an S. coelicolor homologue of afsA
Alignment of the amino acid sequences of AfsA from S.
griseus and its homologue, BarX, from S. virginiae (Kinoshita et al., 1997), revealed two highly conserved regions (corresponding to amino acid residues 217-223 and 277-285 of AfsA). These sequences were used, with codon usage data derived from 64 Streptomyces genes (Wright and Bibb, 1992), to design degenerate oligonucleotides for use as primers in PCR. BamHI sites were incorporated at the 5' end of each primer to allow subsequent cloning of the PCR product. An amplified fragment of the expected size (189 by including the flanking BamHI sites) was obtained using S. coelicolor M145 DNA as template. The PCR product was cleaved with BamHI, and cloned in the BamHI site of the pUCl9 derivative pIJ2925, yielding pIJ6114. Sequencing using universal and reverse primers revealed an afsA homologue of S. coelicolor, which was designated scbA.
The BamHI insert of pIJ6110 was isolated and labelled with 32P by random oligonucleotide priming and used as a hybridisation probe to isolate four cosmids from an unaligned cosmid library of S. coelicolor M145 DNA. The probe failed to hybridise to the ordered cosmid library of Redenbach et al., 1996 (see below). Digestion of the four cosmids with BamHI revealed several restriction fragments of identical mobility, suggesting that each cosmid respresented the same genetic locus. Southern analysis of each of the cosmids using the same probe identified a common 4.5 kb BamHI fragment and a smaller hybridising fragment that ranged in size from 2.5 kb to 3.0 kb. The 4.5 kb fragment and the 3.0 kb BamHI
fragment from cosmid GB10 were cloned in the BamHI site of pIJ2925 to yield pIJ6111 and pIJ6114, respectively.
The restriction map of the contiguous 7.5 kb region is shown in Fig.l. All four hybridising cosmids from the unaligned library were used as probes to identify their position in the combined physical and genetic map of the S. coelicolor chromosome. scbA was localised to the gap that lies at approximately 5 o'clock in the ordered cosmid library, in AseI fragment B, and next to cosmid 2H4 (H.M.Kieser personal communication; Kieser et al., 1992) .
EXAMPLE 2: scbA lies adjacent to genes likely to be involved in y-butyrolactone synthesis and binding The nucleotide sequence of the 7.5 kb scbA region was determined (the sequence has been deposited under EMBL
accession number AJ007731). Frame analysis (Bibb et al., 1983) revealed open reading frames (ORFs) with predicted translation products that showed homology to proteins likely (by analogy to the S. griseus system above) to be involved in both Y-butyrolactone synthesis and perception. ScbA (corresponding to nucleotide positions 2914-1970 of EMBL AJ007731 and 2142-1199 of Fig. 14) shares 64% amino acid sequence identity with ~.fsA, and about 40% identity with other AfsA homologues. The deduced amino acid sequence of ScbR, a protein encoded by a divergent ORF (corresponding to nucleotides 3032-3679 of EMBL AJ007731 and 2261-2908 of Fig. 14) which is adjacent to scbA, shows high levels of similarity to several Y-butyrolactone binding proteins. It is 56%
identical to FarA of S. lavendulae FRI-5, and 45%
identical to ArpA. Each of these homologues possesses an N-terminal DNA-binding domain that is also found in the TetR family of transcriptional repressors. The C-terminal regions of the ScbR family of proteins are relatively poorly conserved, and the inventors propose that this may reflect their ability to bind different Y-butyrolactones.
Downstream of ScbR, and transcribed in the opposite orientation, lies ScbB (nucleotides 3795-4529 of EMBL
AJ007731 and 3024-3758 of Fig. 14), whose predicted product shows 50% amino acid identity to a C-5 ketoreductase from S. avermitilis (Ikeda et al., 1999);
based on BLAST search (Altschul et al., 1997).
EXAMPLE 3: Transcription of scbA and scbR occurs in a growth-phase-dependent manner S1 nuclease protection experiments were carried out to determine the transcriptional start sites of scbA and scbR using RNA isolated from S. coelicolor M145 cultured in SMM to different stages of growth. A 259 by PCR
product (nucleotides 2786-3055 of EMBL, 2015-2284 of Fig.
14) labelled uniquely at the 5' end at position 2786/2015 was used as a probe for scbA transcripts, while a 280 by PCR product (nucleotides 2894-3174 of EMBL, 2123-2403 of Fig. 14) labelled uniquely at the 5' end at position 3174/2403, was used as a probe for scbR. Putative transcriptional start sites were identified 46 nucleotides upstream of the likely translational start site of scbA, and 123-124 nucleotides upstream of that for scbR (Fig 2a), i.e. at nucleotides 2960 and 2909-8, respectively (of the EMBL sequence, 2189 and 2138-7 of Fig. 14). Transcription of scbA, which was undetectable during exponential growth, increased markedly at late transition phase, and fell quickly as the culture entered stationary phase (Fig. 2b). The scbR transcript, while detectable during exponential growth, increased markedly in level in late transition phase, approximately one hour after the increase in the level of the scbA transcript.
It also fell in stationary phase, after the decline in the level of the scbA transcript. The transcript of the major and essential sigma factor gene, hrdB, was used as a control, and was present at essentially constant levels through exponential growth, and fell gradually upon entry into stationary phase.
EXAMPLE 4: Binding of ScbR to the promoter regions of scbA and scbR is prevented by SCB1 scbR was expressed in E. coli JM101 by cloning the l.2kb HincII-PvuII fragment containing scbR (Fig. 1) in pIJ2925, yielding pIJ6120. Extracts of JM101/pIJ6120 were then used in gel retardation assays with a 5' end-labelled PCR product that contained the.scbA and scbR
promoter regions (Fig.3a). Retardation of the scbAR
promoter fragment was readily detected on addition of the JM101/pIJ6120 extract (indicating binding of ScbR to the promoter region); no retardation was observed if the extract was first boiled, or with extract isolated from JM101 (Fig.3b). Addition of an excess of unlabelled PCR
product resulted in a reduction in the proportion of the labelled promoter-containing fragment that was retarded;
however, no competition was apparent when unlabelled Streptomyces DNA (the plasmid pIJ922) was added,.
indicating a specific interaction between ScbR and the promoter DNA.
SCB1 (which, like A-factor, is a GBL) and its three chemically synthesized stereoisomers (Takano et al., 2000) were added to the gel retardation assays to assess their ability to influence the DNA-binding activity of ScbR. Formation of the DNA-protein complex was markedly reduced by addition of lug of SCB1, while addition of equivalent amounts of each of the stereoisomers had little or no effect (Fig 3c). Equivalent amounts of A-factor, IM-2 and VB also failed to inhibit the DNA-binding activity of ScbR (data not shown) indicating that the specificity of ScbR for SCB1, its cognate y-butyrolactone, is high.
DNase I footprinting was used to determine the location of the DNA sites to which ScbR binds. Two protected regions were identified (Fig 4a and b); one lies at nucleotide position -4 to -33 with respect to the scbA
transcriptional start site (i.e. nucleotides 2964-2993 of the EMBL sequence, 2193-2222 of Fig. 14), while the other lies at nucleotide position -41 to -67 with respect to the scbR transcriptional start site (i.e. nucleotides 2867/8-2841/2 of EMBL, 2096/7-2070/1 of Fig. 14).
Dilution of the JM101/pIJ6120 extract suggests that ScbR
has a stronger affinity for the binding site upstream of scbA than for that upstream of scbR.
EXAMPLE 5: Deletion of scbA abolishes y-butyrolactone synthesis, but results in overproduction of Act and Red, while deletion of scbR also abolishes y-butyrolactone synthesis, but causes delayed Red production To assess the role of scbA and scbR in antibiotic production in S. coelicolor, in-frame deletions were made in each gene. Mutant scbA and scbR alleles were constructed in which most of the scbA and scbR coding regions (amino acids 42-276 out of 315, and 33-178 out of 216, respectively) were deleted . The mutant scbA and scbR alleles were cloned in the E. coli plasmid pKC1132, yielding pIJ6140 and pIJ 6134, respectively (Fig. 1), and introduced into S. coelicolor strain M145 by conjugation; selection for apramycin resistance ensured integration of the non-replicating plasmids into the streptomycete chromosome by homologous recombination.
After three rounds of sporulation on non-selective medium, apramycin-sensitive segregants were screened by PCR, and putative scbA (M751) and scbR (M752) deletion mutants further confirmed by Southern analysis.
Confluent lawns of the parental strain M145, M751 (~scbA) and M752 (~scbR) were grown on nitrogen-limited SMMS agar plates to assess the affect of each deletion (Fig. 5). After 20 h, Red production had just begun in M145, while Act synthesis was undetectable. In contrast, M751 had produced large amounts of both Red and Act (detectable by exposing the agar plate to ammonia fumes, which resulted in the blue pigmentation characteristic of Act), and M752 had failed to produce either antibiotic.
