# Patent

## Title
Methods and materials relating to gene expression

## Abstract

An expression cassette for expressing a nucleic acid of interest derived from the regulatory region of the methylenomycin gene cluster of the SCP1 plasmid of Streptomyces coelicolor A3(2), and related materials and methods.

## Background
The present invention relates to material derived from the SCP1 plasmid of Streptomyces coelicolor A3(2) and methods and uses relating thereto, in particular to material derived from the gene cluster for methylenomycin A biosynthesis.
Underlying the invention is work carried out by the inventors in sequencing and deducing the function of various genes in the methylenomycin A biosynthetic gene cluster.
The natural role of the DNA to which the present invention relates is the production of the antibiotic methylenomycin A and its congeners. The whole cluster of methylenomycin production, resistance and regulatory genes (the mmy gene cluster) is known only from studies of Streptomyces coelicolor A3(2) and Streptomyces violaceoruber No. 2416 SANK 95570 (Chater and Bruton, 1985). In these two bacteria the genes concerned with methylenomycin production are present on different plasmids, SCP1 and pSV1 (Aguilar and Hopwood, 1982). No other example is known of plasmid-specified antibiotic production in Streptomyces. Where studied, all naturally occurring Streptomyces plasmids, including SCP1, can be transferred to new Streptomyces hosts by conjugation.
The DNA sequence of a 9.5 kb stretch of this gene cluster has now been discovered, and the inventors have identified several genes and their transcriptional organisation. They found that the transcriptional organisation of this region is significantly different from that suggested previously, for example in Chater and Bruton (1985). The function of certain of the genes has newly been deduced from the discovery that they display high levels of homology with other genes which are involved in the regulation of other, chromosomally located, antibiotic biosynthetic gene clusters in diverse streptomycetes.
This discovery is particularly surprising in view of the fact that methylenomycin is the only Streptomyces antibiotic whose biosynthesis is known to be conferred by a plasmid, rather than native Streptomyces genomic DNA. It implies that these genes should be adapted to function in an appropriately controlled manner in any Streptomyces host to which these plasmids may be transmitted.
Further, the inventors have discovered that the insertion of a gene of interest into a particular transcriptional unit within this 9.5 kb stretch allows that gene to be regulated so as to be expressed only at high cell. density. This transcriptional unit contains three methylenomycin biosynthetic genes. Similar results were obtained for another transcriptional unit of the mmy gene cluster, indicating that other biosynthetic genes are similarly regulated, as well as for a transcriptional unit that is itself part of the regulatory system.
On the basis of these discoveries, the inventors now provide a model of how methylenomycin expression is regulated in Streptomyces. 
This model allows predictions to be made about the effect of disrupting certain portions of the gene cluster. Observations consistent with these predictions have been made.
The inventors have sequenced and identified a block of eight genes, designated mmyR, mmfP, mmfH, mmfL, mmfR, mmyT, mmyO and mmyG. The arrangement of these genes in the sequenced stretch of DNA is shown in FIG. 1d. FIGS. 1a-c show the location of this stretch in the methylenomycin gene cluster and on the SCP1 plasmid.
The authors have further sequenced and identified five more genes, designated mmyK, mmyP, mmyA, mmyC and mmyX, which are part of an incompletely defined transcription unit, mmy . . . XCAPK, in a nearby block. The organisation of these genes, and further nearby genes, is shown in FIG. 1e. The sequence of this whole region, along with deduced amino acid sequences of the gene products, is now available in the GenBank/EMBL database, under accession number AJ276673.
At the heart of the system are the products of the two genes mmyR and mmfR. The inventors have discovered that these two genes encode proteins with very significant similarity to several other proteins from various Streptomyces spp. (FIG. 2). A known model member of this protein sub-family is ArpA, a protein of Streptomyces griseus (see e.g. Onaka and Horinouchi, 1997; Onaka et al., 1997; and Sugiyama et al., 1998). ArpA binds A-factor, an acyl-γ-butyrolactone (GBL), with high affinity. In the absence of A-factor, ArpA binds to specific sequences in the promoters of target genes, and prevents their expression. When ArpA binds A-factor, it loses its DNA-binding activity, and the target genes are expressed.
A-factor-like GBLs are a widespread family of molecules in streptomycetes, which accumulate outside the cells in culture; they appear to be freely exchanged between the cytoplasm and the medium. Only at high cell density does the concentration outside, and therefore inside, the cells become sufficient to cause detachment of cognate binding proteins (such as ArpA) from promoters. The result of this is that the target genes become active only at high cell density. Moreover, at least some of these target genes regulate sporulation and/or antibiotic production, processes which occur only in dense cultures.
The inventors have also discovered that the deduced amino acid sequence of the mmfL gene is very significantly similar to proteins which confer GBL production in other Streptomyces spp. (FIG. 3). It can be seen in FIG. 1 that the mmfL gene is located between the two repressor-encoding genes mmyR and mmfR, along with mmfP and mmfH.
Further, the inventors present experimental results, based on the insertion of a marker gene into the mmyG and mmfH genes, which show that mmyG and mmfH are selectively expressed at high cell density (FIG. 4). The gene chosen was xylE from a plasmid of pseudomonads (Bruton et al, 1991). The xylE gene product is the enzyme catechol oxygenase, which may be detected by colony staining (Ingram et al., 1989).
From sequence analysis, it newly appears that the mmyT, mmyO and mmyG genes are transcribed from a common promoter, within the non-coding region between mmfR and mmyT. Thus, the inventors suggest that all three genes of the mmyTOG region are selectively expressed at high cell density. Similarly, it newly appears that mmfL, mmfH and mmfP are transcribed from a common promoter, within the non-coding region between mmfL and mmfR, and that this promoter is similarly regulated.
Similar regulation of expression was obtained when xylE was inserted into the mmy . . . XCAPK transcription unit (FIGS. 4 and 5), indicating that the promoter for this region is similarly regulated.
Based on their studies, the inventors now provide a model for the regulation of methylenomycin production in Streptomyces. The products of the mmyR and/or mmfR genes, ArpA homologues, bind to the promoter(s) of gene(s) presumed to encode methylenomycin biosynthetic enzymes or positive regulators of methylenomycin biosynthesis, thereby preventing methylenomycin production. MmfL, the product of the mmfL gene, directs the synthesis of a GBL, which binds to the products of the mmyR and/or mmfL genes. At sufficiently high cell density (and hence sufficiently high mmfL-specified GBL concentration in the medium), this latter binding is sufficient to prevent binding of the mmyR and/or mmfR gene products to the promoter(s) whose activation is necessary for methylenomycin production. Included among these are the promoters of mmyTOG, mmfLHP and mmy . . . XCAPK. It is further deduced that the induction of the mmfLHP promoter forms a “gearing” system, amplifying the production of GBL, and “committing” the cells to uninhibited expression from the mmyTOG and mmy . . . XCAPK promoters.
As a result of further experiments, it is further suggested that the product of another gene, mmyB, is also involved in this commitment (see Example 4).
The inventors now predict from this model that disruption of the mmyR and/or mmfR genes will cause increased methylenomycin production, because loss of repressors releases target promoters from repression. Observations have confirmed this prediction and show that none of the mmyR, mmfP and mmfH genes is a positive regulator; that none of them encodes an essential biosynthetic enzyme for methylenomycin biosynthesis; and that mmyR acts negatively.
As a result of further experiments, the inventors also predict that an additional gene, mmyB, is also involved in the operation of the regulatory scheme. Observations reported in Example 4 have supported this prediction.
The inventors also believe that the presence of an extra copy of mmfL would cause increased methylenomycin production, since this should lead to increased GBL synthesis and hence more efficient lifting of repression. Observations have confirmed this prediction also.
The sequencing of the mmfL gene shows that it contains an unusual feature, namely the presence of a TTA codon (position shown in FIG. 1). TTA (=UUA in mRNA) is one of six codons which encode leucine. However, from the fact that there are fully viable mutant strains of Streptomyces which lack the ability to translate this codon, it is known that the TTA codon is not used in any essential genes of Streptomyces spp. Such mutants are defective in the bldA gene which directly encodes the transfer RNA responsible for recognising the UUA codon (Leskiw et al., 1991). Translation of mmfL mRNA into MmfL protein would therefore be severely impaired in a bldA mutant. Observations reported in Example 4 have strongly supported this prediction.
The present inventors also predict that there would be a failure of GBL production in a bldA mutant, which would lead to non-production of methylenomycin, since their model indicates that mmfL confers production of a GBL needed to relieve repression of methylenomycin synthesis. Observations reported in Example 4 have strongly supported this prediction.
Experiments are described herein in which an appropriately orientated foreign gene encoding an easily detectable enzyme (xylE) was inserted into different transcription units in the methylenomycin gene cluster. Expression of xylE was detected at high levels in, for example, bldA+ strains carrying mmyG::xylE, mmfH::xylE or mmy . . . XCAPK::xylE fusions, but was undetectable in bldA mutants carrying the same fusions, confirming the prediction of the model.
The pattern of expression of catechol oxygenase in bldA+ strains carrying mmyG::xylE, mmy . . . XCAPK::xylE or mmfH::xylE fusions was that early in growth there was no detectable activity, whereas later in growth the specific activity rose very sharply (FIG. 4), demonstrating that the promoter driving xylE expression is very specifically regulated.
For comparison when xylE was fused to the redX transcription unit for production of undecylprodigiosin, a different antibiotic, in the chromosome of S. coelicolor, much lower specific activity was obtained, albeit in different growth conditions (Guthrie and Chater, 1990). Thus the promoter driving mmyG::xylE expression is very strong, by comparison to those of genes for other Streptomyces antibiotics. Similarly strong expression is indicated for the promoters driving expression of mmyK and mmfH.
In continuing sequencing of the methylenomycin genes one other target TTA sequence for bldA action has been discovered, indicating that the TTA codon in mmfL may not be the sole reason for bldA-dependence of methylenomycin production. Observations have verified this prediction (see Example 4b).
From their model, the inventors teach that the insertion of a nucleic acid of interest (e.g. a gene or genes) into the mmyTOG region, in the correct orientation, and in the presence of the mmyR-mmf-mmyTOG region left of the insertion site, provides self-regulating, strong expression of the nucleic acid of interest specifically late in culture, to give a high level of expression only in conditions when the main growth phase has been completed (i.e. at high biomass and high mycelium density). This has four main advantages for the expression of the nucleic acid of interest: (1) reduced or no expression earlier in growth, avoiding toxic effects of some gene products on growth; (2) there is no requirement for an exogenously added inducer, avoiding various constraints on the culture medium or problems of the cost of adding inducers to large fermenters or of removing them from the desired end product; (3) the methylenomycin cluster, naturally present on a highly transmissible plasmid, is likely to have evolved to permit properly regulated expression in diverse Streptomyces hosts (see above), which is important commercially because virtually every antibiotic or other Streptomyces product made commercially involves a different strain; and (4) the expression is driven by a strong promoter, leading to high yield of the desired end product. Further experiments appear to indicate that the host strain used for expression should contain those mmy genes to the right of mmr, or at least the mmyB gene.
The inventors similarly teach that similar results could be obtained with the nucleic acid of interest inserted in the appropriate orientation into the mmy . . . XCAPK region, in the presence of the mmy . . . XCAPK region to the right of the insertion site and in the presence of the mmy . . . XCAPK promoter, or with the nucleic acid of interest inserted in the appropriate orientation into the mmfLHP region, in the presence of the mmfLHP region to the right of the insertion site and in the presence of the mmfLHP promoter.
Similar results may also be obtained with the nucleic acid of interest inserted in the appropriate orientation into the mmyBQE region, in the presence of the mmyBQE region to the right of the insertion site and in the presence of the mmyBQE promoter, or with the nucleic acid of interest inserted in the appropriate orientation into the mmyYF region, in the presence of the mmyYF region to the left of the insertion site and in the presence of the mmyYF promoter.
Furthermore, it is believed that certain regions of the 9.5 kb stretch which has been investigated are of greater importance than others. In particular, the model teaches that interplay between the products of the mmfR, mmyR, mmfL and mmyB genes and the promoters of the mmyTOG, mmy . . . XCAPK and mmfLHP regions is key to the regulation of methylenomycin production. Consequently, it is taught that the combination of the nucleic acid of interest and minimal regulatory portions which include an mmfR gene and/or an mmyR gene; an mmfL gene; an mmyB gene; and an mmyTOG promoter and/or an mmy . . . XCAPK promoter and/or an mmfLHP promoter and/or an mmyBQE promoter and/or an mmyYF promoter will also lead to increased expression of the nucleic acid of interest at higher cell density relative to lower cell density.
However, it is also contemplated that the mmfP and mmfH genes may be of importance in regulation of methylenomycin production, for example in some conditions their products may modify the structure of the GBL whose production is conferred by the mmfL gene, resulting in changes in the details of interactions between the GBL and the mmyR and/or mmfR gene products. Consequently these genes may also be present in the regulatory portion.
Moreover, having found that three different promoters of methylenomycin biosynthetic and positive regulatory genes are regulated in the same way, the inventors expect that other promoters of methylenomycin biosynthetic genes will also be similarly regulated.
In a first aspect, therefore, the present invention provides an expression cassette for the expression of a nucleic acid of interest, the expression cassette including:

    
    
        a regulatory portion or portions including:
        
            a first regulatory element which includes either an mmyR gene encoding an MmyR polypeptide, or an mmfR gene encoding an MmfR polypeptide, or both;
            a second regulatory element which includes an mmfL gene encoding an MmfL polypeptide; and
            a promoter (the “repressible promoter”), the function of which is repressed by the expression product of the first regulatory element, that repression being alleviated or removed by a product, the production of which is conferred by the MmfL polypeptide, and
        
        
        the nucleic acid of interest, in operative association with said promoter.
    
    


Such a construct represents the minimal expression cassette which may be predicted by the above model to cause expression of the nucleic acid of interest in Streptomyces at high cell density.
Preferably, the expression cassette is capable of expressing the nucleic acid of interest at increased levels in stationary phase cultures of Streptomyces compared to early exponential phase cultures.
Preferably the regulatory elements, promoter and nucleic acid of interest are provided on a single expression cassette, but it is contemplated that they may be provided separately, for example on two vectors which may be co-introduced into a desired host, or that one or more of the regulatory elements and promoter may be provided by the SCP1 plasmid.
Accordingly in this first aspect, the present invention also provides a set of nucleic acids for the expression of a nucleic acid of interest, the set of nucleic acids together including:

    
    
        a regulatory portion or portions including:
        
            a first regulatory element which includes either an mmyR gene encoding an MmyR polypeptide, or an mmfR gene encoding an MmfR polypeptide, or both;
            a second regulatory element which includes an mmfL gene encoding an MmfL polypeptide;
            a promoter (the “repressible promoter”), the function of which is repressed by the expression product of the first regulatory element, that repression being alleviated or removed by a product, the production of which is conferred by the MmfL polypeptide, and
        
        
        the nucleic acid of interest, in operative association with said promoter.
    
