CA2322105A1 - Antibiotic production (ii) - Google Patents

Abstract

The nucleic acid and amino acid sequences of .alpha.1, .alpha.2, .beta. and .epsilon.
subunits of acetyl-CoA carboxylase (ACCase) from Streptomyces coelicolor are provided. This subunit structure differs from that of known acyl carboxylases. Materials and methods are provided of increasing ACCase activity and production of secondary metabolites (such as polyketides and especially antibiotics) by causing expression in Streptomyces of such nucleic acid.
Also provided are methods of increasing ACCase activity and production of secondary metabolites (such as polyketides and especially antibiotics) by culturing Streptomyces in the presence of exogenous malonate.

Description

ANTIBIOTIC PRODUCTION (II) Introduction Malonyl-CoA is essential as a metabolic substrate in all living organisms studied so far and it also plays a role as a modulator of specific protein activity (for a review see Brownsey et al., 1997). Malonyl-CoA is a substrate for fatty acid synthase (FAS) (Bloch and Vance, 1977), for polyketide synthases (PKS) in plants, fungi and bacteria (Hopwood & Sherman, 1990) and for fatty acid chain-elongation systems (Saggerson, et al., 1992). Understanding the pathways) that lead to the biosynthesis of malonyl-CoA
in Streptomyces might have an outstanding interest, since these micro-organisms are well known to have the ability to synthesize a vast array of pharmaceutically important polyketide compounds (such as antibiotic, antiparasitic, antifungal, immunosuppressant and/or antitumour polyketides), where malonyl-CoA is used as the most common extender unit (Hopwood & Sherman, 1990). Therefore, information gained on the enzymes) involved in the supply of this key metabolite will be relevant, not only for a better understanding of the primary metabolism of Streptomyces, but for improving production of many useful secondary metabolites.
Biosynthesis of malonyl-CoA occurs in most species through the ATP-dependent carboxylation of acetyl-CoA by an acetyl-CoA carboxylase (ACCase) (Bloch & Vance, 1977; Harwood, 1988). The overall reaction catalyzed by ACCase is a two step process that involves ATP-dependent formation of carboxybiotin followed by transfer of the carboxyl moiety to acetyl-CoA. The importance of this biosynthetic pathway is most directly reflected by the fact that ACCase expression is essential for normal growth of bacteria (Perez, et al., 1998; Li and Cronan, 1993), yeast (Hasselacher, et al., 1993) and isolated animal cells in culture (Pfizer, et al. , 1996) .
Several complexes with ACCase activity have been purified from various actinomycetes. Interestingly, these complexes have also shown the ability to carboxylate other substrates like propionyl- and butyryl-CoA (Erfle, 1973; Henrikson and Allen, 1979; Huanaiti and Kolattukudy, 1982). This property has led to these enzyme being called acyl-CoA
carboxylases, and all of them have been shown to consist of two subunits, a larger one (a-chain) with the ability to carboxylate its covalently bound biotin group, and a smaller sub-unit ((3-chain) bearing the carboxyl transferase activity. However, there is no information gained, so far, regarding the physiological role of these enzymes.
In Streptomyces the purification of a complex with ACCase activity has proved to be unsuccessful, probably due to its high instability (Bramwell et al., 1996). However ACCase activity has been readily measured in crude extracts of S.
coelicolor (Bramwell et al., 1996; Rodriguez and Gramajo, 1999), indicating that this enzyme complex was present in this micro-organism.
A pathway for the biosynthesis of malonyl-CoA in S.
aureofaciens has been described that does not involve ACCase (Behal et al., 1977; Laakel et al., 1994). This route involves the anaplerotic enzymes phosphoenolpyruvate carboxylase and oxaloacetate dehydrogenase. In S.
coelicolor A3(2), no evidence for the presence of oxaloacetate dehydrogenase has been found (Bramwell et al., 1993); thus, biosynthesis of malonyl-CoA in this organism seemed to occur exclusively through the ACCase enzyme activity.