S By 40 h, the overproduction of both Act and Red by M751 was very marked, while Red production was noticeably delayed and Act production detected earlier ~.ig. 5A) in M752 as compared to M145. The mutant phenotypes were also observed on rich R5 agar and on phosphate-limited R2 agar, but both mutants resembled the parental strain on rich SFM agar and minimal medium containing mannitol as carbon source. Growth of the strains in SMM liquid medium gave phenotypes that corresponded to those observed with SMMS agar.
To assess thq ability of the mutants to respond to SCB1, 1 ug of chemically synthesised SCB1(Takano et al (2000) supra) was spotted onto confluent lawns of M751, M752 and M145 (Fig. 5b). While M145 responded in the expected way to exogenous SCBl, M752 did not respond. Since M751 precociously overproduced both Act and Red, it was not possible to determine whether it had retained the ability to respond to the the Y-butyrolactone; however, the inhibitory effect of high concentrations of SCB1 on antibiotic production in M145 (the lighter halo surrounding the point of application; Takano et al., 2000) was also observed with M751, suggesting that it had indeed retained the ability to sense SCB1.
The ability of the mutants to produce compounds, including SCB1, with antibiotic stimulatory activity was assessed using the standard bioassay (the ability to induce precocious Act and Red production in a lawn of M145). M751, M752 and M145 were grown on SMMS agar and in SMM liquid medium, and samples of agar and culture supernatant from different growth phases were extracted with ethyl acetate. Neither mutants produced stimulatory activity (Fig. 6) regardless of growth phase or medium .
To confirm that the mutant phenotypes reflected the absence of a functional scbA or scbR, rather than a mutation elsewhere in the genome, scbA or scbR were reintroduced into M751 and M752, respectively. A 1194 by PCR product containing scbA and its promoter (Fig. 1, pIJ6143), and a 1.3 kb BglII fragment containing scbR and its promoter (Fig.l, pIJ6135), were cloned in E. coli in pSET152 yielding pIJ6147 and pIJ6135, respectively. The plasmids were introduced into the corresponding S.
coelicolor mutant by conjugation and selection for apramycin resistance, and integration at the 45C31 attachment site was confirmed by Southern hybridisation.
All of the mutant phenotypes were restored to those observed in M145 (Fig. 6 for restoration of SCB1 synthesis).
EXAMPLE 6: scbR regulates the transcription of both scbR and scbA, and scbA is required for the transcription of scbA
Since ScbR binds in vitro to the scbA and scbR promoter regions, and given that SCB1 is able to prevent such binding, the effect of the scbA and scbR deletions on transcription of each of the genes in vivo was assessed.
RNA was isolated from SMM-grown M145, M751 and M752 cultures at different stages of growth and subjected to S1 nuclease protection experiments (Fig. 7a). While the scbA transcript was readily detected in early transition phase cultures of M145, it was absent in M751(~scbA) and barely detectable in M752 (~scbR) regardless of growth phase, suggesting that both scbA and scbR are required for induction of scbA transcription. While the level of the scbR transcript increased during late transition and early stationary phase in M145, it was markedly diminished in the scbA mutant, and highly abundant in exponential and early transition phase in th~scbR
mutant. These observations suggest that scbR negatively regulates its own transcription, and that relief of this repression requires scbA. Transcription of hrdB, the major and essential sigma factor of S. coeliclor, was monitored as a control. Antibiotic production (Fig. 7a) and production of antibiotic stimulatory factors (Fig.
7b) were also assessed at the times of RNA extraction.
The commencement of factor synthesis in M145 corresponded well with the increase in the scbA transcription.
EXAMPLE 7: Addition of SCB1 to M751 (~scbA) stimulates scbR transcription but fails to restore scbA transcription To assess the effect of addition of exogenous SCB1 on scbA and scbR expression in the ascbA mutant, in which transcription of both genes is markedly impaired, chemically synthesised SCB1 was added at a final concentration of 31ngm1-1 to a mid-exponential phase (ODQSOnm= 0.5) culture of M751. While there was a marked increase in the level of scbR transcription, scbA
transcription in the dscbA mutant was not restored (Fig.
8 ) .
EXAMPLE 8: Deletion of scbA in S. lividans 1326 abolishes gamma-butyrolactone synthesis and results in increased production of Act and Red by strains containing the multi-copy plasmids pIJ68 or pIJ6014.
The mutant scbA allelle from S. coelicolor (described in example 5) was introduced into S. lividans 1326 using pIJ6140. Integration of the non-replicating plasmid was selected using apramycin. After three rounds of non selective growth (on SFM agar) colonies were screened for sensitivity to apramycin (indicating loss of the plasmid due to a second homologous recombination event). 4 apramycin-sensitive colonies were identified among 3,000 colonies screened. PCR analysis of chromosomal DNA
produced amplified DNA fragments consistent with that observed from wild type chromosomal DNA for three colonies, whereas the fourth colony yielded a smaller DNA
fragment consistent with the in-frame deletion allelle.
Southern hybridisation experiments of chromosomal DNA
digested either with Ncol or a mixture of BglII and Pstl produced hybridising bands consistent with the results expected for the wild type arrangement for the first three colonies and the mutant for the fourth colony, which was designated S. lividans M707. When this strain was grown on agar medium no ScbA was detected, whereas it was demonstrably produced by the wild type S. lividans 1326 strain.
Spores of the M707 strain were inoculated into liquid YEME medium (containing 0.5% glycine and 5mM MgCl2) and grown with shaking at 30°C for 2 days. The mycelium was collected by centrifugation and used to produce protoplasts, which were transformed with pIJ68 (actII-orf4) (Passantino R et al (1991) J. Gen Microbiol 137:2059 - 2064), pIJ6014 (redD) (Takano E et al (1992) 6(19): 2797 - 2804) or pIJ486 (vector control) (Ward J M
et al (1986) Mol Gen Genet 203:468 - 478). Thiostrepton-resistant transformants were selected and tested in shake flask fermentation experiments. Spores of the transformed strains were streaked on SFM agar (containing 50 ~g/ml of thiostrepton) and incubated at 30°C for 4-5 days. Spores were harvested and inoculated into spring flasks with 50m1 YEME (containing 50 ~g/ml of thiostrepton). After two days incubation at 30°C the mycelium was collected by centrifugation and resuspended in fresh spring flasks containing phosphate-limited Evans medium with 20 ~tg/ml of thiostrepton. Incubation was continued at 30°C for a further seven days with lml samples being removed for assessment of antibiotic production. The Act or Red production is shown in Figures 12 and 13 and confirmed the findings observed for S. coelicolor that antibiotic synthesis was precocious and elevated. Approximately three to four' times the concentration of Red was observed compared to the S. lividans 1326 strain carrying pIJ6014.
For Act the concentration difference was five to ten fold for the pIJ68-containing strains. Moreover, when dry cell weight (DCW) measurements were made it was noted that the M707/pIJ68 strain produced less mycelial material than the S. lividans 1326/pIJ68 under these conditions. Thus, when expressed as concentration of Act produced per gram DCW, the M707 strain produced 121 compared to the control 1.5.
(Plasmids pIJ68 / pIJ6014 were introduced into S.
lividans to supply the pathway specific transcriptional activator genes for Act / Red production. No equivalent plasmids are required in S. coelicolor.) These strains were further tested for their ability to produce Act in 1 litre liquid batch fermentations in stirred tank bioreactors using a modified phosphate-limited Evans medium (with NH4C1 instead of NaN03). The M707/pIJ68 strain produced 10g/1 of act compared to the S. lividans 1326/pIJ68 control, which made 5g/1.
DISCUSSION
Two genes, scbA and scbR, have been isolated from S.
coelicolor A3(2) and respectively show high homology to the afsA and arpA genes of S. griseus, which encode A-factor synthetase and A-factor binding protein. The in-frame deletion mutant of scbA overproduces both antibiotics, while the in-frame deletion mutant of scbR
is delayed in RED production and does not produce y-butyrolactones which (in the wild-type strain) cause precocious RED and ACT production. These phenotypes are most surprising considering the high homology of the genes to the A-factor system in S. griseus.
Onishi et al. (1999) reported the cascade for the streptomycin production in S. griseus, triggered by A-factor. ArpA (A-factor binding protein) binds to the promoter region of adpA (a transcriptional activator for streptomycin production) and represses the transcription of adpA during early growth cultures. In transition phase, A-factor is synthesised via AfsA and releases the ArpA from the promoter region by binding to it. Thus adpA