    


Preferably the set is an isolated set of nucleic acids.
Preferably both an mmyR gene and an mmfR gene will be provided in the regulatory portion(s) of this aspect.
Preferably an mmyB gene encoding an MmyB polypeptide will be provided within, or in addition to, the expression cassette or set of nucleic acids, as a mediator of the regulatory effects of the regulatory portion. In a preferred embodiment, the mmyB gene is incorporated into a third regulatory element of the regulatory portion(s). Preferably some or all of other mmy genes to the right of the mmr gene (as shown in FIG. 1e) are also provided.
The repression of the function of the repressible promoter by the expression product of the first regulatory element may be direct repression by the expression product, or it may arise from the absence of an activator which is itself repressed by the first regulatory element.
Preferably the regulatory portion(s) comprise at least a portion of the 9.5 kb newly sequenced stretch of nucleic acid as shown in FIG. 7, or a variant thereof.
Preferably, the mmyR gene encodes an MmyR polypeptide having the amino acid sequence of FIG. 8a (optionally excluding the first 6 listed amino acids). More preferably it comprises residues 1407 to 796 of FIG. 7, lower strand (optionally excluding residues 1407 to 1390).
Preferably, the mmfR gene encodes an MmfR polypeptide having the amino acid sequence of FIG. 8e. More preferably it comprises residues 4807 to 5451 of FIG. 7, upper strand.
Preferably, the first regulatory element also includes a promoter operatively linked to the mmyR or mmfR gene. In embodiments where both genes are present, they are preferably operatively linked to respective promoters. Still more preferably, the promoter to which the mmyR gene is linked is an mmyR promoter and/or the promoter to which the mmfR gene is linked is an mmfR promoter. Even more preferably the promoter to which the mmyR gene is linked comprises some or all of residues 1557 to 1390 of FIG. 7, lower strand (optionally excluding residues 1409 to 1390) and/or the promoter to which the mmfR gene is linked comprises some or all of residues 4613 to 4806 of FIG. 7, upper strand.
Preferably, the mmfL gene encodes an MmfL polypeptide having the amino acid sequence of FIG. 8d. More preferably, it comprises residues 4612 to 3551 of FIG. 7, lower strand.
Preferably, the second regulatory element also includes a promoter operatively linked to the mmfL gene. Still more preferably, the promoter to which the mmfL gene is linked is an mmfL promoter. Even more preferably the promoter to which the mmfL gene is linked comprises some or all of residues 4806 to 4613 of FIG. 7, lower strand.
It will be observed that the preferred promoters for mmfL and mmfR both comprise some or all of residues 4806 to 4613 of FIG. 7. This region is thought to include a bi-directional promoter for both these genes. Preferably the promoter(s) for mmfL and/or mmfR include a palindromic sequence having a high degree of sequence identity or complete sequence identity with a palindromic sequence having the half-site 5′-GG(T/C)CGGT(A/T) (T/C)G(T/G)-3′ (SEQ ID NO: 1), which is the consensus sequence for binding of DNA by the ArpA protein. In this context high sequence identity preferably represents sequence identity at seven or more, more preferably 8 or more, 9 or more, or 10 or more corresponding positions within the half-site. More preferably the palindromic sequence has the half-site 5′-GGAAGGTATTA-3′ (SEQ ID NO: 2) (or a variant thereof). In a particularly preferred embodiment, the regulatory portion comprises residues 3551 to 5451 of FIG. 7, upper strand (or a variant thereof).
Preferably the repressible promoter is a promoter of a methylenomycin biosynthetic or regulatory gene.
More preferably the repressible promoter comprises an mmyTOG promoter or an mmy . . . XCAPK promoter or an mmfLHP promoter or an mmyBQE promoter or an mmyYF promoter.
However, it is also thought that other promoters of methylenomycin biosynthetic genes may be regulated in the same way as mmyTOG, mmy . . . XCAPK and mmfLHP. Accordingly, the repressible promoter may comprise a promoter of any other methylenomycin biosynthetic gene which is regulated by the mmyR and/or mmfR genes and the mmfL gene, typically mediated by the mmyB gene.
Preferably the mmyTOG promoter comprises some or all of residues 5452 to 5675 of FIG. 7; upper strand.
Preferably the mmfLHP promoter comprises some or all of residues 4613 to 4806 of FIG. 7, lower strand.
The mmy . . . XCAPK, mmyBQE and mmyYF promoters remain to be accurately located. However, this may be accomplished routinely by sequencing and sequence analysis of, for example, the restriction fragments A4.2 and A3.13 (Chater and Bruton, 1983), which make available different parts of the mmy . . . XCAPK cluster, or by analysis of EMBL accession number AJ276673. It is thought that the mmy . . . XCAPK promoter comprises some or all of residues 15404 to 15977 of EMBL AJ276673 and that the mmyBQE promoter comprises some or all of residues 18892 to 19123 of EMBL AJ276673, or that the mmyBQEDXCAPK genes are all transcribed from a single promoter which comprises some or all of residues 18892 to 19123 of EMBL AJ276673. It is thought that the mmyYF promoter comprises some or all of residues 18892 to 19123 of EMBL AJ276673.
Apart from the repressible promoter, the nucleic acid of interest is preferably not additionally in operative association with any exogenous promoter, i.e. any promoter which is not derived from (or a variant of) a promoter present in the methylenomycin gene cluster. For example, in preparing the expression cassette (or set of nucleic acids) according to this aspect of the invention, the nucleic acid of interest is preferably brought into operative association with the repressible promoter in the functional absence of any promoter with which it may have previously been associated (for example a promoter of a cloning vector).
Preferably, the mmyB gene encodes an MmyB polypeptide having the deduced amino acid sequence shown for this gene in EMBL AJ276673. More preferably, it comprises the complement of residues 18032 to 18892 of the nucleic acid sequence shown in EMBL AJ276673.
Preferably, the third regulatory element also includes a promoter operatively linked to the mmyB gene. Still more preferably, the promoter to which the mmyB gene is linked is an mmyB promoter. Even more preferably the promoter to which the mmyB gene is linked comprises some or all of residues 18892 to 19123 of EMBL AJ276673.
Since it is thought that they may be of importance in directing expression to high cell density cultures in some fermentation conditions, an mmfP gene and/or an mmfH gene, preferably both, are preferably included in the regulatory portion(s). However in some embodiments, in which an mmyLHP promoter is operatively linked to the nucleic acid of interest, one or more of these genes may be replaced or disrupted by the nucleic acid of interest.
When an mmyTOG promoter is used, at least part of at least one of an mmyT, an mmyO and/or an mmyG gene is suitably present in the regulatory portion, in operative association with the mmyTOG promoter. However, there may in particular be a 3′ (right hand end) truncation of the mmyTOG coding region.
When an mmfLHP promoter is used, at least part of at least one of an mmfH and/or an mmfP gene is suitably present in the regulatory portion, in operative association with the mmfLHP promoter. However, there may in particular be a 3′ (left hand end) truncation of the mmfLHP coding region. Preferably an intact mmfL gene is also in operative association with the same mmfLHP promoter.
When an mmy . . . XCAPK promoter is used, at least part of at least one of an mmyX, an mmyC, an mmyA, an mmyP, an mmyK gene, and/or an mmyD gene (mmyD is a co-transcribed gene located between mmyx and the putative mmy . . . XCAPK promoter) is suitably present in the regulatory portion, in operative association with the mmy . . . XCAPK promoter. However, there may in particular be a 3′ (left hand end) truncation of the mmy . . . XCAPK coding region.
When an mmyBQE promoter is used, at least part of at least one of an mmyB, an mmyQ and/or an mmyE gene is suitably present in the regulatory portion, in operative association with the mmyBQE promoter. However, there may in particular be a 3′ (left hand end) truncation of the mmyBQE coding region.
The same applies mutatis mutandis to the situation where an mmyBQEDXCAPK promoter is used.
When an mmyYF promoter is used, at least part of at least one of an mmyY and an mmyF gene is suitably present in the regulatory portion, in operative association with the mmyYF promoter. However, there may in particular be a 3′ (right hand end) truncation of the mmyYF coding region.
The location and sequence of the individual genes with the mmyDXCAPK, mmyBQE (or mmyBQEDXCAPK) and mmyYF regions is given in EMBL AJ276673. The sequences of the corresponding promoters may be deduced by sequence analysis and/or routine experimentation on the basis of this sequence information (e.g. using DNase footprinting experiments).
Desirably, the nucleic acid of interest is inserted into the regulatory portion, preferably within an mmyT, mmyO or mmyG gene (when an mmyTOG promoter is used) or within an mmyD, mmyX, mmyC, mmyA, mmyP or mmyK gene (when an mmY . . . XCAPK promoter is used) or within an mmfH or mmfP gene (when an mmfLHP promoter is used) or within an mmyQ or mmyE gene (when an mmyBQE promoter is used) or within an mmyQ, mmyE, mmyD, mmyX, mmyC, mmyA, mmyP or mmyK gene (when an mmyBQEDXCAPK promoter is used) or within an mmyY or mmyF gene (when an mmyYF promoter is used).
The nucleic acid of interest may be inserted into the regulatory portion by means of homologous recombination (e.g. using a vector containing a fragment of the mmyTOG, mmy . . . XCAPK, mmyBQE, mmyBQEDXCAPK, mmyYF or mmfLHP coding region). Accordingly, the regulatory portion may contain an entire mmyTOG, mmy . . . XCAPK, mmyBQE, mmyBQEDXCAPK, mmyYF or mmfLHP coding region, which is interrupted by the nucleic acid of interest.
Preferably, the mmyT gene encodes an MmyT polypeptide having the amino acid sequence of FIG. 8f, or a variant thereof. More preferably, it comprises residues 5676 to 6401 of FIG. 7, upper strand.
Preferably, the mmyO gene encodes an MmyO polypeptide having the amino acid sequence of FIG. 8g. More preferably, it comprises residues 6432 to 7553 of FIG. 7, upper strand.
Preferably, the mmyG gene encodes an MmyG polypeptide having the amino acid sequence of. FIG. 8h. More preferably, it comprises residues 7636 to 8817 of FIG. 7, upper strand.
Preferably, the mmfL gene encodes an MmfL polypeptide having the amino acid sequence of FIG. 8d. More preferably, it comprises residues 3551 to 4612 of FIG. 7, lower strand.
Preferably, the mmfH gene encodes an MmfH polypeptide having the amino acid sequence of FIG. 8c. More preferably, it comprises residues 3554 to 2352 of FIG. 7, lower strand.
Preferably, the mmfP gene encodes an MmfP polypeptide having the amino acid sequence of FIG. 8b. More preferably, it comprises residues 3554 to 1558 of FIG. 7, upper strand.
The same applies, mutatis mutandis, to the mmyDXCAPK, mmyBQE and mmyYF genes, based on the respective amino acid and nucleic acid sequences given in EMBL AJ276673.
Preferably the nucleic acid of interest is heterologous, i.e. having a sequence not present in or derived from the 9.5 kb stretch of newly sequenced DNA, more preferably not present in or derived from the methylenomycin biosynthetic gene cluster.
In one preferred embodiment, the regulatory portion(s) include(s) an mmfR gene and an mmfL gene, with the mmfL promoter as the repressible promoter (for example the nucleic acid may be inserted into the mmfH or mmfP gene, downstream of mmfL under the control of the mmfLHP promoter).
In a more preferred embodiment, the regulatory portion(s) include(s) an mmfR gene, an mmfL gene and an mmrR gene, with the mmyB promoter as the repressible promoter (for example the nucleic acid may be inserted into the mmyB, mmyQ or mmyE gene, under the control of the mmyB promoter).
In a still more preferred embodiment, the regulatory portion(s) include(s) an mmfR gene, an mmfL gene, and mmyR gene, with the mmyTOG promoter, the mmyDXCAPK or the mmyYF promoter as the repressible promoter, such regulatory portion(s) being suitable for use in the presence (either as a third regulatory portion or as another part of the expression system) of an mmyB gene.
In a highly preferred embodiment, the regulatory portion(s) may include nucleic acid having the sequence from residue 796 to a residue between 5676 and 8817 (inclusive) of FIG. 7 (or a variant thereof). When double stranded, this nucleic acid contains mmyR, mmfP, mmfH, mmfL and mmfR genes and intergenic regions between those genes and at least a region containing an mmyTOG promoter (i.e. the region upstream of mmyT), and optionally some or all of an mmyTOG coding region. The nucleic acid of interest may then be inserted into or downstream of the mmyTOG region (or part thereof) in operative association with the mmyTOG promoter.
In another highly preferred embodiment, the regulatory portion(s) may include nucleic acid having the sequence from residue 796 to residue 5451 (inclusive) of FIG. 7 (or a variant thereof). When double stranded, this nucleic acid contains mmyR, mmfP, mmfH, mmfL and mmfR genes and intergenic regions between those genes. The nucleic acid of interest may then be inserted into the mmfHP region in operative association with the mmfLHP promoter.
It is contemplated that elements contributing to promoter activity (e.g. enhancer elements) may lie outside the non-coding intergenic regions specified above. Accordingly, it is particularly preferred that the intergenic promoter regions are located in their natural immediate context, i.e. between genes (or parts of genes) which normally flank them. Accordingly embodiments using a cassette containing an intact mmyR-mmf-mmyTOG region, optionally with a disruption of or 3′ (right-hand end) truncation of the mmyTOG region, are particularly preferred.
In a second aspect, the present invention provides a vector comprising an expression cassette according to the first aspect of the invention. Further it provides a set of vectors comprising a set of nucleic acids according to the first aspect of the invention.
Suitable vectors comprising nucleic acid for introduction into bacteria can be chosen or constructed, containing appropriate additional regulatory elements if necessary, including additional promoters, terminator fragments, enhancer elements, marker genes and other elements as appropriate. Vectors may be plasmids, viral e.g. “phage”, or “phagemid”, as appropriate. For further details see, for example, Sambrook et al, (1989). Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. (1992). Many aspects of the employment of these techniques in the context of Streptomyces spp. are described in detail in Hopwood et al (1985) and Kieser et al (2000). The disclosures of Sambrook et al, Ausubel et al, Hopwood et al and Kieser et al are all incorporated herein by reference for these and all other purposes.
In a third aspect, the present invention provides an expression system comprising an expression cassette or set of nucleic acids according to the first aspect of the invention and an expression system comprising a vector according to the second aspect of the invention.
Preferably the expression system is a host cell, although cell-free expression systems are also contemplated. Preferably the host cell is a bacterium, more preferably an actinomycete, further preferably a streptomycete. In particular, it has been shown (see Examples) that the invention can be applied successfully in streptomycete strains other the S. coelicolor, as expected on the basis of the transmissibility of the plasmid SCP1.
Preferably the expression system (usually a native or genetically modified host cell) contains an mmyB gene, more preferably as an additional part of the vector system used to introduce the expression cassette/set f nucleic acids. However, it is also contemplated that the mmyB gene may be present e.g. as part of the host cell genome and/or on a plasmid, e.g. SCP1 or pSV1 also present within the expression system. This is a less preferred embodiment as other methylenomycin gene promoters may sequester mmyB gene product, reducing its effectiveness in mediating expression from the expression cassette/set of nucleic acids.
In one preferred embodiment, the expression system lacks the ability to translate the codon TTA (UUA in mRNA), and the expression cassette, set of nucleic acids or vector(s) lacks TTA codons, and/or has been modified to eliminate one or more (preferably all) naturally occurring TTA codons. For example the expression system is preferably a cell of a bldA mutant strain of Streptomyces and the expression cassette preferably contains a variant of the mmfL gene in which the naturally occurring TTA codon has been altered (e.g. by site-directed mutagenesis) into another leucine-encoding codon. Preferably the mmyB gene included in this system (whether as part of an expression cassette or part of the host cell genome or on a plamsid also present in the system) has also been similarly altered so that its TTA codon has been changed to another leucine-encoding codon (see Example 10). This provides the advantage, particularly in Streptomyces spp., that expression of the nucleic acid of interest can be achieved with reduced expression of other products (for example antibiotics) which would otherwise also be expressed at high cell density via bldA-dependent mechanisms; i.e. this system provides a clean background for expression of the nucleic acid of interest. One or more TTA codons are present in the biosynthetic gene clusters for most streptomycete antibiotics, particularly in pathway-specific regulatory genes. TTA codons should not be present in genes whose expression is being carried out in a bldA mutant host. In some circumstances it may be necessary to use site-directed mutagenesis to convert a TTA codon to another leucine codon.
In another preferred embodiment the number of repressible promoters present in the expression system is limited. This may reduce expression-limiting sequestration of mmyB gene product by promoters other than the repressible promoter controlling expression of the nucleic acid of interest. Such limitation may involve the absence from the expression system of the SCP1 plamid and/or the pSV1 plasmid (though in such cases the mmyB gene is preferably otherwise present in the expression system). Additionally or alternatively, the expression cassette/set of nucleic acids may lack repressible promoters other than the promoter controlling expression of the nucleic acid of interest (however, promoters controlling expression of other genes of the expression cassette/set of nucleic acids may for this purpose be regarded as not being repressible promoters, e.g. the mmyTOG promoter may control the expression of the nucleic acid of interest and the mmfL and mmyB genes may be controlled by their native promoters). Lacking may be relative (i.e. there are fewer repressible promoters than in the native methylenomyin cluster), substantial or complete.
The introduction of the expression cassette, set of nucleic acids or vector(s) into a host cell, which may (particularly for in vitro introduction) be generally referred to without limitation as “transformation”, may employ any available technique. For bacterial cells, suitable techniques may include calcium chloride transformation, polyethyleneglycol assisted transformation, electroporation, conjugation and transfection or transduction using bacteriophages.
In a fourth aspect, the present invention provides a method of expressing a nucleic acid of interest, the method comprising providing a host cell (or other expression system) according to preferred embodiments of the third aspect and culturing the host cell, so as to express the nucleic acid of interest.
Preferably the nucleic acid of interest is expressed substantially only when the host cell culture reaches high cell density, more preferably at or close to the stationary phase of host cell culture. Cell cultures at or close to stationary phase may have OD650 values in the range of 1-20.
Known methods of culturing cells are well known in the art, for example from Sambrook et al (1989), Ausubel et al (1992), and (in particular for Streptomyces spp.) Hopwood et al (1985) and Kieser et al (2000).
In a fifth aspect, the present invention provides a method of expressing a nucleic acid of interest, the method comprising:

    
    
        providing in an expression system a regulatory portion or portions as defined in the first aspect;
        providing in the expression system the nucleic acid of interest;
        operatively associating the nucleic acid of interest with the repressible promoter of the regulatory portion(s); and
        expressing the nucleic acid of interest in the expression system.
    