Attempts carried on in S. coelicolor to characterize enzymes with carboxylase activity, have led to the characterization of two complexes exhibiting exclusively PCCase activity. The PCCase purified by Bramwell et al., (1996) comprises a biotinylated protein of 88 kDa, PccA, and a non-biotinylated component, the carboxyl transferase, of 66 kDa. More recently the inventors have also.
characterized at both the genetic and biochemical levels, the components of a second PCCase in this bacterium. In vitro reconstitution experiments have shown that an active complex could be obtained by mixing a carboxyl transferase component of 59 kDa (deduced MW, though it runs anomalously in SDS-PAGE, with an apparent MW of 65 kDa), PccB, with either of the two almost identical biotinylated components named AccAl and AccA2 (Rodriguez and Gramajo, 1999).
Recently a gene cluster encoding malonyl-CoA decarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putative decarboxylate carrier protein (MatC) has been proposed as the pathway for malonate metabolism in Rhizobium trifolii (An and Kim, 1998). After the transport of the malonate by MatC, the malonate is converted into malonyl-CoA by MatB
and finally decarboxylated to acetyl-CoA by MatA. However, the fact that the F~, of the malonyl-CoA decarboxylase for malonyl-CoA is relatively high has led the inventors to speculate that malonyl-CoA synthesised from malonate by malonyl-CoA synthetase (rather than malonyl CoA synthesised by ACCase) is the major source for fatty acid biosynthesis in the bacterioid R. trifolii. Interestingly, genes with very high identity to MatC and MatB have been recently reported in the S. coelicolor genome project, suggesting that malonyl-CoA could also be synthesized from malonate in this micro-organism.

The inventors have identified an essential acyl-CoA
carboxylase of S. coelicolor, and provide detailed genetic and biochemical characterization. The enzyme complex contains a unique sub-unit composition and appears to be the main pathway for the biosynthesis of malonyl-CoA, one of the key metabolites in the linkage between primary and secondary metabolism. An alternative pathway for the biosynthesis of malonyl-CoA is also proposed for cultures growing in malonate, and it most probably involves the matB
and matC homologues of R. trifolii. However, even in these growing conditions, the acyl-CoA carboxylase seems to be essential for the viability of the micro-organism.
Suaanary of invention Two genes accB and accE, forming a single operon, have been cloned from Streptomyces coelicolor A(3)2. The deduced amino acid sequence of AccB showed high similarity to carboxyl transferase of several propionyl- or acyl-CoA
carboxylases of different actinomycetes. By contrast, AccE
did not show any significant homology with protein sequences deposited in the GenBank data base. Heterologous expression of accB and accE in Escherichia coli and in vitro reconstitution of enzyme activity in the presence of the biotinylated component AccAl or AccA2 confirmed that AccB was the carboxyl transferase subunit of an acyl-CoA
carboxylase.
These experiments also established that AccE was a necessary component to obtain a fully active enzyme complex, whose subunit composition seems to be unique within this type of carboxylase. Gene disruption experiments clearly determined that AccB was essential for S. coelicolor viability. This protein together with AccA2, a biotinylated component essential for the .viability of this micro-organism (Rodriguez and Gramajo, 1999), are the best candidates to form an acyl-CoA carboxylase, whose main physiological role is, most probably, the biosynthesis of malonyl-CoA.
Transcriptional studies of accBE, accA2 and acct have shown that accBE and accA2 are mainly expressed during vegetative and transition phase of growth, although some expression of these genes also occurred during stationary phase where they should provide the malonyl-CoA subunits for secondary metabolites biosynthesis. accA1 is only expressed during the transition phase of growth and its role in the formation of a carboxylase complex involved in providing the substrate for polyketide compounds of S.
coelicolor is discussed.
Finally, an alternative route for the biosynthesis of malonyl-CoA is proposed when malonate is used as a carbon source. However, this route seems unable to substitute the main one, determined by the acyl-CoA carboxylase.
Accordingly, in a first aspect, the present invention provides a nucleic acid comprising a nucleic acid sequence which encodes an AccB polypeptide and/or an AccE
polypeptide, or a nucleic acid sequence complementary thereto.
In a second aspect, the present invention provides a nucleic acid comprising a nucleic acid sequence which encodes an AccAl and/or AccA2 polypeptide, or a nucleic acid sequence complementary thereto. It is believed that such nucleic acid was not made available to the public S