is transcribed and activates the streptomycin biosynthetic cluster via strR (streptomycin pathway-specific activator) and the antibiotic is produced. To corroborate their model, the afsA mutant (equivalent to the scbA mutant of the present work) produces neither streptomycin nor A-factor. Also the arpA mutant (equivalent to the scbR mutant of the present work) overproduces antibiotics; A-factor production is not effected. These are the reverse phenotypes compared to those of the in-frame deletion mutants of the present work using S. coelicolor. The inventors propose that Y-butyrolactones are involved in antibiotic production differently in S. coelicolor, compared with the known GBL
model of S. griseus.
The two genes scbA and scbR are located next to each other in the S. coelicolor genome, which is not the case for the equivalent genes of S. griseus (afsA and arpA).
afsA is located at the end of the linear chromosome (Lezhava et al., 1997) thus being easy to mutate to obtain deletion mutants and arpA is located elsewhere on the chromosome (Ohnishi et al., 1999). On the other hand, like the arrangement in S. coelicolor, the homologues of afsA in S. virginiae (barX) and S.fradiae (farX) are located next to genes encoding Y-butyrolactone binding proteins (barA and farA respectively) (Nakano et al. , 1998; Waki et a1. , 1997) . In S. virginiae, a mutation in barA (a homologue of arpA) results in precocious virginiamycin production, consistent with the role of arpA in S. griseus, yet it abolishes VB
production (Nakano et al., 1998). The inventors propose, therefore, that the juxtaposition of streptomycete genes encoding GBL synthetases and GBL binding proteins may be reflective of a different antibiotic regulatory system from that of the S. griseus model (possibly in some cases additional to such a regulatory system), namely one in which functional deletion of the gene encoding the GBL
binding protein leads not to overproduction of antibiotic (as in the S. griseus system), but under- or delayed production. Moreover, they suggest that in such systems, functional deletion of the GBL synthetase leads to overproduction of the antibiotic (in contrast to abolition of streptomycin production in S. griseus).
MATERIALS AND METHODS
S
Bacterial strains, plasmids, and growth conditions S coelicolor A3(2) strain M145 (Hopwood et a1.,1985) , M751 and M752 (this study) were manipulated as previously described (Hopwood et al., 1985). E.coli K-12 strains JM101( Sambrook et al., 1989) and ET12567 (MacNeil et al., 1992) were grown and transformed according to Sambrook et al., (1989). Vectors used were pIJ2925 (Janssen and Bibb., 1993), pKC1132 (Bierman et al., 1992), pset152 (Bierman et al, 1992), pBluescript SK+
(Stratagene), pGEM-T vector (Promega). SMM is the modified minimal medium of Takano et al., (1992); it lacks (NHQ) 2S04 and has 0 .25 mM NaH2P04, 0 .25mM KZHP04 instead of 0.5mM each. SMMS is a modified solid version of SMM, as described above. SFM medium was used to make spore suspensions and for use in conjugation with E.coli ET12567 containing the RP4 derivative pUZ8002 (Flett et al., 1997).
PCR
The synthetic oligonucleotides oligol; 5'-GACCACGT(CG)CC(CG)GGCATG and oligo2; 5'-GTCCTG (CG) TGGCC (CG) GT (CG) AC (CG) CG (CG) AC (bracketed nt indicate positions of degeneracy) were used in the PCR
(Erlich, 1989) to amplify the internal segment of scbA
from S. coelicolor M145 total DNA (Fig. 2a). The reaction mixture contains: lOx reaction mixture supplied by Boehringer Mannheim, 200uM final concentration of four dNTPs, 5% final concentration of DMSO, 50pmo1 of each primer, SOng of chromosomal DNA in a final volume of 100u1. After denaturation by boiling 5 min, 2.5U of Taq polymerase was added and subjected to 30 cycles of denaturation at 94°C for 50 sec, annealing at 55°C for 40 sec and extension at 72°C for 40 sec, and then incubated at 72°C for 10 min. PCR products were analyzerl-on a 2% w/v agarose gel electrophoresis.
To complement M751, scbA coding sequence with its promoter region was amplified by PCR from S. coelicolor M145 cosmid GB10 DNA. Two synthetic oligonucleotides 5'-GCCAGCAGGTGGGCGACCTGAC,(SEQ.ID.N0.:3)~l~gSnt position) and' 5'-GATCGCCCGGTCCTGCTTGGCCATG (SEQ.ID.N0.:4)(3055nt position) were used.
The PCR conditions were as stated above except the High Fidelity Kit ,(Beoringher Mannheim) was used and the PCR
cycle was reduced to 20. The PCR product was purified by a Sephadex G-50 (Pharmacia) spin column then ligated to the pGEM easy vector (Promega) and transformed to JM101.The sequence of the transformant was confirmed by using the ABI automated sequencer and Big Dye dye terminator cycle sequencing kit (Perkin Elmer).
Nucleotide sequencing The nucleotide sequencing of the 7.5kb scbA region was sequenced by the ABI automated sequencer and using the Big Dye dye terminator cycle sequencing kit (Perkin Elmer) as recommended by the suppliers, except in the PCR
reaction, final concentration of 5% DMSO was added to the reaction mixture. The sequence was submitted to the databases (EMBL AJ007731) and sequenced on both strands.
S1 nuclease mapping For each S1 nuclease reaction, 30 or 90ug of RNA were hybridized in NaTCA buffer (Murray, 1986; Solid NaTCA(Aldrichi was dissolved to 3M in 50mM PIPES, 5mM
EDTA, pH7.0) 'o about 0.002pmol (approximately 104Cerenkov counts min 10-') of the following probes. For scbA the synthetic oligonulceotide 5'-TATCCAGCTGACCGGGAACGCGTC csEQ.iD.rro.:5), corresponding to the region within the coding region of scbA was labelled with ['2P]-ATP using T4 polynucleotide kinase uniquely at the 5' end of the oligonucleotide, then used in the PCR reaction with the unlabelled oligonucleotide s'-ATCGCCCGGTCCTGCTTGGCCATG (SEQ.ID.b10.:6) which corresponds to a region upstream of the scbA promoter region to generate a 259bp probe. For scbR, the synthetic oligonulceotide s'-AAGTAGAGGGCTCCCTTGGTCA (SEQ.ID.NO.:7), corresponding to the region within the coding region of scbR was labelled with ['2P]-ATP using T4 polynucleotide kinase uniquely at the 5' end of the oligonucleotide, then used in the PCR reaction with the unlabelled oligonucleotide 5~-C~CTACTGCTTCGGGCATG cs~.ID.NO.:s> which corresponds to a region upstream of the scbR promoter region to generate a 280bp probe. Both PCR reactions were done using M145 total DNA as template. For hrde, the probe was made as previously described (Buttner et al., 1990). Subsequent steps were as described by Strauch et al. (1991) .
Gel retardation assays and Dnase 1 footprinting studies 50pmo1 of the synthetic oligonucleotides 5'-CTGCACCCTGGTCCGGTGGACA
(SEQ.ID.N0.:9) arid 5'-ATCGCCCGGTCCTGCTTGGCCATG (SEQ.ID.NO.:10) were both labelled with (''P]-ATP using T4 polynucleotide kinase uniquely at the 5' end of the oligonucleotide, then used in the PCR reaction with the unlabelled synthetic oligonucleotide corresponding to the other primer to generate a 244bp DNA fragment. The SCR
amplified fragment was further purified by Qiagen PCR
purification kit. The gel retardation assay reaction mixture contains; 5x gelretardation buffer(125mM HEPES
pH7.5, 20mM DTT, lOmM ATP, 20% glycerol) 200mM KC1, 0.16ug/ul calf thymus DNA, and 0 to 15u1 of JM101 crude extract containing ScbR protein in a final volume of 12.5 to 25 u1. The final concentration of DNA fragments used was 2.5 ng/ml. The mixture was incubated at room temperature for 10 min then 2 u1 of dye(50%(w/v) glycerol with BPB in TE) was added to the mixture and 10u1 was loaded to a 5%(w/v) non-denaturing polyacrylamide gel buffered with TBE. SCB1 was added to the reaction mixture either prior to incubation, or after 10 min of incubation then incubated for further 10 min.
Dnase I footprinting studies were performed as described by Drapal and Sawer, (1995). 25 ng/ml of DNA fragments 2~ were incubated in gel retardation assay mixture (final total volume 25u1) with varying concentration of protein.
After incubation, 25u1 of lOmMMgCl and SmMCaClz was added.
After 1 min O.lunit of Dnase I (Boerhinger Mannheim) was added and incubated for 45 sec then the reaction terminated by adding 30u1 of stop solution (20mMEDTA, 200mM NaCl, 1% SDS(w/v), 250ug ml-' tRNA). The DNA
fragments were purified by phenol/chloroform extraction and precipitated with three volumes of ethanol. The precipitants were resuspended in loading buffer and ran on a 6%(w/v) sequencing gel. Sequencing reactions were performed using the synthesised oligonucleotides as primers on double strand DNA and by using a dideoxy sequencing kit (Taq Track, Progema).
S
Crude extract isolation An overnight culture of E. coli JM101 harboring pIJ6120 was diluted 1/100 and innoculated into 25m1 LB media. The culture was grown at 37°C for approximately 3.5 hr or until the cultures were at 1.0 OD6oonm. The culture was then induced with final concentration of 1mM IPTG. After further 3 hr of growth, the cells were harvested by centrifugation and the cell pellet was washed twice with buffer (50mM Tris pH7.0, 1mM EDTA, 1mM DTT, 100mM PMSF), resuspended in 500u1 of buffer and disrupted by sonication. The cell lysate was then clarified by centrifugation and the supernatant was used as crude extracts.
Isolation of y-butyrolactones, bioassay and HPLC
analysis y-butyrolactones were isolated from liquid or solid media by extracting the culture supernant or the agar with ethylacetate. The ethylacetate was evaporated and the sample was resuspended in 100% methanol for use in a bioassay or for HPLC analysis. Bioassay and HPLC
analysis were conducted as described previously(Takano et al., 2000).
Construction of an in-frame deletion mutant of scbA and scbR