    


The steps of the method need not be performed in the order recited. In particular, the operative association may occur prior to introduction regulatory portion(s) and nucleic acid of interest into the expression system.
The preferred features specified for the regulatory portion(s) in the context of the first aspect of the invention may also be present in the regulatory portion(s) used in this aspect. Preferably the expression system is a cell, more preferably a bacterium, further preferably an actinomycete and most preferably a streptomycete. Preferably the expression system contains an mmyB gene which may be introduced together with the regulatory cassette. The cell is preferably cultured for expression of the nucleic acid of interest.
The nucleic acid of interest may be brought into operative association with the repressible promoter in a variety of ways. For example, the nucleic acid of interest may be inserted into a nucleic acid molecule which contains the repressible promoter, downstream of the repressible promoter (FIGS. 6c and 6d). In a preferred example, the repressible promoter is an mmyTOG promoter and the insertion site is within an mmyTOG region.
Alternatively, the regulatory portion may be inserted into nucleic acid containing the nucleic acid of interest, for example by homologous recombination (FIG. 6b). Thus a fragment from the 5′ end of the nucleic acid of interest may be included downstream of and in operative association with the repressible promoter to permit homologous recombination of the regulatory portion into the nucleic acid of interest.
This method may be advantageously used in a bldA mutant Streptomyces or other actinomycete host cell, with a regulatory portion or portions (and preferably mmyB gene) lacking TTA codons, to regulate the expression of a nucleic acid of interest which is native to the host cell and which preferably confers production of an antibiotic. This embodiment has the advantage that other antibiotics encoded by the host cell will generally not be expressed, since the pathway-specific regulatory genes for production of such other antibiotics in Streptomyces typically include a TTA codon. For example, in one preferred expression system, S. coelicolor A(3)2, major pathway-specific regulatory genes of each of two known chromosomally located antibiotic pathways contain TTA codons, and a bldA mutant therefore makes neither of these antibiotics in typical culture media (Fernandez-Moreno et al. 1991, White and Bibb 1997).
The expression products of the nucleic acids of interest of the fourth and fifth aspects may be collected and purified. This may be achieved by conventional methods. See for example McDaniel et al. (1993).
Where the nucleic acid of interest is for example a biosynthetic gene cluster, both the end product of the biosynthesis and the biosynthetic enzymes themselves may be regarded to be the expression product, but more usually the end product will be regarded to be the expression product.
In a sixth aspect, therefore, the present invention provides an expression product produced according to the method of either of the fourth or fifth aspects of the invention.
The nucleic acid of interest may be any nucleic acid. Preferred nucleic acids are genes, the expression of which is desired, or gene clusters, for example which encode the enzymes necessary for the biosynthesis of e.g. antibiotics. Gene clusters may have a plurality of genes within the same transcriptional promoter, so as to allow expression of all genes of the cluster from the repressible promoter. Alternatively, the nucleic acid of interest may be an unknown nucleic acid which it is desired to investigate, for example nucleic acid derived from a sample e.g. of soil.
In a seventh aspect, the present invention provides a nucleic acid molecule comprising an mmyR and/or an mmfR gene, an mmfL gene, and a repressible promoter, all as defined in the first aspect, wherein the molecule is capable of regulating the expression of a nucleic acid of interest when that nucleic acid is arranged in operative association with the repressible promoter.
The same preferred and optional features apply to this aspect as they apply to the first aspect.
Preferably the molecule of this aspect is other than pIJ519, as disclosed in Chater and Bruton (1985) and/or does not consist of or include the 350 kb SCP1 plasmid of Streptomyces coelicolor and/or the pSV1 plasmid of Streptomyces violaceoruber. However, such plasmids may be used in conjunction with the molecule of this aspect (e.g. to supply an mmyB gene).
Preferably, the molecule consists essentially of an approximately 4.8 to 8 kb stretch of DNA including mmyR, mmfP, mmfH, mmfL and mmfR genes and at least a portion of at least one of an mmyT gene, an mmyO gene and an mmyG gene. More preferably, the molecule includes the entire mmyT and mmyO genes and at least a portion of the mmyG gene.
Preferably the molecule consists essentially of the nucleic acid having the sequence from residue 796 to a residue between 5676 and 8817 (more preferably between 7636 and 8817) of FIG. 7.
In an alternative embodiment, the molecule consists essentially of a stretch of nucleic acid including mmyR, mmfP, mmfH, mmfL and mmfR genes in combination with a stretch of nucleic acid including an mmy . . . XCAPK promoter and at least a portion of at least one of an mmyD gene, an mmyX gene, an mmyC gene, an mmyA gene, an mmyP gene, an mmyK gene.
In an alternative embodiment, the molecule consists essentially of an approximately 5 kb stretch of DNA including mmyR, mmfP, mmfH, mmfL and mmfR genes.
In addition to the defined stretches of DNA, the molecule preferably comprises an mmyB gene. p In an eighth aspect, the present invention provides a nucleic acid molecule consisting essentially of one or more of an mmyB gene in which a naturally occurring TTA codon has been changed into another (preferably leucine encoding) codon, an mmyR gene, an mmfP gene, an mmfH gene, an mmfL gene (preferably in which a naturally occurring TTA codon has been changed into another (preferably leucine encoding) codon), an mmfR gene, an mmyT gene, an mmyO gene, and an mmyG gene, optionally with a respective upstream region or respective upstream regions. Where present, the upstream region(s) may comprise promoters (preferably as previously defined) for the genes. Where two or more genes are present, an upstream region for one or more of those genes may be provided in an intergenic region.
The genes may be as previously defined.
In a ninth aspect, the present invention provides the use of one or more nucleic acid molecules as defined in any one of the seventh or eighth aspects, in or for the regulation of expression of a nucleic acid of interest in an expression system. p In a tenth aspect, the present invention provides a polypeptide encodable by one of the following genes: an mmyR gene, an mmfP gene, an mmfH gene, an mmfL gene, an mmfR gene, an mmyT gene, an mmyO gene and an mmyG gene. Preferably the polypeptide is substantially isolated from other proteins with which it is naturally associated.
The polypeptide preferably has an amino acid sequence as shown in one of FIGS. 8a to 8h. However, this aspect also provides polypeptides which are variants of those amino acid sequences.
In an eleventh aspect, the present invention provides a vector including a nucleic acid according to the seventh or eighth aspect. In embodiments of the seventh and eighth aspects in which the nucleic acid lacks a promoter or promoters for the gene or genes it contains, the vector preferably includes a promoter in operative association with that gene or those genes.
In a twelfth aspect, the present invention provides an expression system containing one or more nucleic acids according to the seventh or eighth aspects and an expression system containing a vector according to the eleventh aspect.
Preferably the expression system is a cell. Where it is desired merely to express the polypeptide encoded by the nucleic acid, rather than for example to regulate the expression of another nucleic acid of interest, any appropriate cell may be used (e.g. a standard E. coli overexpression system). See for example Sambrook et al (1989) and Ausubel et al (1992). Otherwise, bacterial, actinomycete and streptomycete cells are preferred as previously indicated.
In a thirteenth aspect, the present invention provides a method of producing a polypeptide according to the tenth aspect, the method comprising producing the polypeptide in an expression system according to the twelfth aspect.
The polypeptide may be purified from the expression system by conventional methods.
References herein to genes, coding regions and nucleic acids are not to be interpreted as being restricted to genes, coding regions and nucleic acids having the specific nucleic acid sequences disclosed herein or in EMBL AJ276673. Rather, genes, coding regions and nucleic acids having variants of those sequences are also included. Genes, coding regions and nucleic acids having such specific sequences are preferred embodiments. Thus, for example, a reference to “an mmfR gene” is not to be interpreted as being restricted to a gene having the sequence from residue 4807 to residue 5451 of FIG. 7, but also includes variants.
Similarly, references herein to polypeptides are not to be interpreted as being restricted to polypeptides having the specific amino acid sequences disclosed herein or in EMBL AJ276673. Rather, polypeptides having variants of those sequences are also included. Polypeptides having such specific sequences are preferred embodiments. Thus, for example, a reference to “an MmfR polypeptide” is not to be interpreted as being restricted to a polypeptide having the amino acid sequence shown in FIG. 8e, but also includes variants.
References herein to promoters are not to be interpreted as being restricted to nucleic acids having the sequence of all or part of a specific intergenic region disclosed herein or in EMBL AJ276673. Again, promoters having variants of those intergenic sequences are also included and the specific intergenic sequences (or parts thereof) are preferred embodiments. Thus, for example, a reference to “an mmyTOG promoter” is not to be interpreted as being restricted to the specific mmyTOG promoter disclosed herein (i.e. a nucleic acid having all or part of the sequence from residues 5452 to 5675 of FIG. 7, upper strand), but also includes variants.
In all cases, where a preferred embodiment of a gene, nucleic acid, polypeptide or promoter is defined by reference to a specific sequence, the invention in its broader sense is intended to include embodiments having variants of that specific sequence.
The term “variant” as used herein in relation to a particular nucleic acid (the reference nucleic acid) denotes: any nucleic acid having a sequence which is different from that of the reference nucleic acid, but which is its complement or which shows significant nucleic acid sequence identity with, or hybridisation under stringent conditions to, the reference nucleic acid or its complement or a fragment of the reference nucleic acid or its complement; or any nucleic acid which encodes an amino acid sequence having significant amino acid sequence identity with the amino acid sequence encoded by the reference nucleic acid, or a fragment of that nucleic acid. The term “variant” also refers to nucleic acids which differ from each other due only to the degeneracy of the genetic code, and which therefore encode identical deduced amino acid sequences.
The term “variant” as used herein in relation to a particular polypeptide (the reference polypeptide) denotes: any polypeptide having an amino acid sequence which is different from, but which shows significant amino acid sequence identity with, the amino acid sequence of the reference polypeptide, or a fragment of that polypeptide.
Unless otherwise specified, significant amino acid sequence identity is preferably at least 80%, more preferably 85%, 90% or 95%, still more preferably 98% or 99% and/or significant nucleic acid sequence identity is preferably at least 50%, more preferably 60%, 70%, 80% or 90%, still more preferably 95%, 98% or 99%.
Significant amino acid sequence identity is preferably shown between the variant polypeptide (or a portion thereof) and a fragment of at least 10 amino acids of the reference polypeptide, more preferably a fragment of a least 20, 30 or 40 amino acids, still more preferably a fragment of 60, 80 or 100 amino acids, more preferably the entire reference polypeptide.
Significant nucleic acid sequence identity is preferably shown between the variant nucleic acid (or a portion thereof) and a fragment of at least 30 residues of the reference nucleic acid, more preferably a fragment of a least 60, 90 or 120 residues, still more preferably a fragment of 180, 240 or 300 residues, more preferably the entire reference nucleic acid.
In relation to variants of the specific mmyR gene disclosed herein, or of its product, MmyR, significant amino acid sequence identity is preferably shown with residues 40 to 49 of FIG. 8a, more preferably residues 38 to 49, more preferably residues 38 to 56, more preferably residues 38 to 59, still more preferably residues 21 to 59, still more preferably residues 3 to 59 and/or significant nucleic acid sequence identity is preferably shown with corresponding portions of FIG. 7 which encode the above amino acid residues.
In relation to variants of the specific mmfR gene disclosed herein, or of its product, MmfR, significant amino acid sequence identity is preferably shown with residues 61 to 70 of FIG. 8e, more preferably residues 59 to 70, more preferably residues 59 to 77, more preferably residues 59 to 80, still more preferably residues 42 to 80, still more preferably residues 24 to 80 and/or significant nucleic acid sequence identity is preferably shown with corresponding portions of FIG. 7 which encode the above amino acid residues.
In relation to variants of the specific mmfL gene disclosed herein, or of its product, MmfL, significant amino acid sequence identity is preferably shown with residues 77 to 87 and/or residues 240 to 255 of FIG. 8d, more preferably residues 77 to 95 and/or residues 231 to 255, more preferably residues 77 to 107 and/or residues 223 to 255 and/or significant nucleic acid sequence identity is preferably shown with corresponding portions of FIG. 7 which encode the above amino acid residues.
“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.
In relation to variants of the promoters used in the present invention, nucleic acid sequence identity is preferably assessed over a sequence of at least 30 residues, more preferably 40 or 50 residues, still more preferably 60 residues. Thus, for example, preferred variants of the embodied mmyTOG promoter may have sequences which show 80% (or more) sequence identity over a 30 (or more) residue sequence within residues 5452 to 5675 of FIG. 7, upper strand.
“Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.
When a nucleic acid of interest is in “operative association” with a promoter, the promoter is able to direct transcription of the nucleic acid of interest in an appropriate expression system, with the nucleic acid of interest in the correct reading frame for translation. Preferably when a nucleic acid of interest is in operative association with a promoter, the transcript of the nucleic acid of interest contains an appropriately located ribosome binding site for expression in an appropriate expression system of the polypeptide encoded by the nucleic acid of interest. See for example Sambrook et al. (1989) and Ausubel et al. (1992).
Variants of the genes, coding regions, nucleic acids, polypeptides and promoters specifically disclosed herein preferably have the same function as those specifically disclosed. In relation to the mmyR, mmfR and mmfL genes such function may be encoding an MmyR, MmfR and MmfL polypeptide, respectively; in relation to the MmyR, MmfR and MmfL polypeptides and the repressible promoter (e.g. the mmyTOG promoter, the mmy . . . XCAPK promoter or the mmfLHP promoter) such function may be the ability to interact with each other according to the model proposed above.
As used herein “mmyTOG” denotes nucleic acid including MmyT, mmyO and mmyG genes. The same applies mutatis mutandis to “mmy . . . XCAPK”, the dots indicating the previously unidentified mmyD gene upstream of mmyX in the same transcription unit, and possibly the mmyB, mmyQ and mmyE genes, although these may form a separate transcription unit under the control of a different (mmyBQE) promoter. “mmyBQE” denotes nucleic acid including mmyB, mmyQ and mmyE genes. “mmyBQEDXCAPK” denotes nucleic acid including mmyBQE and mmyDXCAPK. “mmyYF” denotes nucleic acid including mmyY and mmyF genes. “mmf” denotes nucleic acid including mmfP, mmfH, mmfL and mmfR genes. “mmfLHP” denotes nucleic acid including mmfL, mmfH, and mmfP genes. “mmyR-mmf-mmyTOG” denotes nucleic acid including an mmyR gene and mmf and mmyTOG. “mmy . . . XCAPK promoter” may be a promoter which controls the transcription of mmyBQEDXCAPK, or a promoter which controls the transcription of mmyDXCAPK only.



The invention, in its various aspects, will now be described in detail, with reference to the following figures, in which:
FIG. 1 shows the origin of the regulatory portion.

    
    
        (a) The genome of Streptomyces coelicolor A3 (2) consists of a linear chromosome and two plasmids—the circular SCP2 and the linear SCP1.
        (b) The methylenomycin production genes form a large gene cluster on the SCP1 plasmid (Chater and Bruton, 1985; Redenbach et al., 1998).
        (c) About 25 kb of DNA includes regulatory, resistance and biosynthetic genes associated with methylenomycin production.
        (d) The leftmost ca. 8 kb of the gene cluster comprises regulatory portion of the present invention, i.e. genes involved negatively and positively in regulating levels of methylenomycin production, and promoters under the control of those regulatory genes (it also includes other genes which are transcribed from these promoters).
        (e) DNA to the right of the regulatory portion shown in (d), comprising the mmr (methylenomycin resistance) gene and genes further to the right, indicating the location of mmyB.
    