The in-frame deletion mutant of scbA was constructed by digesting pIJ6136 which contains a l.4kb flanking DNA of scbA in pIJ2925 (Fig. 1) with BamHI and end filled using Klenow fragment and ligated with a l.lkb PvuII -HincII
fragment from pIJ6111. The transformants were analysed to find the PwII -HincII fragment was inserted with the internal PstI site at the EcoRI side of the multiple cloning site of pIJ6136 and designated pIJ6137. The BgIII
fragment of pIJ6137 was inserted into the BamHI site of pKC 1132 (Bierman et al . , 1992) to give pIJ6140 (Fig. 1) .
The in-frame deletion mutant of scbR was constructed by PCR using the High Fidelity Kit (Beoringher Mannheim) with a universal primer and s'-CATCTGCAGCGTGATCGTGGCAGCTTGGTAG
csEQ.zD.NO.:ii> (3130nt position) primer designed to give a 1.059kb DNA fragment flanking scbA as described earlier. A PstI site was designed into the end of this fragment to enble ligation with a PstI site internal of scbR. pIJ6111 was used as template for the PCR reaction and the amplified product was cloned into pGEM-T vector (Promega) to give pIJ6148. The sequence of the PCR amplified insert of pIJ6148 was confirmed by ABI
automated sequencing. The BamHI-KpnI 3kb fragment of pIJ6111 was cloned into pBluescript SK" (Stratagene) to give pIJ6131. The 1.059kb BamHI-PstI fragment was isolated from pIJ6148 and cloned into the BamHI-PstI
digested pIJ6131 to give pIJ6152. pIJ6152 was then digested with KpnI and blunt ended then further digested with BamHI. This 2.48kb DNA fragment was cloned into pKC1132 digested with BamHI and EcoRV to give pIJ6134 (Fig. 1). Both plasmids were introduced into the methylation deficient E. coli strain ET 12567 containing the RP4 derivative pUZ8002 (Paget et al., 1999) and transferred into S. coelicolor M145 by conjugation.
Single-crossover exconjugants were selected on SFM

containing apramycin. Three such single colonies were then taken through three rounds of non-selective growth on SFM to promote the second crossover. Spores were then plated for single colonies which were scored for S apramycin sensitivity. Deletions within scbA and scbR
were confirmed by PCR using primers correponding to flanking sequences, and by Southern hybridisation. For scbA, nine out of 20 apramycin sensitive colonies were deleted for scbA while 11 had reverted to wildtype. For scbR, 4 out of 20 apramycin sensitive colonies were deleted for scbR while 16 reverted to wildtype. The scbA
and scbR deletion mutants were called M751 and M752, respectively.
To complement the mutants, a 1194bp PCR product (subsequently sequenced) containing the entire scbA
coding region with its promoter (pIJ6143) and a l.3kb BglII fragment containing the entire region of scbR with its promoter (pIJ6135) (Fig. 1) was cloned into a conjugative vector pset152 (Bierman et al, 1992), which integrates into the chromosome of S. coelicolor by site-specific recombination at the bacteriophage ~C31 attachment site, attB (Kuhstoss, E. et al 1991). The resulting plasmids, pIJ 6147 and pIJ6135 (Fig. 1), respectively were transferred into S. coelicolor by conjugation via the E. coli donor ET 12567 containing the RP4 derivative pUZ8002 (Paget et al., 1999). Exconjugants were purified by single-colony isolation, and the plasmid integration were confirmed by southern hybridization.
Other methods Antibiotic production was determined by extracting actinorhodin and undecylprodigiosin as described previously (Strauch et al., 1991). RNA was isolated as described in Strauch et al., (1991). Southern hybridisation was done as previously described (Hopwood et al., 1985). Probes for southern hybridisation were S made by labelling DNA fragments or PCR products with 32P
by random oligolabelling (Pharmacia).
Further protocols are performed according to standard reference texts, such as Hopwood et al. (1985) and Sambrook et al. (1989), or later editions thereof.