    


FIG. 2 shows a comparison of the products of methylenomycin regulatory genes with GBL-binding proteins from various Streptomyces spp. The products of mmyR (mmyrep, SEQ ID NO: 12) and mmfR (mmyrep2, SEQ ID NO: 11) are aligned with GBL-binding proteins associated with the production of virginiamycin (bara, SEQ ID NO: 9; barb, SEQ ID NO: 3), streptomycin (arpa, (SEQ ID NO: 10) and showdomycin and mimimycin (fara, (SEQ ID NO: 7). Other probable GBL-binding proteins from S. coelicolor A3(2) (cpra, (SEQ ID NO: 5; cprb, SEQ ID NO: 6; scbr, SEQ ID NO: 8) and the jadomycin biosynthesis gene cluster (jadr2, SEQ ID NO: 4) are also shown.
FIG. 3 shows that the product of mmfL (abbreviated here as mmy, SEQ ID NO: 17) is homologous with Streptomyces proteins implicated in the biosynthesis of GBLs. The latter proteins are for biosynthesis of A-factor (afsa, SEQ ID NO: 13), a GBL of S. coelicolor (scba, SEQ ID NO: 14), virginiae butanolide (barx, SEQ ID NO: 15) and IM-2 (farx, SEQ ID NO: 16).
In FIGS. 2 and 3, dots (“.”) denote the absence of an amino acid at that position, or the insertion of a gap for optimal sequence alignment and asterisks. (“*”) denote the end of an amino acid sequence.
FIG. 4a shows the expression of a foreign gene (xylE) from an expression cassette which comprises the regulatory portion and the foreign gene.

    
    
        Lower panel: organisation of the methylenomycin gene cluster in a strain engineered to express xylE from the transcription unit containing mmyG. The vector KC861 (Bruton et al, 1991) was engineered to contain the PstI fragment C2.18 (Chater and Bruton, 1983) which is now known to extend from within mmyT to within mmyG: the insert permitted integration of the phage in the configuration indicated. mmyG' and mmy'T denote 3′ and 5′ truncated copies of those genes, respectively.
        Upper panel: time course for catechol oxygenase activity in a strain (based on J1507: Bruton and Chater, 1983) carrying the fusion illustrated. Samples grown on cellophane membranes overlaid on R2YE were harvested after the indicated culture times, and extracts were made and assayed as in Guthrie and Chater (1990).
    
    


FIG. 4b shows the expression of a foreign gene (xylE) by fusing the foreign gene to different transcription units in the methylenomycin biosynthesis cluster. The fusions were made exactly as in FIG. 4a (lower panel), but with reg or A3.13 fragments (FIG. 9) replacing the C2.18 fragment.

    
    
        Upper panel: fusion to the mmfLHP transcription unit via the reg fragment;
        Lower panel: fusion to the mmy . . . XCAPK transcription unit via fragment A3.13.
    
    


FIG. 5 summarises the results of a Southern blot, demonstrating the extensive deletion of methylenomycin biosynthetic DNA from the R333 mutant. The probe, pIJ518 (Chater and Bruton, 1985) contains a large segment from the centre of the methylenomycin cluster. In a R333 digest (not shown), most of the PstI fragments are missing. Also shown is the organisation of genes within the methylenomycin biosynthetic gene cluster and to the right of the newly sequenced region. Also shown is an EcoRI segment of SCP1 DNA which, when sub-cloned and introduced into an SCP1− S. coelicolor host, stimulated methylenomycin production by an adjacent culture of the indicator “convertor” strain R39 described by Kirby and Hopwood (1977).
FIG. 6, parts a-d show examples of situations in which the regulatory portion and/or expression cassette could be used to enhance production of useful products. See text for explanations. Preferably in parts b, c and d, either the host strain used contains the mmyB gene, or the mmyB gene is present in the vector containing the expression cassette.
FIG. 7 shows the entire double-stranded sequence of an approximately 9.5 kb stretch of nucleic acid from the SCP1 plasmid, containing the mmyR, mmfP, mmfH, mmfL, mmfR, mmyT, mmyO, mmyG and mmyJ genes and the start of the mmr (methylenomycin resistance) gene. Some restriction sites are shown. The top strand is SEQ ID NO: 18 and the bottom strand is SEQ ID NO: 19. Deduced start and end residues of the genes are as follows:


















Starts at
Ends at residue



Gene
Strand
residue number
number









MmyR
bottom
1389 or 1407
 796



MmfP
bottom
2352 or 2355
1558



MmfH
bottom
3554
2352



MmfL
bottom
4612
3551



MmfR
top
4807
5451



MmyT
top
5676
6401



MmyO
top
6432
7553



MmyG
top
7636
8817



MmyJ
bottom
9115 or 9151
8780



Mmr
top
9333
not shown










    
    
        In the figure:
        ← denotes the start of a gene;
        [ denotes the end of a gene; and
        * denotes one of two possible start sites for a gene.
    
    


FIG. 8, parts a-h show the deduced amino acid sequences of the mmyR (SEQ ID NO: 12), mmfP (SEQ ID NO: 20), mmfH (SEQ ID NO: 21), mmfL (SEQ ID NO: 17), mmfR (SEQ ID NO: 11), mmyT (SEQ ID NO: 221), mmyO (SEQ ID NO: 23), and mmyG (SEQ ID NO: 24) genes, respectively.
FIG. 9 shows restriction sites flanking DNA fragments used to guide insertion of foreign DNA (such as xylE; FIG. 4) into the mmfHLP, mmyTOG, and mmy . . . XCAPK gene clusters (information from Chater and Bruton, 1993 and 1995, and FIG. 7). Only relevant sites are shown.
FIG. 10 shows the restriction sites used in a restriction analysis of KC861::C2.18 phages to determine orientation of the C2.18 insert. P=PstI, B=BamHI, SI=SstI, Bg=BglII, r=right hand end, 1=left hand end. Only relevant sites are shown.
FIG. 11 shows the construction of φG-UP vectors.
FIG. 12 shows the restriction sites and oligonucleotides (top: SEQ ID NO: 27; bottom: SEQ ID NO: 28) used to provide the salIR gene as a BglII fragment suitable for expression in a φG-UP vector.
FIG. 13 shows in part a the construction of pG-UP. Part b shows the preferred form, pG-UP*, which contains mmvB. The HindIII site of the illustrated version of pG-UP is used to introduce mmyB in a form that leaves a unique HindIII site between the expression cassette and mmyB.
FIG. 14 shows the use of pG-UP for the expression of the J21 gene set. Numbers on primers (forward: SEQ ID NO: 25; SEQ ID NO: 26) refer to base positions in FIG. 7.
FIG. 15 shows in part a a fragment of cosmid cos73, containing the genes from mmyR to mmyG; part b shows this region engineered to terminate with the mmyTOG promoter, for use to control expression of a nucleic acid of interest; and part c shows the construction strategy



EXAMPLE 1
High Levels of Expression of a Foreign Gene Under the Control of Promoters in the Methylenomycin Biosynthetic Gene Cluster
In order to determine expression levels at different points in the methylenomycin biosynthetic gene cluster (here termed the mmy cluster), derivatives of bacteriophage C31 containing the foreign gene xylE (a gene originating from Pseudomonas: Zukowski et al., 1983) were used to place xylE in defined positions and orientations within the mmy cluster contained in S. coelicolor strains J1507 (which contains the mmy cluster within SCP1NF, a chromosomally integrated copy of SCP1; Bruton and Chater, 1983), or J1506 (a derivative of J1501 (Chater et al. 1982) which has an autonomous copy of SCP1).
Because KC861, the vector used, lacks the attP site normally used by C31 to permit its integration into the host chromosome during the establishment of lysogeny, it cannot form a prophage by the normal route (Bruton et al., 1991). It is, however possible to provide an alternative integration route by inserting a piece of Streptomyces host DNA into KC861, so that homologous recombination can integrate the prophage at the corresponding position in the host's DNA. Such events, which are quite rare, can be detected if the prophage carries a selectable resistance gene such as vph (viomycin resistance) or tsr (thiostrepton resistance) (Chater & Bruton, 1983; Bruton et al., 1991). In the present case, the mmy inserts placed in KC861 permitted the vector to integrate into particular positions in the mmy DNA of SCP1NF in J1507 or of SCP1 in J1506, with orientations that depended on the orientation of the insert in the vector. pBR327 and pBR322 recombinants (Chater & Bruton, 1983, 1985) were the DNA source for cloning the fragments reg, C2.18, mmr, A4.2 and A3.13 (FIG. 9) into the BamHI site of KC861. The reg fragment was a SstI-BglII subfragment of a larger insertion in pIJ519 (Chater and Bruton, 1985). The other four fragments had PstI boundaries. In order to provide them with BamHI compatible ends, they were introduced into the E. coli plasmid pIJ2925 (Janssen and Bibb, 1993). The mmy inserts were separated from the pBR327/322 vectors by digestion with PstI (or SstI and BglII for reg) and gel electrophoresis and were then ligated to suitably cut pIJ2925. JM101 was transformed with the ligations. The plasmid-host combination allowed blue/white screening for recombinants. For each fragment plasmid DNA of several white colonies was examined by BglIl digestion to show whether it contained the insertion. A second enzyme was used to determine its orientation in relation to the polylinker of pIJ2925 (Table 1). This helped later to determine the orientation relative to the xylE gene in the phage vector.







TABLE 1







The orientation of mmy fragments inserted into


pIJ2925














Enzyme used





Derivative of
to determine



Fragment
pIJ2925
orientation
Orientation*







reg
pIJ560
XhoI
r



C2.18
pIJ561
EcoRI
r



mmr
pIJ562
PvuII
l



A4.2
pIJ563
SstI
l



A3.13
pIJ564
EcoRI
r







*“r” indicates that the right end of the insert DNA is located at the right end of the pIJ2925 polylinker; “l” indicates that the left end of the insert DNA is located at the right end of the pIJ2925 polylinker.






The plasmids pIJ560-pIJ564 were cut with BglII and the purified digested DNA was ligated to KC861 DNA cut with BamHI. Protoplasts of S. lividans 1326 were transfected with the ligated DNA. 100-200 well separated plaques were picked to masterplates of 50 plaques each.
Phages with the desired insertion of mmy-DNA fragments were identified by a hybridisation signal on plaque lifts. The phage DNA was transferred from the plaques on the masterplate onto nitrocellulose (Benton & Davis, 1977). From the plasmids pIJ560-pIJ564 the inserted DNA fragments were isolated and labelled non-radioactively by the digoxigenin system of Boehringer Mannheim to prepare probe DNA for the hybridisation. Four positive plaques on the masterplate for each of the five insertions were purified to get phage suspensions from single plaques. Phage DNA was prepared and analysed by digestion with restriction enzymes to verify that it contained the expected insertion and to determine its orientation. As an example the situation is demonstrated for the insertion of C2.18 in FIG. 10. In this example restriction digests (not shown) of each sample with respectively PstI and SstI were used to determine the orientation of the insert in the sample. Table 2 shows the results of the DNA analysis and gives the names of the constructed phages.







TABLE 2







Structure of KC861 recombinants with mmy-DNA


fused to xylE.















Number of





Orientation
representatives in



Inserted
Phage
of inserted
four analysed



fragment
designation
fragment
phages







reg
KC133
r
3 (A, B, C)



(2.2 kb)
KC134
l
1



C2.18
KC135
r
2 (A, B)



(2.05 kb)
KC136
l
2 (A, B)



mmr
KC137
r
3 (A, B, C)



(2.5 kb)
KC138
l
1



A4.2
KC139
r
1



(2.75 kb)
KC140
l
3 (A, B, C)



A3.13
KC141
r
3 (A, B, C)



(2.28 kb)
KC142
l
1







A, B and C name the different isolated phages;



r indicates that the right end of the insert DNA is to the right of KC861; and



l indicates that the left end of the insert DNA is to the right of KC861.






The orientation was determined using SstI, for which there is a unique site in the polylinker regions of KC861 and pIJ2925. Information about the orientation of the cloned fragment in pIJ2925 was necessary (see Table 1).
Phage suspensions from selected single plaques were used to lysogenise the strains J1506 (SCPI+) and J1507 (SCPINF). The integration of the phage conferred thiostrepton resistance on the lysogens and placed the xylE fusions into the mmy gene cluster of the host strain (FIG. 4a; FIG. 6a). To prepare suitable lysogens, 10-20 l of a phage suspension from a single plaque of each of KC133-KC142 was spotted on an R5 plate spread with 107-108 spores of J1507 or J1506. After 5-7 days the cultures had sporulated and were replicated to minimal medium containing 50 gml−1 thiostrepton. After c. four days resistant colonies were streaked on R5 plates containing 5 gml−1 thiostrepton to get single colonies, which were then spread on the same medium to obtain spores of purified lysogens.
To check that the prophages had integrated at the expected locations, Southern blotting was done. Genomic DNA was prepared from 25 ml YEME cultures of the lysogens containing 4 gml−1 thiostrepton. In each case an XhoI digestion of the DNA was used to investigate disruption in the mmy gene cluster. There is a unique XhoI site in KC861 upstream of the tsr gene. As the XhoI sites flanking or within the cloned fragments were located asymmetrically to their ends, it was possible deduce the orientation of the integrated phage and to confirm the results of the phage analysis. The same non-radioactive DNA probes as for the plaque lifts were used. Table 3 lists the results of the Southern analysis.







TABLE 3







Southern analysis of lysogens of J1507 and J1506.














Distinctive



Cloned
Distinctive
Integrated
band for
Obtained with












fragment
band for wt
phage
disruption
J1507
J1506





reg
3.2 kb, 5.2
KC133
2.35 kb
yes
—



kb
KC134
 3.7 kb
yes
—


C2.18
5.25 kb
KC135
4.55 kb
yes
yes




KC136
 6.7 kb
yes
yes


mmr
 6.1 kb
KC137
7.75 kb
—
yes




KC138
4.75 kb
yes
yes


A4.2
 6.1 kb
KC139
5.25 kb
—
yes




KC140
7.55 kb
yes
yes


A3.13
 2.7 kb
KC141
 4.4 kb
yes
yes




KC142
 4.5 kb
—
yes









Lysogens with the expected hybridisation pattern were obtained with every type of phage, though some of the strains tested did not show the expected pattern. Thus, J1507::KC137(A),137(B),139,142(A),142(B) were thiostrepton resistant, but the integration was not in the right place. As lysogens with the correct construction were obtained for these phages with J1506, no more J1507 lysogens were analysed.
The BamHI restriction site of KC861 into which the mmy DNA was inserted is part of a multiple cloning site (MCS) located close to, and just upstream of, the promoterless xylE gene. As illustrated by Guthrie and Chater (1990) and Bruton et al. (1991), this has the effect, upon integration of the phage by homologous recombination, of placing xylE under the control of the transcription unit from which the particular insert originated, provided that the transcription unit and xylE have the same orientation. Therefore, the level of expression of the relevant transcription unit is indicated by the level of xylE expression. Expression of xylE is readily monitored because the xylE gene product is an enzyme (catechol 2,3 dioxygenase) that converts colourless catechol into a yellow compound, 2-hydroxymuconic semialdehyde. This can be detected by eye as a yellow zone round colonies after spraying with catechol (the Ylo+ phenotype), or quantitatively by spectrophotometry after cell-free extracts have been prepared (Zukowski et al., 1983; Ingram et al., 1989).
The in vivo assay for xylE activity of the lysogens (i.e. Ylo+ phenotype) was carried out by spraying catechol onto colonies (Ingram et al., 1989; Bruton et al., 1991). Complete medium (CM; Hopwood et al., 1985), which gave strongest expression, was used throughout the experiments and HMM (Hobbs et al., 1992; solidified with 1% agar), in which the Ylo phenotype was more easily scored but which gave lower expression, was used only occasionally for comparison. Table 4 shows the combined results of repeated xylE tests for all fusion points. Cultures had been sprayed with catechol solution at ages of 42 h and 72 h.







TABLE 4







Plate assays for xylE activity in J1507 and


J1506 lysogens (see following page).