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Yamada, Y., et al. (1987) J Antibiot 40:496-504.
All of the above references (and any later editions thereof) are hereby incorporated by reference in their entirety, individually and for all purposes.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Plant Bioscience Limited (B) STREET: Norwich Research Park, Colney Lane (C) CITY: Norwich (D) STATE: Norfolk (E) COUNTRY: UK
(F) POSTAL CODE (ZIP): NR4 7UH
(ii) TITLE OF INVENTION: Antibiotic Production (iii) NUMBER OF SEQUENCES: 19 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Bereskin & Parr (B) STREET: Box 401, Scotia Plaza, 40 King Street West (C) CITY: Toronto (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): M5H 3Y2 (v) COMPUTER READABLE FORM:
(A) COMPUTER: IBM PC compatible (B) OPERATING SYSTEM: PC-DOS/MS-DOS
(C) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,322,241 (B) FILING DATE: 23-OCT-2000 (viii) PATENT AGENT INFORMATION:
(A) NAME: Micheline Gravelle (B) REGISTRATION NUMBER: 4189 (C) REFERENCE NUMBER: 420-359 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:

(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID N0: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (xi) SEQUENCE
DESCRIPTION:
SEQ
ID NO:
12:

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH: 401 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
double (D) TOPOLOGY:
circular (xi) SEQUENCE DESCRIPTION:SEQ ID 13:
NO:

(2) INFORMATION FOR SEQ ID N0: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 14:
Met Pro Glu Ala Val Val Leu Ile Asn Ser Ala Ser Asp Ala Asn Ser Ile Glu Gln Thr Ala Leu Pro Val Pro Met Ala Leu Val His Arg Thr Arg Val Gln Asp Ala Phe Pro Val Ser Trp Ile Pro Lys Gly Gly Asp Arg Phe Ser Val Thr Ala Val Leu Pro (2) INFORMATION FOR SEQ ID N0: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 15:
Met Ala Lys Gln Asp Arg Ala Ile Arg Thr Arg Gln Thr Ile Leu Asp Ala Ala Ala Gln Val Phe Glu Lys Gln Gly Tyr Gln Ala Ala Thr Ile Thr Glu Ile Leu Lys (2) INFORMATION FOR SEQ ID N0: 16:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Ala Lys Gln Asp Arg Ala Ile Arg Thr Arg Gln Thr Ile Leu Asp Ala Ala Ala Gln Val Phe Glu Lys Gln Gly Tyr Gln Ala Ala Thr Ile Thr Glu Ile Leu Lys Val Ala Gly Val Thr Lys Gly Ala Leu Tyr Phe His Phe Gln Ser Lys Glu Glu Leu Ala Leu Gly Val Phe Asp Ala Gln Glu Pro Pro Gln Ala Val Pro Glu Gln Pro Leu Arg Leu Gln Glu Leu Ile Asp Met Gly Met Leu Phe Cys His Arg Leu Arg Thr Asn Val Val Ala Arg Ala Gly Val Arg Leu Ser Met Asp Gln Gln Ala His Gly Leu Asp Arg Arg Gly Pro Phe Arg Arg Trp His Glu Thr Leu Leu Lys Leu Leu Asn Gln Ala Lys Glu Asn Gly Glu Leu Leu Pro His Val Val Thr Thr Asp Ser Ala Asp Leu Tyr Val Gly Thr Phe Ala Gly Ile Gln Val Val Ser Gln Thr Val Ser Asp Tyr Gln Asp Leu Glu His Arg Tyr Ala Leu Leu Gln Lys His Ile Leu Pro Ala Ile Ala Val Pro Ser Val Leu Ala Ala Leu Asp Leu Ser Glu Glu Arg Gly Ala Arg Leu Ala Ala Glu Leu Ala Pro Thr Gly Lys Asp (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 313 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Met Pro Glu Ala Val Val Leu Ile Asn Ser Ala Ser Asp Ala Asn Ser Ile Glu Gln Thr Ala Leu Pro Val Pro Met Ala Leu Val His Arg Thr Arg Val Gln Asp Ala Phe Pro Val Ser Trp Ile Pro Lys Gly Gly Asp Arg Phe Ser Val Thr Ala Val Leu Pro His Asp His Pro Phe Phe Ala Pro Val His Gly Asp Arg His Asp Pro Leu Leu Ile Ala Glu Thr Leu Arg Gln Ala Ala Met Leu Val Phe His Ala Gly Tyr Gly Val Pro Val Gly Tyr His Phe Leu Met Thr Leu Asp Tyr Thr Cys His Leu Asp His Leu Gly Val Ser Gly Glu Val Ala Glu Leu Glu Val Glu Val Ala Cys Ser Gln Leu Lys Phe Arg Gly Gly Gln Pro Val Gln Gly Gln Val Asp Trp Ala Val Arg Arg Ala Gly Arg Leu Ala Ala Thr Gly Thr Ala Thr Thr Arg Phe Thr Ser Pro Gln Val Tyr Arg Arg Met Arg Gly Asp Phe Ala Thr Pro Thr Ala Ser Val Pro Gly Thr Ala Pro Val Pro Ala Ala Arg Ala Gly Arg Thr Arg Asp Glu Asp Val Val Leu Ser Ala Ser Ser Gln Gln Asp Thr Trp Arg Leu Arg Val Asp Thr Ser His Pro Thr Leu Phe Gln Arg Pro Asn Asp His Val Pro Gly Met Leu Leu Leu Glu Ala Ala Arg Gln Ala Ala Cys Leu Val Thr Gly Pro Ala Pro Phe Val Pro Ser Ile Gly Gly Thr Arg Phe Val Arg Tyr Ala Glu Phe Asp Ser Pro Cys Trp Ile Gln Ala Thr Val Arg Pro Gly Pro Ala Ala Gly Leu Thr Thr Val Arg Val Thr Gly His Gln Asp Gly Ser Leu Val Phe Leu Thr Thr Leu Ser Gly Pro Ala Phe Ser Gly (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Arg Ala His Gly Thr Arg Tyr Gly Arg Pro Leu Glu Gly Lys Thr Ala Leu Val Thr Gly Gly Ser Arg Gly Ile Gly Arg Gly Ile Ala Leu Arg Leu Ala Ala Asp Gly Ala Leu Val Ala Val His Tyr Gly Ser Ser Glu Ala Ala Ala Arg Glu Thr Val Glu Thr Ile Arg Ser Ser Gly Gly Gln Ala Leu Ala Ile Arg Ala Glu Leu Gly Val Val Gly Asp Ala Ala Ala Leu Tyr Ala Ala Phe Asp Ala Gly Met Gly Glu Phe Gly Val Pro Pro Glu Phe Asp Ile Leu Val Asn Asn Ala Gly Val Ser Gly Ser Gly Arg Ile Thr Glu Val Thr Glu Glu Val Phe Asp Arg Leu Val Ala Val Asn Val Arg Ala Pro Leu Phe Leu Val Gln His Gly Leu Lys Arg Leu Arg Asp Gly Gly Arg Ile Ile Asn Ile Ser Ser Ala Ala Thr Arg Arg Ala Phe Pro Glu Ser Ile Gly Tyr Ala Met Thr Lys Gly Ala Val Asp Thr Leu Thr Leu Ala Leu Ala Arg Gln Leu Gly Glu Arg Gly Ile Thr Val Asn Ala Val Ala Pro Gly Phe Val Glu Thr Asp Met Asn Ala Arg Arg Arg Gln Thr Pro Glu Ala Ala Ala Ala Leu Ala Ala Tyr Ser Val Phe Asn Arg Ile Gly Arg Pro Asp Asp Ile Ala Asp Val Val Ala Phe Leu Ala Ser Asp Asp Ser Arg Trp Ile Thr Gly Gln Tyr Val Asp Ala Thr Gly Gly Thr Ile Leu (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4346 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (xi) SEQUENCE DESCRIPTION: SEQ 19:
ID NO:

GCGGTACTCG

GACGTCGGCT

CATCTCCCAC

GCCACGTCCG

CCAGATGCGG

CGCCTTGGCC

AACGCGTCGG

AGCGCCATCG

CGCAGAATTG
ATCAAAACTA

AACGTTAAGA
TACAGACTGA
GCGGTTTTTT

CCCGGGGGAG
ACATGAACAA
GGAGGCAGGC
ATGGCCAAGC
AGGACCGGGC

CGGCAGACGA
TCCTGGACGC
CGCGGCGCAG
GTCTTCGAGA
AGCAGGGCTA

ACGATCACGG
AGATCCTCAA
GGTGGCCGGG
GTGACCAAGG
GAGCCCTCTA

CAGTCCAAGG
AAGAACTGGC
GCTGGGCGTC
TTCGACGCCC
AGGAACCACC

Claims (24)

1. A method of modifying an antibiotic-producing strain of a Streptomyces species to increase antibiotic production in said strain, the method comprising functionally deleting in said strain a gene which is the scbA gene of S. coelicolor or a homologue thereof, with the proviso that the functional deletion is not of the afsA gene of S. griseus.