Conclusion about



Cloned

Ylo
transcription



fragment
Lysogen
phenotype
(see FIG. 9)







reg
J1507::KC133
−−
not rightward across






BglII at pos. 13.0




J1507::KC134
+++
leftward across SstI






at pos. 10.8



C2.18
J1507::KC135
+++
rightward across






PstI at pos. 16.0




J1506::KC134
+++
rightward across






PstI at pos. 16.0




J1507::KC136
−−
not leftward across






PstI at pos. 13.9




J1506::KC136
−−
not leftward across






PstI at pos. 13.9



mmr
J1506::KC137
−−
not rightward across






PstI at pos. 19.3




J1507::KC138
+
leftward across PstI






at pos. 16.8




J1506::KC138
+
leftward across PstI






at pos. 16.8



A4.2
J1506::KC139
−−
not rightward across






PstI at pos. 22.1




J1507::KC140
++
leftward across PstI






at pos. 19.3




J1506::KC140
−−
leftward across PstI






at pos. 19.3



A3.13
J1507::KC141
−−
not rightward across






PstI at pos. 25.0




J1506::KC141
−−
not rightward across






PstI at pos. 25.0




J1506::KC142
+++
leftward across PstI






at pos. 22.7










Of particular relevance was the finding that the insert in KC135 gave rise to a strong Ylo+++ phenotype in strain J1507::KC135. This was examined in closer detail by inoculating ca. 108 J1507::KC135 spores onto each of a series of plates containing CM (Hopwood et al., 1985) supplemented with histidine (50 g ml−1) and uracil (7.5 g ml−1) overlaid with a cellophane disc as described by Tan and Chater (1993), then incubating at 30° C. for the times indicated in FIG. 4a, upper panel, before scraping off the mycelial growth. For catechol 2,3 dioxygenase assays, the mycelium from one cellophane disk was suspended in 0.5 ml extraction buffer, and cell-free extracts were prepared by sonication and clarified by centrifugation as in Ingram et al. (1989). Catechol 2.3 dioxygenase specific activities were determined by spectrophotometry, as above. The results are shown in FIG. 4a, upper panel. The specific activities in samples harvested before 30 h were not significantly above the lowest reliably measurable levels, but between 30 and 40 h, as the growth on the cellophane became dense and morphological differentiation began, there was a very rapid increase in activity up to 30 mU mg−1 protein.
In an earlier experiment in which xylE was fused to the redX gene, which is involved in the synthesis of another antibiotic in S. coelicolor, Guthrie and Chater (1990) reported peak values of 2 mU mg−1 protein. The mmy-driven transcription of xylE in J1507::KC135 was therefore very strong.
Further experiments (FIG. 4b) showed a similar pattern and level of xylE expression when suitable fusions were made at other points in the mmy cluster. The positions of these fusions are shown in FIG. 9. These results show that a foreign gene (in this example, xylE) can be expressed to quite high levels specifically late in growth when inserted at the illustrated locations in the mmy cluster, without the addition of any inducing agent.
EXAMPLE 2
A Diffusible Substance, Capable of Eliciting Methylenomycin Production from Certain Methylenomycin Non-producing Mutants, is Produced by a Mutant Containing the Leftmost 6.5 kb of the Methylenomycin Biosynthetic Gene Cluster, but Little if Any Other mmy DNA
(a) In previous work (Kirby et al., 1975; Kirby and Hopwood, 1977) it was shown that mutants unable to make methylenomycin could be isolated after different procedures. The ability of these mutants to produce and/or respond to extracellular substances relevant to methylenomycin production was tested by growing strains close together on the surface of CM agar (Kirby and Hopwood, 1977). R333 was one of ten mutants that produced an extracellular substance that elicited methylenomycin production by another methylenomycin non-producing mutant, R39. In the work of Kirby and Hopwood (1977), it was considered likely that the substance produced by R333 was converted into methylenomycin.
In the present example, DNA was isolated from R333 and digested with the restriction enzymes PstI and PvuII. The digested DNA was subjected to agarose gel electropheresis and blotted onto a nitrocellulose membrane (Southern, 1975). The membrane was then hybridised with a 32P-labelled probe derived by nick translation of pIJ518, a plasmid containing much of the mmy cluster (Chater and Bruton, 1985; FIG. 5). R333 contained a deletion extending rightwards from a position about 6.5 kb inside the left end of the mmy cluster, and ending beyond the righthand end of the KC518 insert. Thus, R333 contains about 6.5 kb of DNA from the left end of the mmy region, and little (perhaps no) other methylenomycin-related DNA (FIG. 5).
Having discovered the magnitude of the deletion of biosynthetic genes in R333, the present inventors suggest that this 6.5 kb region confers biosynthesis of an extracellular signalling molecule and that this, not an intermediate of methylenomycin biosynthesis, is the substance which is secreted by R333 and which is capable of stimulating R39 to produce methylenomycin.
This is consistent with the observation that only one of 16 mmy mutants studied by Kirby and Hopwood (1977) could be induced to produce methylenomycin when grown near other mmy mutants.
The present inventors further suggest that biosynthetic intermediates accumulating in blocked mutants are generally not freely released and exchangeable between strains, but observe that an immediate precursor (desepoxymethylenomycin) is produced by the wild-type and is convertible by SCP1+ strains into methylenomycin (Hornemann and Hopwood, 1978). They further suggest that nearly the whole of the biosynthetic pathway has to be completed before a precursor capable of functioning in cosynthesis is produced—a requirement that would probably necessitate much more than ca. 6.5 kb of biosynthetic genes.
These suggestions, and other results indicating a regulatory role for some of this DNA (see above), are consistent with the deduced function of genes discovered by sequencing this region of the mmy cluster. The results of this sequence analysis are given in Example 3.
(b) In further confirmation of these predictions, an 8.3 kb EcoRI fragment of SCP1 containing the genes from mmyR to mmyT, with part of mmyO (FIG. 5), was sub-cloned from cosmid 73 of Redinbach et al (1998) into pSET152 (Bierman et al. 1992) and the resulting plasmid was introduced into the φC31 attB site of the SCP1− Streptomyces coelicclor strain J1501 (Kieser et al 2000). The resulting strain, when used in “co-synthesis” tests with R39 (Kirby and Hopwood 1977), elicited methylenomycin production in the R39 strain (as judged by inhibition of the SCP− indicator strain J1501).
EXAMPLE 3
DNA Sequence of the Left End of the Methylenomycin Biosynthetic Gene Cluster
The DNA sequence of the region from the left end of the mmy region to the previously sequenced (Neal and Chater, 1987) 2.55 kb PstI fragment containing mmr (see FIG. 5) was determined in three sections. The leftmost XhoI fragment (ca. 3.2 kb) was sequenced by dideoxy-sequencing using the method of Sanger et al. (1977), adapted as described in Bruton and Chater (1987). Random sonicated fragments were cloned into M13mp19 to provide the templates for sequencing, using M13 forward primer (Norrander et al., 1983). The overlapping SstI/PstI (ca. 5 kb) and PvuII (ca. 2.2 kb) fragments were sequenced by automated fluorescence sequencing on an ABI automated sequencer, using templates cloned into pbluescript vectors. For one orientation, templates were generated by the exonuclease III procedure of Henikoff (1984), and in the other, oligonucleotides were designed for “primer walking”. All sequences were determined on both strands, and each base position was read through in at least two sequencing reactions. The sequence is given in FIG. 7.
Use of the FRAME programme (Bibb et al., 1984) led to the recognition that there are nine methylenomycin-related genes to the left of the resistance gene mmr (FIGS. 1 and 5). Only one of these, mmyJ, had previously been sequenced (Neal and Chater, 1987).
Predicted Functions of Genes Identified by Sequencing
Using the BLAST (Altschul et al., 1990; 1996) and TFASTA (Pearson and Lipmann, 1988) programs to search the major protein and DNA databases, similarities of the deduced products of the eight newly sequenced genes to proteins of known function were discovered. Three of the eight—mmfL, mmfR and mmyR—were of particular interest, because they gave crucial clues about the nature of the regulation of the mmy genes and the putative extracellular signalling molecule, relevant to the design and use of the expression system that is the subject of this patent application. In this section we give details of these similarities. The products of the other five genes all showed some degree of resemblance to various enzymes, and are likely to be directly involved in enzymatic reactions leading to the biosynthesis or metabolism of methylenomycin or the GBL factor involved in regulating methylenomycin biosynthesis.
mmfL
The predicted product of mmfL (MmfL) showed significant similarity to only four proteins: AfsA, ScbA, BarX and FarX. These proteins are all from other Streptomyces spp., and they are all intimately associated with the production of GBL signalling molecules involved in the regulation of antibiotic production (they are generally believed to be the enzymes responsible for GBL synthesis: Horinouchi et al., 1985, 1989; E. Takano, personal communication). The similarity of MmfL to these proteins is essentially end-to-end, and is very highly significant (FIG. 3). The discovery of such a gene in a small region of DNA that encodes production of an extracellular substance that stimulates methylenomycin production (See Example 2) makes it highly likely that the signal is a GBL, and that MmfL protein encodes a critical step in its biosynthesis.
mmfR and mmyR
The MmfR and MmyR proteins show significant alignment with a large family of bacterial regulatory proteins (the TetR “superfamily”), principally in a substantial region near the N-terminus of the proteins. This region has been shown in a few of these proteins (Hillen and Berens, 1994) to assume an α-helix-turn-α-helix organisation that permits it to bind to specific sequences in double-stranded DNA.
The most similar proteins to MmfR and MmyR form a particular sub-group (or “family”). All are from Streptomyces spp., and where sufficiently studied, all are associated with the regulation of antibiotic production and/or morphological differentiation (Onaka and Horinouchi, 1997; Onaka et al., 1997, 1998; Sugiyama et al., 1998; Nakano et al., 1998). The alignments are shown in FIG. 2.
The greatest similarity is in the region of the DNA-binding domain expected to make sequence-specific contacts with target DNA sequences, leading to the expectation that MmfR and MmyR bind sequences similar to those recognised by the other members of the family (see below). Although the central and C-terminal regions of these proteins are less well conserved, nevertheless there is evidence of similarity throughout these regions, whereas other members of the TetR superfamily do not show similarity to MmfR beyond the N-terminal region. The aligned proteins have a very significant further feature in common—the ones studied to-date all act as specific receptors for different GBL signalling molecules.
The present inventors have newly discovered that mmfR and the putative GBL biosynthesis gene mmfL are arranged in a similar way, and close inspection of the sequence between them reveals a palindromic sequence (half-site 5′-GGAAGGTATTA-3′; SEQ ID NO: 2) that resembles the consensus sequence (half-site 5′-GG(T/C)CGGT(A/T) (T/C)G(T/G)-3′; SEQ ID NO: 1) defined for binding of DNA by the ArpA protein that is the archetype of these factor-binding proteins (Onaka and Horinouchi, 1997).
The present inventors therefore suggest that the chemically undefined extracellular factor secreted by R333 (see Example 2) is a GBL whose extracellular concentration builds up slowly as hyphal density in the cell culture increases, to some critical threshold at which it is effectively perceived by the binding protein encoded by mmfR. This interaction releases MmfR from its location in the bidirectional promoter region, leading to derepression of mmfL and hence of factor biosynthesis. This in turn, it is -proposed, causes an acceleration in the rate of factor production, sufficient to interact with, and inactivate, repressor(s) bound either to the mmyTOG and mmy . . . XCAPK promoters or to the promoter of another regulatory gene (e.g. mmyB) needed to activate the mmyTOG and mmy . . . XCAPK promoters, permitting the mmyTOG and mmy . . . XCAPK genes to be expressed. A convenient hypothesis would be that the mmyR gene product (MmyR, see below) is this repressor and that MmyR requires a higher GBL concentration for its repressor function to be inactivated than does MmfR.
EXAPLE 4
Prevention of Translation of mmfL mRNA is Associated with Failure to Transcribe mmy DNA
(a) In the DNA sequencing in Example 3 mmfL is the only gene to contain a TTA (leucine) codon. Such codons are unexpressed in severe bldA mutants, because bldA encodes the only tRNA capable of translating UUA codons (Leskiw et al., 1991). A prediction of the model for regulation of methylenomycin production is that mmfL mRNA would be untranslatable in a bldA mutant, leading to inability to make the GBL factor and hence to inability to transcribe the genes for methylenomycin production. To test this, xylE fusions into various parts of the mmy cluster of a bldA mutant were constructed using the C31KC861 derivatives described in Example 1. The ability of the resulting strains to express xylE was then tested by the qualitative plate method. Catechol oxygenase activity is not detected, confirming the prediction, and indicating that a TTA-containing gene is involved in regulating mmy gene expression.
(b) In verification of this, the TTA codon of mmfL was changed to CTC in the 0.9 kb SunI internal fragment of mmfL (which was sub-cloned into pFA6a (Wach et al. 1994, replacing the KanMX module), using the “Quick Change” system of Stratagene, followed by reinsertion of the SunI fragment into the 1.7 kb XhoI-BglII fragment containing mmfL (in pIJ2925, Kieser et al. 2000) to give mmfLCTC. This fragment was sub-cloned into pSET151 (Bierman et al. 1992) and introduced into J1703 (bldA−, SCP1NF:Lawlor 1997). Most of the resulting strains acquired the ability to stimulate R39 to produce methylenomycin in co-synthesis, proving that non-translation of the TTA codon of mmfL is responsible for factor non-production in the bldA mutant. However, none of the strains produced methylenomycin, indicating that an additional bldA-dependent step intervenes between factor production and methylenomycin production. This was further verified, since J1703 was not stimulated to produce methylenomyin by growth adjacent to the GBL factor-producing strain R333. The likely intervening step is a gene (mmyB) located about 9 kb to the right of the sequence given in FIG. 7. This gene is predicted to encode a DNA-binding protein and contains a TTA codon.
EXAMPLE 5
Construction of a Vector (φG-UP) Permitting the Easy Insertion of Small Transcription Units Into the Expression Region of the Methylenomycin Gene Cluster
In order to facilitate expression of foreign genes, the vector φG-UP is first constructed. Using the procedures in Hopwood et al. (1985) and Kieser et al. (2000), DNA is extracted from large-scale preparations of φC31 KC889 (FIG. 11). 5 μg of this DNA is digested by EcoRI and—separately—5 μg is digested by XboI plus SstI. Completeness of digestion is checked by agarose gel electrophoresis (1% agarose) of 0.5 kg of each digested DNA, immediately after heating it to 70° C. for 10 min and cooling it on ice (to separate cohesive ends of the phage DNA).
After phenol extraction, the two digests are mixed and co-precipitated with ethanol, washed once with 70% ethanol, then dissolved in 100 μl Klenow buffer. The solution is heated at 70° C. for 10 min in a waterbath, which is then turned off and allowed to cool down overnight (this permits φC31 cos ends to join together). The dissolved DNA is then subjected to filling in of the 5′ ends generated by XhoI and EcoRI, using Klenow enzyme, before phenol extraction, ethanol precipitation, washing with 70° ethanol, and redissolving in 200 μl ligation buffer, prior to ligation overnight using conditions suitable for blunt end ligation (low ATP, high T4 DNA ligase). After ligation, the DNA is precipitated with ethanol, washed in 70% ethanol, and dissolved in 100 μl TE buffer, prior to being used for transfection of Streptomyces lividans 1326 essentially as described by Hopwood et al. (1985) and Kieser et al. (2000).
Most plaques are expected to have phages with the desired DNA structure (deletion of the XhoI-EcoRI fragment that contains vph), so screening is done by restriction analysis of DNA isolated from the progeny of 12 of the transfectant plaques. The use of PstI, BglII and/or BamHI provides diagnostic digestion patterns.
A phage with the correct organisation is identified. A large-scale DNA preparation from this phage is digested with PstI, ethanol precipitated, washed with 70% ethanol and redissolved in TE buffer, before being ligated (at 100-200 μg ml−1 DNA) with an equimolar amount of the 2.05 kb PstI fragment C2.18 (Bruton and Chater, 1983), obtained from pIJ518 by PstI digestion followed by separation by agarose gel electrophoresis and isolation from the gel. After ligation, the DNA is ethanol precipitated, washed and redissolved in 20 μl TE buffer. This solution is used to transfect S. lividans, and the resulting plaques are arrayed on master plates prior to analysis by filter hybridisation (Benton and Davis, 1978) to identify candidates with the desired insertion. The probe for this analysis is the C2.18 PstI fragment, labelled non-radioactively with the digoxigenin system. Twelve candidate plaques are used to propagate phage for small-scale DNA preparation. The DNA is analysed by restriction analysis, using two enzyme combinations: BamHI plus PvuII, and BglII plus PvuII. A phage with each orientation of the insert is retained. Of these, the phage in which the BglII plus PvuII digest gives a 1.2 kb fragment and the BamHI plus PvuII digest gives a 0.8 kb fragment, is termed φG-UP (R) and that in which the BamHI plus PvuII digest gives a 1.2 kb fragment and the BglII plus PvuII digest gives a 0.8 kb fragment is termed φG-UP (L) (FIG. 11).
EXAMPLE 6
Production of the SalI Restriction Enzyme by Placing the salIR Gene Under the Control of the Expression Cassette in a φG-UP Vector
SalI is a restriction enzyme with substantial use for molecular biology, and therefore with substantial sales. The genes for SalI and the SalI methylase were cloned by Rodicio and Chater (1988) from the producing organism, Streptomyces albus G, and sequenced by Rodicio et al. (1994). They are arranged in tandem, and are expressed as a bicistronic operon (salIR preceding salIM) (Rodicio et al., 1994; Alvarez et al., 1993). In addition, salIM is expressed from its own promoter (Alvarez et al., 1993). Expression of these genes is usually at a very low level, a very high specific activity of SalI being generated by a small amount of protein (of the order of 106 units μg−1 protein). Here we show how the expression cassette in φG-UP can be used to overproduce SalI.
The sa1IRM genes are present in pIJ4430 (Rodicio and Chater, 1988). To introduce the gene pair into φG-UP, pIJ4430 is first cleaved with BclI and then with RcaI to generate a fragment with protruding 5′ GATC and 5′ CATG single-stranded ends. This fragment is inserted into the intermediate vector pIJ2925 (Janssen and Bibb, 1993), using the oligonucleotide adaptors shown in FIG. 12. The fragment is then excised from the intermediate vector by digestion with BglII, and ligated with φG-UP (L) cleaved with BglII. The ligation mixture is used to transfect S. lividans, and the resulting plaques are screened by plaque hybridisation, using the digoxigenin-labelled salIRM BclI-RcaI fragment as a probe. Phages from twelve hybridizing plaques are used for small-scale DNA preparations, and the resulting DNA samples are analysed by digestion with BglII to demonstrate the presence of the full-length 2.9 kb insert, and PstI to determine the orientation of the insert. An additional 2.9 kb PstI fragment would indicate the incorrect orientation, and the absence of such a fragment would indicate the correct orientation.
Once identified, the desired phage is used to prepare a high titre stock, spots of which are placed on an R2YE plate spread with J1507 spores. After 4-6 days at 30° C., when sporulation has taken place, the plate is replicated to MM (supplemented as necessary for the growth of the auxotrophic J1507) containing thiostrepton (50 μg ml−1), and colonies present after 4 days are purified by single colony isolation then used to prepare confluent plates for the harvesting of dense spore suspensions.
In order to obtain the desired SalI enzyme, initially on a demonstration scale, the spores are used to inoculate 50 ml CM in a baffled 500 ml flask and the culture is incubated with shaking at 30° C. until stationary phase. At this time, the expression cassette is auto-activated, and the SalI RM genes expressed. The cloned salIM gene will achieve two purposes: the use of its own promoter during early growth will have permitted modification of the host DNA, so rendering it immune to cleavage when SalI is eventually produced; and the expression of SalIM during the main expression period will ensure the optimal production of salIR. Further extraction and purification of SalI follows standard procedures for restriction enzymes.
EXAMPLE 7
(a) Construction of pG-UP, an integrative plasmid vector for the activation of cryptic gene clusters
As shown in FIG. 6(b), it is possible to cause the expression cassette to integrate into desired positions in a Streptomyces genome, and thereby to elicit stationary phase expression of adjacent genes. In Example 8, this is put to use in the expression of cryptic genes potentially encoding a new secondary metabolite. Here we describe the construction of pG-UP, an E. coli plasmid containing the expression cassette and capable of transfer into Streptomyces. Insertion of appropriate Streptomyces DNA into pG-UP will permit the use of the construct for gene expression of this kind. The vector is based on pSET151 (Bierman et al., 1992), though any E. coli replicon with a marker permitting selection in Streptomyces and lacking Streptomyces replication or chromosomal integration machinery could be used.
To construct pG-UP, the 6.2 kb BamHI and BstZ17I fragment of pIJ519 (Chater and Bruton, 1985; and FIG. 7) is isolated from an agarose gel and its ends are blunted by filling in with Klenow enzyme. It is ligated with pSET151 (precleaved with EcoRI and blunted by filling in with Klenow enzyme). After transformation of E. coli strain JM101 transformants are analysed by colony hybridisation using the BamHI and BstZ17I 6.2 kb fragment of pIJ519 as probe to detect the desired insert, and plasmid DNA is extracted from 12 positive colonies. The DNA is digested with BamHI plus BglII to determine the orientation of the insert. An example with a 2.3 kb fragment (rather than a 4 kb fragment) is chosen as pG-UP (FIG. 13a).
In the form shown in FIG. 13a, the effective use of pG-UP would be guaranteed I na host containing SCP1, to supply the additional genetic component (mmyB) revealed by experiments outlined in Example 4. The use of pG-UP in SCP1− strains will benefit from the further incorporation of mmyB into pG-UP or into the host genome (see Example 7b).
(b) Incorporation of mmyB Into pG-UP to Give pG-UP*
In order to make the effective use of pG-UP independently of a separately provided mmyB gene, mmyB is obtained from the SCP1 plasmid by PCR amplification using the following primers, with cosmid 73 (Redinbach et al 1998) as template:










5′ TATAAGCTTGGTGAACTCCTTCGGCGAGTGGTTCGGA 3′




(SEQ ID NO: 29)



 



5′ TATGGTACCGGGGAGAACTCCTTGGGATACTTCCTG 3′



(SEQ ID NO: 30)






After amplification and conventional preparation for digestion, the PCR product is digested with KpnI, then purified before ligation with the unphosphorylated oligonucleotide 5′ AGCTGTAC 3′. After gel purification, the linear DNA is cleaved with HindIII, repurified, and ligated with HindIII-cleaved pG-UP. After transformation of E. coli strain DH5 alpha, transformants are screened by colony hybridisation with a mmyB-specific probe, and plasmid is isolated from suitable colonies for verification by restriction analysis for constructs corresponding to FIG. 13b. 
EXAMPLE 8
Use of the Expression Cassette in pG-UP in Forced Expression of Genes Apparently Encoding an Unknown Polyketide Molecule
The project to sequence the genome of Streptomyces coelicolor A3(2) (www.sanger.ac.uk/Projects/S—coelicolor) has revealed several genes and gene clusters that encode proteins related to some known to be involved in the production of valuable antibiotics. One example is found in cosmid J21, which contains (inter alia) a series of six or seven genes (here termed “the J21 gene set”) that appear to form a single transcription unit of perhaps 18 kb, among which two encode probably multidomain β-ketoacyl synthases of the type involved in the biosynthesis of erythromycin, rapamycin, tylosin, avermectin and other macrocyclic polyketides (Hopwood, 1997). No such compound is known to be made by S. coelicolor A3(2). In order to force expression of the J21 gene set, so that culture fluids can be screened for novel compounds, the J21 gene set is placed under the control of the new expression cassette. For this purpose, a PCR-amplified fragment of J21 DNA is cloned into the pG-UP vector as indicated in FIG. 14. In outline, the fragment is amplified from J21 DNA with the use of an oligonucleotide permitting the start codon of the first gene to be maximally accessible to ribosomes translating mmyG from the expression cassette, and a reverse primer oligonucleotide permitting amplification of a c. 1 kb fragment. Primers include features permitting ready subcloning into G-UP. After transformation of E. coli JM101 and arraying of colonies as patches on a masterplate, colony hybridisation is used to identify colonies containing the J21 insert (with, as probe, the amplified PCR fragment labelled non-radioactively). Plasmid DNA is prepared from candidate colonies by the standard alkaline lysis procedure, and checked by restriction analysis with the enzymes BamHI and HindIII. An example of such a plasmid, in which the orientation of the insert permits its “sense” transcription from the expression cassette, is introduced by transformation into the non-methylating E. coli strain ET12567 (MacNeil et al., 1992) containing the mobilising plasmid, UZ8002 (Flett et al., 1997); and transformants are used in conjugal mating with S. coelicolor M145, selecting thiostrepton-resistant, nalidixic acid-resistant exconjugants (Bierman et al., 1992). Most transformants are expected to contain pG-UP integrated, by homologous recombination, at the start of the J21 gene set. After culture of five representative transformants on R2YE (Hopwood et al., 1985) containing thiostrepton (5 μg/ml), spores are harvested and used to inoculate both liquid and surface CM cultures, which are then incubated for 3 days before extraction with ethyl acetate prior to conventional HPLC and mass spectrometry to determine the structures of any new compounds (e.g. McDaniel et al., 1993).
In an improved version of this strategy, mmyB is inserted into the polylinker HindIII site of pG-UP, preferably in a form regenerating a HindIII site only between the regulatory cassette and the mmyB gene (see Example 7b and FIG. 13b).
EXAMPLE 9
Fusing Genes to the Cassette in Vectors that can be Maintained in Streptomyces Hosts
Many antibiotic pathways are highly dependent for expression on pathway-specific transcriptional activators. Additional copies of these genes often stimulate substantial overproduction of the cognate antibiotic (e.g. Chater, 1990). The use of the expression cassette to express such genes would permit over-expression to be confined to dense cultures, thereby minimising antibiotic production during earlier stages of growth: premature production would diminish overall yield and might be lethal if the antibiotic were a novel compound made by a genetically engineered hybrid pathway for which no self-resistance mechanism had evolved. The ability to express such hybrid gene sets in a standard host-vector system would permit the ready screening of large combinational libraries. Examples might include recombinant libraries of type I polyketide synthase genes. In another use of this kind of vector, DNA isolated directly from the environment (e.g. soil) can be expressed from the expression cassette, permitting screening for novel compounds (FIG. 6d). To facilitate this approach, the cassette, prepared as above, can be combined with different vectors capable of stable maintenance in Streptomyces hosts. Examples of such vectors include: those that are maintained as autonomous plasmids, at low copy or medium copy number (usually based on SCP2), or at high copy number (often based on pIJ101); and those that integrate efficiently into the chromosome by site-specific recombination involving the att sites of prophages (such as C31: see FIG. 6d for an example), integrative plasmids (such as pSAM2) or site-specific transposons (such as IS117).
EXAMPLE 10
Providing a Clean Background.
The bldA gene, which can be inactivated without interfering with growth, encodes the tRNA for the rare codon UUA (TTA in the DNA). TTA codons are present in most antibiotic gene clusters, but not in genes for growth. For this reason, bldA mutants make no antibiotics. The expression cassette contains a TTA codon, but a TTA codon-free version has been engineered to permit expression of mmfL in a bldA mutant (see Example 4).
To allow the effects of this to be full manifested, the TTA codon of mmyB is similarly engineered to an alternative leucine codon, using the Stratagene “Quick ChangeTM” system, and the altered gene is introduced by standard procedures into the bldA host strain, along with the expression cassette coupled to the genes to be expressed. In one preferred case, the TTA-free mmyB gene is introduced into a pG-UP vector as indicated in FIG. 13b. However, it can be introduced separately from the expression cassette, e.g. as part of plasmid SCP1.
Because nearly all the TTA codons in antibiotic clusters are in regulatory genes, newly discovered sets of genes for biosynthetic enzymes are usually TTA-free. Therefore, expression of such genes from the TTA-free expression cassette will usually be effective in a bldA host. Any new metabolite will be made in the absence of other antibiotics, making it easier to study a range of biological and chemical aspects of the new metabolite without the need to separate it from other bioactive metabolites. Accordingly, the vectors described in examples 5, 7 and 9 would also be constructed with the TTA-free version of the cassette, to permit their use in bldA mutants hosts such as J1703, which contains an integrated copy of SCP1 [for the kind of vector described in example 5], or J1700, which does not contain any DNA from the methylenomycin cluster [for the kinds of vector described in examples 7 and 9].
EXAMPLE 11
Evaluation of the Methylenomycin Promoter.
SUMMARY
The methylenomycin cluster is borne on the large linear plasmid, SCP1, of Streptomyces coelicolor. Previous work has indicated that the promoters within this cluster are strong and could be used commercially.
This example describes a series of experiments to evaluate the strength of the methylenomycin promoter PmmyTOG in S. coelicolor and heterologous hosts such as S. lividans and S. erythraea. 
In order to evaluate the promoter, a number of test vectors were engineered in which a reporter gene is placed under the control of the methylenomycin promoter. This fragment was then placed into a suitable vector for introduction into an appropriate host. The reporter gene used was the DEBS1-TE encoding gene. The strength of the promoter can be assessed on the basis of yield of triketide lactone. The actinorhodin promoter PactI was used as a positive control.
A gene cassette containing 5 genes, mmyR, mmfP, mmfH, mmfL and mmfR, and the promoter for mmyT, was constructed and is called the promoter cassette. In addition, a separate gene has been identified, mmyB, which contains a rare TTA codon, and is also thought to be involved in the regulation of this promoter. Only trace amounts of triketide lactone are observed when the promoter cassette is used alone in S. coelicolor. Yields increase by 40-100-fold when the plasmid SCP1 is present in the cells. SCP1 harbours the methylenomycin cluster, which contains mmyB. We therefore suggest that the observed increase is due to the presence of the mmyB gene product.
The experiments carried out in the evaluation of the methylenomycin promoter are described in detail below.
(a) Isolation of the Promoter Cassette
The final expression vector contains the promoter cassette as a SpeI/NdeI fragment with the NdeI site located such that the ATG start codon of the gene of interest is optimally spaced from the ribosomal binding site of the promoter. This was achieved by amplifying each end of the cassette by PCR using oligonucleotide primers designed to incorporate the sequence for the appropriate restriction enzyme, and cloning in the central region using existing sites (FIG. 1).
The promoter cassette was isolated from cos73 (Redenbach et al 1998) as follows (see FIG. 15):
The 8366 bp EcoRI fragment of cos73 was cloned into the unique EcoRI site of pUC18 to give plasmid pCJR332.
The same EcoRI fragment was used as a template for PCR amplification of the ends of the promoter cassette. Oligonucleotide primer CR343 was designed to introduce the SpeI site at 2401-2406 bp (numbering from the beginning of the EcoRI fragment), this is 200 bp after the end of mmyR. Oligonucleotide primer CR344 is fully complementary to the wild-type sequence and binds at a SanDI site (3429-3435 bp—numbering from the beginning of the EcoRI fragment).











ATTACTAGTTCGCCGAGCGGCTGCGCTCGCTCCGTC
CR343




 



CCGCCGACGCGGGACCCCGCTGTGCAT
CR344






The 1050 bp PCR product was phosphorylated by treatment with T4 polynucleotide kinase and cloned into pUC18 previously digested with SmaI and dephosphorylated. The orientation was determined by restriction enzyme digestion and inserts from a number of clones of the desired orientation were sequenced to check for errors incorporated during in vitro polymerisation. None of the clones were error-free, all errors are substitutions and are within the primer binding region and after the stop codon of mmyR. It was considered that these would not affect the promoter cassette, but as a precaution two of these were selected to carry forward with the experiments and designated pCJR331A and pCJR331B, the errors are described below:

    pCJR331A contains a G at 2415 bp where the wild-type sequence has a C
    pCJR331B contains a G at 2418 bp where the wild-type sequence has a C and a G at 2425 bp where the wild-type sequence has a C


The 4750 bp SanDI fragment (3431-8180 bp numbering from start of 8366 bp EcoRI fragment) from pCJR332 was cloned into the unique SanDI site in each of pCJR331A and pCJR331B to give plasmids pCJR334A and pCJR334B which were confirmed by restriction analysis.
A second PCR was used to introduce the NdeI site for cloning of genes under the PmmyTOG. Oligonucleotide primer CR346 was designed to introduce the NdeI site at 7481-7486 bp (numbering from the beginning of the EcoRI fragment). Oligonucleotide primer CR345 is fully complementary to the wild-type sequence and binds at an XcmI site 6844-6855 bp (numbering) from the beginning of the EcoRI fragment).









AATCACTGGCCATCGCCGTGGTGGAGGAGCACT
CR345



 


TTTCATATGCGCCCGCGCTCCCAGTCTCTTCTGCCA
CR346






The 657 bp PCR product was phosphorylated by treatment with T4 polynucleotide kinase and cloned into pUC18 previously digested with SmaI and dephosphorylated. The orientation was determined by restriction enzyme digestion and inserts from a number of clones of the desired orientation were sequenced to check for errors incorporated during in vitro polymerisation. One correct clone was selected and designated pCJR328.
The plasmids pCJR334A and pCJR334B were digested with HindIII and XcmI and the 4491 bp inserts isolated. These were used to ligate into pCJR328 digested with HindIII and XcmI. Correct clones were identified using restriction analysis and designated pCJR335A and pCJR335B.
These two plasmids, pCJR335A and pCJR335B contain the promoter cassette as defined previously on a SpeI/NdeI fragment. In order to test the utility of this promoter cassette it was introduced into different backbone vectors, which could be used in a number of different hosts, see (b) and (c).
(b) Construction of Plasmids pCMS100 and pCMS101
The backbone for the first expression vector is pCJR30 (Rowe et al 1998), which has been used previously for the production of the DEBS1-TE triketide lactone, from the actinorhodin promoter PactI, in Streptomyces coelicolor. The plasmid pCJR30 is therefore the positive control for these experiments and will also provide the backbone for the expression vectors with the methylenomycin promoter.
pCJR30 was digested with NdeI and SpeI and the promoter cassettes from pCJR335A and pCJR335B isolated as SpeI/NdeI fragments. The promoter cassettes were ligated to the backbones, correct clones identified by restriction analysis, and a single clone from each ligation designated pCMS100A and pCMS100B as appropriate.
pCMS100A and pCMS100B are final constructs for testing the strength of the methylenomycin promoter PmmyTOG. These plasmids can be used to assess levels of DEBS1-TE triketide lactone production in actinomycete hosts which can maintain the SCP2* origin of replication.
The backbone for the second expression vector is pCJR65 (pCJR65 is pCJR24 (Rowe et al 1998) containing DEBS1-TE as an NdeI/XbaI fragment), which has been used previously for the production of the DEBS1-TE triketide lactone, from the actinorhodin promoter PactI, in Saccharopolyspora erythraea. The plasmid pCJR65 contains no origin of replication for actinomycetes and relies on the presence of the DEBS1-TE encoding gene as homologous DNA to allow integration into the chromosome.
pCJR65 was digested with NdeI and SpeI and the promoter cassettes from pCJR335A and pCJR335B isolated as SpeI/NdeI fragments. The promoter cassettes were ligated to the backbones, correct clones identified by restriction analysis and a single clone from each ligation designated pCMS101A and pCMS101B as appropriate.
pCMS101A and pCMS101B are final constructs for testing the strength of the methylenomycin promoter PmmyTOG. These plasmids are used to assess levels of DEBS1-TE triketide lactone production in S. erythraea JC2 (Rowe et al 1998), or the level of erythromycin production in S. erythraea wild-type.
(c) Construction of Plasmids pCMS104 and pCMS105
Incorporation of the mmyB gene into pCMS100 and pCMS101 was engineered as follows;
The mmyB gene was amplified from cos73 with primers CR349 and CR350, which have the following sequences;











TATAAGCTTGGTGAACTCCTTCGGCGAGTGGTTCGGA
CR349




(SEQ ID NO: 29)



 



TATAAGCTTGGGGAGAACTCCTTGGGATACTTCCTG
CR350



(SEQ ID NO: 35)






Each of the oligonucleotide primers has a HindIII site (AAGCTT) incorporated at the 5 prime ends.
In the published database sequence AJ276673, the mmyB gene is located on the complementary strand between 18032 bp and 18892 bp—the oligonucleotide primers bind in the following positions;

    17854-17890 binding region of CR350
    19095-19122 binding region of CR349


The total fragment then covers the region 17854-19122 bp with HindIII sites directly flanking this. The entire non-coding DNA from either end of the gene is included in this fragment. It is anticipated that upstream of this gene there will be a promoter and this strategy should ensure that any promoter sequences are incorporated and if any terminator sequences are present these should also be included within the fragment. The PCR fragment was cloned into pUC18 previously digested with SmaI and dephosphorylated, and insert containing clones identified by restriction analysis. The insert was sequenced to confirm that no errors had been incorporated during PCR amplification and the resulting plasmid was called pMMYBH.
The mmyB gene was then isolated on a HindIII fragment and cloned into HindIII digested, dephosphorylated pCMS100A, pCMS100B, pCMS101A and pCMS101B to give pCMS104A, pCMS104B, pCMS105A and pCMS105B.
(d) Production of the DEBS1-TE Triketide Lactone from the PmmyTOG Promoter in Streptomyces coelicolor 
The following S. coelicolor strains were transformed with pCMS100A and pCMS100B

    S. coelicolor J1501 (SCP1−, SCP2−)
    S. coelicolor J1506 (SCP1+, SCP2−)
    S. coelicolor J1508 (SCP1NF, SCP2−)


Transformants were selected by resistance to thiostrepton and the presence of the plasmid confirmed by re-isolating the plasmid and analysing by restriction digestion.
Production and analysis experiments were carried out as follows:
6 ml of YEME + additives (Glycine, MgCl2, uracil and histidine, as recommended) + thiostrepton at a final concentration of 5 μg/litre was inoculated with cells from a plate and grown for 48 hours at 30° C. (in flasks with springs, 250 rpm, 2 inch throw). 300 μl was used as a 5% inoculum into 6 ml of each of YEME and modified Complete Media (as described in Kieser et al 2000 but without the yeast nucleic acid hydrolysate). Cultures were incubated for 88 hours at 30° C. 3 ml of each culture was acidified with formic acid to pH 3 and extracted twice with an equal volume of ethyl acetate, and the organic phase evaporated to dryness using a Büchi rotor evaporator. This crude material was resuspended in 100 μl methanol and 1 μl applied to a Gas Chromatography instrument with a Mass Spectrometry detector. Yields are calculated by comparison to a synthetic standard and the numbers given represent the quantity of triketide lactone in the 3 ml samples removed from the culture.
Results for production in complete medium:

















Colony
S. coelicolor
S. coelicolor



Plasmid
isolate
J1501
J1506









pCMS100A
2
No product





3
Trace amount



pCMS100B
4
Trace amount



pCMS100A
6

 17 mg/litre




7

2.3 mg/litre



pCMS100B
8

0.5 mg/litre










These results indicate that there is considerable colony to colony variability in production level, this is to be expected following protoplast transformation. In the absence of SCP1, very little product is observed, a trace amount indicates less than 0.1 mg/litre. We are confident that the trace amounts we see are the triketide lactone as the behaviour of the triketide lactone on GC-MS is predictable and the mass-spectroscopy fragmentation is characteristic. The production levels when cultures were grown in complete medium are comparable to when the same colonies are grown in YEME (full data not yet available). For example, S. coelicolor J1506[pCMS100A] colonies 6 and 7 yielded 20 and 4 mg/litre respectively when cultured in YEME.
Further experiments were carried out as follows. Cultures were grown in 6 ml of an appropriate medium and this was used as a 5% inoculum for production cultures. In this way the production cultures for a single isolate should be comparable, and comparison of production levels between isolates should be relatively robust. In all cases yields are based on 3 or 4 ml of culture withdrawn from the flasks, this does not in any of the cases take into account evaporative loss of liquid during fermentation. This means that the yields quoted are yields in the final cultures rather than production levels. Yields are calculated using mass spectrometry, which has an associated error, but such errors should be similar for all samples, with the largest errors being in calculation of the lowest yields due to ill-defined small peaks.
It should also be noted that the results are always given for the propionate starter triketide lactone [1]. There is a second product from the DEBS1-TE system and this is the equivalent acetate starter triketide lactone [2] which is mainly seen in high producers, or when propionate is limiting in the system. In general, in these experiments this represents less than 5% of the overall product. However, from plates and from some of the complex media there is a significant percentage of is product and in these cases a yield has been calculated based on the propionate starter standard.




(e) Production of the DEBS1-TE Triketide Lactone from the PmmyTOG Promoter in Streptomyces coelicolor in Different Media

















Culture
Yeme
Complete
Hobbs
SSDM





















1
J1501 [pCJR30]_12
No
trace
0.7
no





(0.7)


2
J1501 [pCMS100A]_11
1
trace
1.1
no


3
J1501 [pCMS100A]_12
trace
trace
1.4
no


4
J1501 [pCMS100B]_7
trace
trace
no
no


5
J1506 [pCJR30]_10
No
trace
trace
no





(0.07)


6
J1506 [pCMS100A]_3
20
17
0.02
0.5


7
J1506 [pCMS100A]_9
4
2.3
0.03
0.6


8
J1506 [pCMS100B]_7
trace
0.5
trace
no





Yeme = Yeme + appropriate additives: glycine, MgCl2, uracil and histidine, as recommended.


Complete = As described in Kieser et al (2000) but without the yeast nucleic acid hydrolysate.


Hobbs = As described in Hobbs et al (1992)


SSDM = As described in Caffery et al (1992)






In all cases yield of triketide lactone are given as mg/litre on the basis of comparison to a known quantity of a synthetic by mass spectrometry. Quantitation by mass spectrometry is subject to a certain amount of error in that the concentration of a molecule in a mixture and the composition of the mixture will effect the ionisation of the subject compound. No indicates that no product was observed by mass spec, and trace indicates that a small peak is observed, but it is not well enough defined to accurately integrate.
The variability of production levels from one medium to another is significant; and it is proposed that maximal production levels may not have been attained. This can be achieved by statistical analysis production levels as different components of the media are used, and at different concentrations.
In Hobb's medium, which is adapted for methylenomycin production (i.e. 10 g/litre glucose is added instead of 2), there appears to be higher production in the absence of SCP1.
The increase in production with SCP1 present appears to be a real effect, despite the small sample size. It is suggested that by providing the mmyB gene in isolation the absolute yield may increase.
(f) Production of the DEBS1-TE Triketide Lactone from the PmmyTOG Promoter in Saccharopolyspora erythraea in Different Media
Saccharopolyspora erythraea NRRL2338 JC2 (Rowe et al 1998) was transformed with pCMS100A, pCMS100B, pCMS105A and pCMS105B. Transformants were selected using thiostrepton (final concentration 50 μg/litre) and a secondary round of selection, involving isolating spores and filtering onto fresh selection plates was performed to insure that the isolates contained the resistance from the plasmid.
Three isolates from each of S. erythraea JC2/pCMS100A and S. erythraea JC2/pCMS100B were used to inoculate 6 ml of TSB + tsr (final concentration 5 μg/litre). These precultures were used to inoculate 6 ml of each of two different production media which have previously been shown to yield good levels of erythromycin, SSDM is a defined minimal medium, and SM3 (Ranganathan et al 1999) is more complex. Production cultures were grown for 7 days and 4 ml of each taken and extracted. Analysis by GC-MS gave the following results.

















SSDM
SM3













Culture
[1]
[1]
[2]







S. ery
0.5
8.4
3.0



JC2 [pCMS100A]_1



S. ery
0.3
8.2
0.2



JC2 [pCMS100A]_2



S. ery
0.6
4.0
0.2



JC2 [pCMS100A]_3



S. ery
0.8
7.3
0.2



JC2 [pCMS100B]_1



S. ery
0.9
5.5
0.2



JC2 [pCMS100B]_2



S. ery
0.5
5.0
0.2



JC2 [pCMS100B]_3










[1] and [2] refer to the two triketide starter lactones, as described above.
This experiment demonstrates that the expression cassette can be used to drive expression of a nucleic acid of interest in host cells other than S. coelicolor. Some expression has also been demonstrated in S. lividans. 
Again it is observed that the composition of the media has a significant effect on production level and it is expected that higher yields may be obtained upon optimisation and/or in the presence of mmyB.
(g) A Quick Look at Comparative Yields of Triketide Lactone from Patches on Plates
To try to get a quick indication of whether or not the mmyB gene would affect production levels, plugs were taken from patches on R2YE plates and extracted to look for triketide lactone product.
Using strain J1501, which lacks the SCP1 plasmid (and hence a native mmyB gene), a dramatic (up to orders of magnitude) increase in yield was shown between pCMS100A (which lacks mmyB) and pCMS104 (the equivalent plasmid also possessing mmyB).
Using strain J1506 (which possesses native SCP1 and mmyB), more expression was shown with pCMS100A than was shown using this plasmid in J1501, but less expression was shown with pCMS104 in J1506 than in J1501. Expression in J1506 was similar with both plasmids.
This is consistent with the mmyB gene product being advantageous for expression, but also with such product being sequestered by (e.g. promoters of) the native SCP1 plasmid when present, rather than acting to increase expression from the expression cassette.
Yields represent the total product obtained from the whole agar plug (μg).














Yield (μg)











R2YE
Culture
[1]
[2]





26
J1501 [pCMS100A]_6
trace
none


27
J1506 [pCMS100A]_8
1.0
0.4


31
J1501 [pCMS104]_2
12
20


32
J1501 [pCMS104]_6
1.5
2.5


33
J1506 [pCMS104]_1
2.3
2.6


34
J1506 [pCMS104]_7
1
1.0









In some cases the second product was the predominant product, probably reflecting substrate availability, which may therefore be a limiting factor.
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## Claims

1. An expression cassette for the expression of a nucleic acid of interest, the expression cassette including:
a. at least one regulatory portion including:
i. a first regulatory element which includes a nucleic acid sequences mmyR encoding an MmyR polypeptide and/or a nucleic acid sequence mmfR encoding an MmfR polypeptide;
ii. a second regulatory element which includes the nucleic acid sequence mmfL encoding an MmfL polypeptide; and
iii. a repressible promoter, the function of which is repressed by the expression product of the first regulatory element, that repression being alleviated or removed by a product, the production of the last-mentioned product being conferred by the MmfL polypeptide; and

b. a heterologous nucleic acid of interest, in operative association with said promoter; wherein:
said MmyR polypeptide shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 12, optionally excluding the first 6 listed amino acids;
said MmfR polypeptide shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 11 said MmyR polypeptide and/or said MmfR polypeptide being effective to repress the function of said repressible promoter; and
said MmfL polypeptide shows at least 80% 90% amino acid sequence identity with the sequence of SEQ ID NO: 17 said MmfL polypeptide being effective to produce said product which alleviates or removes repression of said repressible promoter by said MmyR polypeptide and/or said MmfR polypeptide.

2. A set of nucleic acids for the expression of a nucleic acid of interest, the set of nucleic acids together including:
a. at least one regulatory portion including:
i. a first regulatory element which includes a nucleic acid sequence mmyR encoding an MmyR polypeptide, and/or a nucleic acid sequence mmfR encoding an MmfR polypeptide;
ii. a second regulatory element which includes the nucleic acid sequence mmfL encoding an MmfL polypeptide;
iii. a repressible promoter, the function of which is repressed by the expression product of the first regulatory element, that repression being alleviated or removed by a product, the production of the last-mentioned product being conferred by the MmfL polypeptide; and

b. a heterologous nucleic acid of interest, in operative association with said promoter; wherein:
said MmyR polypeptide shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 12, optionally excluding the first 6 listed amino acids;
said MmfR polypeptide shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 11 said MmyR polypeptide and/or said MmfR polypeptide being effective to repress the function of said repressible promoter; and
said MmfL polypeptide shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 17 said MmfL polypeptide being effective to produce said product which alleviates or removes repression of said repressible promoter by said MmyR polypeptide and/or said MmfR polypeptide.

3. The expression cassette according to claim 1, wherein the regulatory portion(s) comprises both an mmyR nucleic acid sequence and an m13mfR nucleic acid sequence as defined in claim 1.

4. The expression cassette according to claim 1 further including a third regulatory element, which includes a nucleic acid sequence mmyB encoding an MmyB polypeptide, wherein said MmyB polypeptide shows at least 90% amino acid sequence identity with the MmyB amino acid sequence set out in EMBL AJ276673 said MmyB polypeptide being effective to bind DNA.

5. The expression cassette according to claim 1, wherein the regulatory portion(s) comprise(s) at least a portion of SEQ ID NO: 18 or SEQ ID NO: 19, or a variant thereof having at least 90% nucleic acid sequence identity to the nucleic acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19.

6. The expression cassette according to claim 1, wherein the repressible promoter is a promoter of a methylenomycin biosynthetic gene or a promoter of a methylenomycin regulatory gene.

7. The expression cassette according to claim 1, wherein the repressible promoter comprises an mmyTOG promoter or an mmy . . . XCAPK promoter or an mmfLHP promoter or an mmyYF promoter or an mmyBQE promoter, wherein:
a. the mmyTOG promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 5452 to 5675 of SEQ ID NO: 18;
b. the mmfLHP promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4716 to 4909 of SEQ ID NO: 19;
c. the mmy . . . XCAPK promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of the complement of residues 18892 to 19123 or 15404 to 15977 of EMBL AJ276673;
d. the mmyYF promoter comprises a fragment of at least 30 residues of residues 18892 to 19123 of EMBL AJ276673; and
e. the mmyBQE promoter comprises a fragment of at least 30 residues of the complement of residues 18892 to 19123 of EMBL AJ276673.