2. A method of modifying an antibiotic-producing strain of a Streptomyces species to alter the timing of antibiotic production in said strain, the method comprising functionally deleting in said strain a gene which is the scbR gene of S.
coelicolor or a homologue thereof, with the proviso that the functional deletion is not of the arpA gene of S. griseus, nor of the barA gene of S. virginiae.

3. A modified strain of a Streptomyces species, the modified strain having a functional deletion of a gene which is the scbA gene of S. coelicolor or a homologue thereof, whereby production of at least one antibiotic in said modified strain is increased compared to a wild-type strain of said Streptomyces species, with the proviso that the functional deletion is not of the afsA gene of S. griseus.

4. A modified strain of a Streptomyces species, the modified strain having a functional deletion of a gene which is the scbR gene of S. coelicolor or a homologue thereof, whereby the timing of production of at least one antibiotic in said modified strain is altered compared to a wild-type strain of said Streptomyces species, with the proviso that the functional deletion is not of the arpA gene of S. griseus, nor of the barA gene of S. virginiae.

5. A method of producing an antibiotic, the method comprising providing a modified Streptomyces strain of any preceding claim, and culturing said strain under conditions suitable for production of antibiotic.

6. The method of claim 5, further comprising the step of purifying the antibiotic from the culture medium

7. The method of claim 6, further comprising the step of formulating the antibiotic as a pharmaceutical.

8. The method or modified strain of any preceding claim, wherein the strain is selected from the species S. coelicolor, S. violaceoruber, S. lividans and S. parvulus.

9. The method or modified strain of claim 8, wherein the strain is selected from the species S. coelicolor and S.
lividans.

10. The method or modified strain of claim 9, wherein the strain is S. coelicolor A3(2) or S. lividans 66.

11. The method or modified strain of any preceding claim, as dependent from claim 2 or claim 4, wherein the strain is of the species S. coelicolor.

12. The method or modified strain of any preceding claim, wherein the strain is of a species which possesses adjacent and divergent scbA and scbR genes of S. coelicolor or adjacent and divergent homologues thereof.

13. A method for identifying Streptomyces species in which antibiotic production is increased by functionally deleting the scbA gene of S. coelicolor or a homologue thereof, the method comprising functionally deleting in an antibiotic-producing strain of a Streptomyces species the scbA gene of S.
coelicolor or a homologue thereof, culturing said strain under conditions suitable for the production of antibiotic, and determining whether antibiotic production in said strain is increased.

14. A method for identifying Streptomyces species in which the timing of antibiotic production is altered by functionally deleting the scbR gene of S. coelicolor or a homologue thereof, the method comprising functionally deleting in an antibiotic-producing strain of a Streptomyces species the scbR
gene of S. coelicolor or a homologue thereof, culturing said strain under conditions suitable for the production of antibiotic, and determining whether the timing of antibiotic production in said strain is altered.

15. A method for producing an antibiotic, the method comprising, following identification of a Streptomyces species according to claim 13, providing a strain of said species having a functional deletion of said scbA gene of S.
coelicolor or homologue thereof, and culturing said strain under conditions suitable for antibiotic production.

16. A method for producing an antibiotic, the method comprising, following identification of a Streptomyces species according to claim 14, providing a strain of said species having a functional deletion of said scbR gene of S.
coelicolor or homologue thereof, and culturing said strain under conditions suitable for antibiotic production.

17. The method of claim 15 or claim 16, further comprising the step of purifying the antibiotic from the culture medium

18. The method of claim 17, further comprising the step of formulating the antibiotic as a pharmaceutical.

19. The method or strain of any preceding claim, as dependent from claim 1, wherein the scbA gene or homologue thereof has a nucleotide sequence which:

(a) is the complement of nucleotides 2914 to 1970 of EMBL
AJ007731;
(b) is the complement of nucleotides 2142-1199 of Fig.
14;
(c) encodes a polypeptide having at least about 35%
sequence identity with the amino acid sequence of Fig. 10;
and/or (d) is capable of specific hybridisation with the amplification product obtained using the primers:
oligo1 (5'-GACCACGT(CG)CC(CG)GGCATG) and oligo2 (5'-GTCCTG(CG)TGGCC(CG)GT(CG)AC(CG)CG(CG)AC) to amplify total DNA of said species or strain.

20. The method or strain of any one of claims 1-18, as dependent from claim 2, wherein the scbR gene or homologue thereof:

(a) has a nucleotide sequence which is nucleotides 3032 to 3679 of EMBL AJ007731;
(b) has a nucleotide sequence which is nucleotides 2261-2908 of Fig. 14;
(c) has a nucleotide sequence which encodes a polypeptide having at least about 35% sequence identity with the amino acid sequence of Fig. 9; and/or (d) is adjacent to and divergent from a gene which is capable of specific hybridisation with the amplification product obtained using the primers:
oligo1 (5'-GACCACGT(CG)CC(CG)GGCATG) and oligo2 (5'-GTCCTG(CG)TGGCC(CG)GT(CG)AC(CG)CG(CG)AC) \

to amplify total DNA of said species or strain.

21. The method or strain of claim 19 or claim 20, wherein the level of sequence identity is at least about 50%.

22. The method or strain of claim 21, wherein the level of sequence identity is at least about 65%.

23. The method or strain of claim 22, wherein the level of sequence identity is at least about 80%.

24. The method or strain of claim 23, wherein the level of sequence identity is at least about 95%.