8. The expression cassette according to claim 7 wherein:
a. at least part of at least one of the nucleic acid sequences mmyT, mmyO and mmyG is present in the regulatory portion(s), in operative association with the mmyTOG promoter;
b. at least part of at least one of the nucleic acid sequences mmyD, mmyX, mmyC, mmyA, mmyP and mmyK, and optionally at least part of at least one of the nucleic acid sequences mmyB, mmyQ, and mmyE, is present in the regulatory portion(s) in operative association with the mmy . . . XCAPK promoter;
c. at least part of at least one of the nucleic acid sequences mmfP and mmfH is present in the regulatory portion(s), in operative association with the mmfLHP promoter;
d. at least part of at least one of the nucleic acid sequences mmyY and mmyF is present in the regulatory portion(s), in operative association with the mmyYF promoter; and
e. at least part of at least one of the nucleic acid sequences mmyB, mmyQ and mmyE is present in the regulatory portion(s) in operative association with the mmyBQE promoter;

and wherein:
said mmyT nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 22 and retains mmy polypeptide function;
said mmyO nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 23 and retains mmy polypeptide function;
said mmyG nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 24 and retains mmy polypeptide function;
said mmfP nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 20 and retains mmf polypeptide function;
said mmfH nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 21 and retains mmf polypeptide function;
said mmyD, mmyX, mmyC, mmyA, mmyP, mmyK, mmyB, mmyQ, mmyE, mmyY and mmyF nucleic acid sequences each encode a polypeptide which show at least 90% amino acid sequence identity with the corresponding amino acid sequences set out in EMBL AJ276673 and retains mmy polypeptide function.

9. The expression cassette according to claim 8, further including a third regulatory element which includes a nucleic acid sequence mmyB encoding an MmyB polypeptide, wherein:
(a) the mmfL nucleic acid sequence of the second regulatory element is also in operative association with the mmfLHP repressible promoter; or
(b) the mmyB nucleic acid sequence of the third regulatory element is in operative association with the same mmyBQE repressible promoter; or
(c) the mmfL nucleic acid sequence of the second regulatory element is also in operative association with the mmfLHP repressible promoter and the mmyB nucleic acid sequence of the third regulatory element is in operative association with the same mmyBQE repressible promoter; and wherein:
the MmyB polypeptide shows at least 90% amino acid sequence identity with the MmyB amino acid sequence set out in EMBL AJ276673 said MmyB polypeptide being effective to bind DNA;
the mmfLHP repressible promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4716 to 4909 of SEQ ID NO: 19; and
the mmyBQE repressible promoter comprises a fragment of at least 30 residues of the complement of residues 18892 to 19123 of EMBL AJ276673.

10. The expression cassette according to claim 1, wherein the regulatory portion(s) include(s) at least one of the nucleic acid sequences mmfP and mmfH, and wherein:
said mmfP nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with SEQ ID NO: 20 and is effective to regulate methylenomycin production; and
said mmfH nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with SEQ ID NO: 21 and is effective to regulate methylenomycin production.

11. The expression cassette according to claim 1, wherein the first regulatory element also includes a promoter operatively linked to at least one of the mmyR nucleic acid sequence and the mmfR nucleic acid sequence.

12. The expression cassette according to claim 11 wherein (i) the mmyR nucleic acid sequence is operatively linked to an mmyR promoter; or (ii) the mmfR nucleic acid sequence is operatively linked to an mmfR promoter; or (iii) both the mmyR nucleic acid sequence is operatively linked to an mmyR promoter and the mmfR nucleic acid sequence is operatively linked to an mmfR promoter, wherein:
a. the mmyR promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 7965 to 8132 of SEQ ID NO: 19, optionally excluding residues 8113 to 8132; and
b. the mmfR promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4613 to 4806 of SEQ ID NO: 18.

13. The expression cassette according to claim 1, wherein the second regulatory element includes a promoter operatively linked to the mmfL nucleic acid sequence.

14. The expression cassette according to claim 13, wherein the promoter is an mmfL promoter, wherein the mmfL promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4716 to 4909 of SEQ ID NO: 19.

15. The expression cassette according to claim 1 comprising at least one of an mmfL promoter and an mmfR promoter, the mmfL and/or the mmfR promoter comprising a palindromic sequence having the half-site 5′-GG(T/C)CGGT(A/T)(T/C)G(T/G)-3′ (SEQ ID NO: 1), or a variant thereof having sequence identity at seven or more corresponding positions within the half-site, wherein:
said mmfL promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4716 to 4909 of SEQ ID NO: 19; and
said mmfR promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4613 to 4806 of SEQ ID NO: 18.

16. The expression cassette according to claim 15, wherein the palindromic sequence has the half-site 5′GGAAGGTATTA-3′ (SEQ ID NO: 2).

17. The expression cassette according to claim 1 having a bi-directional mmfL/mmfR promoter, in operative association with the nucleic acid sequences mmfL and mmfR, wherein:
said mmfL promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4716 to 4909 of SEQ ID NO: 19; and
said mmfR promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4613 to 4806 of SEQ ID NO: 18.

18. The expression cassette according to claim 5 comprising a sequence having at least 90% nucleic acid identity with a fragment of at least 30 residues of residues 3551 to 5451 of SEQ ID NO: 18.

19. The expression cassette according to claim 1, wherein the nucleic acid of interest is inserted into the regulatory portion.

20. The expression cassette according to claim 19, wherein the insertion is by means of homologous recombination.

21. The expression cassette according to claim 1, wherein, apart from the repressible promoter, the nucleic acid of interest is not additionally in operative association with any promoter not derived from, or a variant of, a promoter of the methlenomycin gene cluster.

22. The expression cassette according to claim 1, wherein the regulatory portion(s) include(s) nucleic acid having at least 90% nucleic acid sequence identity with the sequence from residue 796 to a residue between 5676 and 8817 inclusive of SEQ ID NO: 18, and wherein the nucleic acid sequence of interest is inserted into or downstream of the mmyTOG region, or part thereof, in operative association with the mmyTOG promoter,
wherein:
said mmyTOG promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 5452 to 5675 of SEQ ID NO: 18; and
said mmyTOG region comprises the mmyT, mmyO and mmyG nucleic acid sequences,

and wherein:
a. the mmyT nucleic acid sequence compnses a sequence having at least 90% nucleic acid identity with the sequence of residues 5676 to 6401 of SEQ ID NO: 18;
b. the mmyO nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 6432 to 7553 of SEQ ID NO: 18; and
c. the mmyG nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 7636 to 8817 of SEQ ID NO: 18.

23. The expression cassette according to claim 1, wherein the regulatory portion(s) include(s) nucleic acid having at least 90% nucleic acid sequence identity with the sequence from residues 796 to residue 5451 inclusive of SEQ ID NO: 18, and wherein the nucleic acid of interest is inserted into or downstream of the mmfHP region, in operative association with the mmfLHP promoter, wherein:
said mmfLHP promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4716 to 4909 of SEQ ID NO: 19; and
said mmfHP region comprises the mmfH and mmfP nucleic acid sequence, and wherein:
a. the mmfH nucleic acid sequence compnses a sequence having at least 90% nucleic acid identity with the sequence of residues 5968 to 7170 of SEQ ID NO: 19; and
b. the mmfP nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 7167 to 7964, optionally excluding residues 7167 to 7169, of SEQ ID NO: 19.

24. The vector or set of vectors comprising an expression cassette according to claim 1.

25. The expression system comprising an expression cassette according to claim 1.

26. The expression system according to claim 25 which comprises a bacterial cell.

27. The expression system according to claim 26, wherein the bacterium is a streptomycete.

28. The expression system according to claim 25 further comprising a nucleic acid sequence mmyB, encoding an MmyB polypeptide wherein said MmyB polypeptide shows at least 90% amino acid sequence identity with the MmyB amino acid sequence set out in EMBL AJ276673 said MmyB polypeptide being effective to bind DNA.

29. The expression system according to claim 28, wherein the mmyB nucleic acid sequence is present within the expression cassette.

30. The expression system according to claim 28, wherein the mmyB nucleic acid sequence is present as part of the host cell genome.

31. The expression system according to claim 28, wherein the mmyB nucleic acid sequence is located on an SCP1 or pSV1 plasmid.

32. The expression system according to claim 25 which lacks the ability to translate the codon TTA (UUA in mRNA).

33. The expression system according to claim 28, wherein at least one of the nucleic acid sequences mmfL and mmyB is modified to lack a naturally occurring TTA codon.

34. A method of expressing a nucleic acid of interest, the method comprising providing an expression system according to claim 25 and maintaining the expression system under conditions suitable for expression of the nucleic acid of interest.

35. The method according to claim 34 for expressing the nucleic acid of interest substantially only when the host cell culture reaches high cell density.

36. The method according to claim 35 for expressing the nucleic acid of interest substantially only at or close to the stationary phase of host cell culture.

37. The method of expressing a nucleic acid of interest, the method comprising:
a. providing in an expression system at least one regulatory portion as defined in claim 1;
b. providing in the expression system the nucleic acid of interest;
c. operatively associating the nucleic acid of interest with the repressible promoter of the regulatory portion(s); and
d. maintaining the expression system under conditions suitable for the expression of the nucleic acid of interest.

38. The method according to claim 37, wherein the step of operative association occurs prior to introducing the regulatory portion(s) and nucleic acid of interest into the expression system.

39. The method according to claim 34, wherein the nucleic acid of interest is brought into operative association with the repressible promoter by inserting the nucleic acid of interest downstream of the repressible promoter.

40. The method according to claim 34, wherein the nucleic acid of interest is brought into operative association with the repressible promoter by inserting the repressible promoter into nucleic acid containing the nucleic acid of interest.

41. A method comprising providing a nucleic acid expression product produced by the method according to claim 34 and subjecting it to one or more purification steps.

42. An expression system comprising a vector or set of vectors according to claim 24.

43. An expression cassette according to claim 1 wherein:
a. the MmyR polypeptide shows at least 90% amino acid sequence identity with residues 40 to 49 of SEQ ID NO: 12; and/or
b. the MmfR polypeptide shows at least 90% amino acid sequence identity with residues 61 to 70 of SEQ ID NO: 11; and/or
c. the MmfL polypeptide shows at least 90% amino acid sequence identity with at least one of (i) residues 77 to 87 and (ii) residues 240 to 255 of SEQ ID NO: 17.

44. An expression cassette according to claim 1 wherein:
a. the mmyR nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 8115 to 8726 of SEQ ID NO: 19 (optionally excluding residues 8115 to 8132); and/or
b the mmfR nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 4807 to 5451 of SEQ ID NO: 18; and/or
c. the mmfL nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 4910 to 5971 of SEQ ID NO: 19.

45. An expression cassette according to claim 1 wherein:
a. the mmyR nucleic acid sequence shows at least 90% nucleic acid sequence identity with the sequence of SEQ ID NO: 19 which corresponds to amino acid resides 40 to 49 of SEQ ID NO: 12; and/or
b. the mmfR nucleic acid sequence shows at least 90% nucleic acid sequence identity with the sequence of SEQ ID NO: 18 which corresponds to amino acid residues 61 to 70 of SEQ ID NO: 11; and/or
c. the mmfL nucleic acid sequence shows at least 90% nucleic acid sequence identity with the sequence of SEQ ID NO: 19 which corresponds to at least one of (i) amino acid residues 77 to 87 and (ii) amino acid residues 240 to 255 of SEQ ID NO: 17.

46. An expression cassette according to claim 8 wherein:
a. the mmfP nucleic acid sequence encodes an MmfP polypeptide having the amino acid sequence of SEQ ID NO: 20; and/or
b. the mmfH nucleic acid sequence encodes an MmfH polypeptide having the amino acid sequence of SEQ ID NO: 21; and/or
c. the mmyT nucleic acid sequence encodes an MmyT polypeptide having the amino acid sequence of SEQ ID NO: 22; and/or
d. the mmyO nucleic acid sequence encodes an MmyO polypeptide having the amino acid sequence of SEQ ID NO: 23; and/or
e. the mmyG nucleic acid sequence encodes an MmyG polypeptide having the amino acid sequence of SEQ ID NO: 24; and/or
f. the mmyB nucleic acid sequence encodes an MmyB polypeptide having the amino acid sequence set out in EMBL AJ276673.

47. An expression cassette according to claim 8 wherein:
a. the mmfH nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 5968 to 7170 of SEQ ID NO: 19; and/or
b. the mmfP nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 7167 to 7964, optionally excluding residues 7167 to 7169, of SEQ ID NO: 19; and/or
c. the mmyT nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 5676 to 6401 of SEQ lID NO: 18; and/or
d. the mmyO nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 6432 to 7553 of SEQ ID NO: 18; and/or
e. the mmyG nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of residues 7636 to 8817 of SEQ ID NO: 18; and/or
f. the mmyB nucleic acid sequence comprises a sequence having at least 90% nucleic acid identity with the sequence of the complement of residues 18032 to 11892 of EMBL AJ276673.

48. The expression cassette according to claim 8, wherein the nucleic acid of interest is inserted into the regulatory portion.

49. The expression cassette according to claim 48, wherein the nucleic acid of interest is inserted within:
a. a nucleic acid sequence mmyT, mmyO or mmyG, in operative association with an mmyTOG promoter;
b. a nucleic acid sequence mmyD, mmyX, mmyC, mmyA, mmyP or mmyK or optionally a nucleic acid sequence mmyB, mmyQ or mmyE, in operative association with an mmy . . . XCAPK promoter;
c. a nucleic acid sequence mmfH or mmfP, in operative association with an mmfLHP promoter;
d. a nucleic acid sequence mmyY or mmyF, in operative association with an mmyYF promoter; and/or
e. a nucleic acid sequence mmyB, mmyQ or mmyE, in operative association with an mmyBQE promoter; wherein:
said mmyT nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 22 and retains mmy polypeptide function;
said mmyO nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 23 and retains mmy polypeptide function;
said mmyG nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 24 and retains mmy polypeptide function;
said mmfP nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 20 and retains mmf polypeptide function;
said mmfH nucleic acid sequence encodes a polypeptide which shows at least 90% amino acid sequence identity with the sequence of SEQ ID NO: 21c and retains mmf polypeptide function;
said mmyD, mmyX, mmyC, mmyA, mmyP, mmyK, mmyB, mmyQ, mmyE, mmyY and mmyF nucleic acid sequence each encodes a polypeptide which shows at least 90% amino acid sequence identity with the corresponding amino acid sequences set out in EMBL AJ276673 and retains mmf polypeptide function; and wherein:
a. the mmyTOG promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 5452 to 5675 of SEQ ID NO: 18;
b. the mmfLHP promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of residues 4716 to 4909 of SEQ ID NO: 19;
c. the mmy . . . XCAPK promoter comprises a sequence having at least 60% nucleic acid identity with a fragment of at least 30 residues of the complement of residues 18892 to 19123 or 15404 to 15977 of EMBL AJ276673;
d. the mmyYF promoter comprises a fragment of at least 30 residues of residues 18892 to 19123 of EMBL AJ276673; and
e. the mmyBQE promoter comprises a fragment of at least 30 residues of the complement of residues 18892 to 19123 of EMBL AJ276673.

50. An expression cassette for the expression of a nucleic acid of interest, the expression cassette including:
a. at least one regulatory portion including:
a first regulatory element which includes a nucleic acid sequence mmyR encoding an MmyR polypeptide and/or a nucleic acid sequence mmfR encoding an MmfR polypeptide;
ii. a second regulatory element which includes the nucleic acid sequence mmfL encoding an MmfL polypeptide; and
iii. a repressible promoter, the function of which is repressed by the expression product of the first regulatory element, that repression being alleviated or removed by a product, the production of the last-mentioned product being conferred by the MmfL polypeptide; and

b. a heterologous nucleic acid of interest, in operative association with said promoter; wherein:
a. said MmyR polypeptide has the amino acid sequence of SEQ ID NO: 12 (optionally excluding the first 6 listed amino acids);
b. said MmfR polypeptide has the amino acid sequence of SEQ ID NO: 11; and
c. said MmfL polypeptide has the amino acid sequence of SEQ ID NO: 17.