before 24 October 1999, when the amino acid sequences of these polypeptides were disclosed in an oral presentation.
Exemplary nucleic acid sequences encoding the AccB, AccE, AccAl and AccA2 polypeptides are given herein. Preferred embodiments of the invention include such sequences.
However, it would be a matter of routine for the skilled person to obtain other nucleic acid sequences encoding these polypeptides, e.g. by introducing mutations which do not alter the encoded amino acid sequence, by virtue of the degeneracy of the genetic code, or by introducing mutations which alter the encoded amino acid sequence, within limits as defined below. Moreover, nucleic acids encoding variants of the polypeptides may be obtained e.g. by screening different strains of S. coelicolor or closely related species of Streptomyces using degenerate probes based on the sequences given herein.
Preferred nucleic acids of the first and second aspects encode AccB and AccE polypeptides along with an AccAl and/or an AccA2 polypeptide (preferably AccA2).
The nucleic acid sequences encoding Acc polypeptides are preferably in operative association with regulatory sequences, e.g. sequences which enable constitutive or inducible expression in Streptomyces species. Examples of plasmids which include such regulatory sequences and of suitable promoters are given herein. A suitable inducible promoter is tipA (inducible by thiostrepton); suitable constitutive promoters are ermE and the optimised ermE*.
Alternatively, naturally occurring nucleic acid sequences may be in operative association with the regulatory sequences with which they are normally associated, or corresponding regulatory sequences from homologous genes in other strains or species. For example, the nucleic acid sequences may be in operative association with the corresponding regulatory (e. g. promoter) sequences defined herein.
For detailed protocols relevant to this and other aspects, see standard reference texts, such as Sambrook et al.
(1989) and Hopwood et al. (1985).
In a third aspect, the present invention separately provides AccB, AccE, AccAl and AccA2 polypeptides having amino acid sequences encoded or encodable by the respective nucleic acid sequences referred to in the first and second aspects.
In a fourth aspect, the present invention provides: vectors containing the nucleic acids of the first and second aspects (preferably vectors, e.g. plasmids, suitable for transforming Streptomyces species for expression therein) and cells, particularly Streptomyces cells, transformed with such vectors. Furthermore, the present invention provides a method of producing a secondary metabolite of a Streptomyces species, the method comprising culturing such transformed Streptomyces cells and extracting the secondary metabolite from the cell culture. The metabolite may be purified and/or formulated as a commercial product according to standard procedures.
In a fifth aspect, the invention provides a method of modifying a secondary metabolite-producing strain of a Streptomyces species to increase production of said secondary metabolite, the method comprising modifying said strain to express, or to increase expression of, nucleic acid encoding one or more polypeptides selected from the group consisting of AccB, AccE, AccAl and AccA2.
In a sixth aspect, the present invention provides a method of modifying a strain of a Streptomyces species to increase ACCase and/or PCCase activity, the method comprising modifying said strain to express, or to increase expression of, nucleic acid encoding one or more polypeptides selected from the group consisting of AccB, AccE, AccAl and AccA2.
In a seventh aspect, the present invention provides a modified strain of a Streptomyces species, produced or producible according to the method of the fifth or sixth aspect. Also provided are cells of said strain, methods of producing secondary metabolites comprising culturing said cells and extracting the secondary metabolite, which may be purified and/or formulated as a commercial product.
In an eighth aspect, the invention provides a method of increasing production of a secondary metabolite in cells of a Streptomyces species, the method comprising culturing said cells in the presence of exogenous malonate, preferably at a concentration of at least about 0.1%, more preferably at least about 0.2%, 0.4%, 0.5%, 0.75% or 1%, though higher concentrations may be used. 1% represents 1g per 100 ml of medium.
Detailed Description In relation to the fifth and sixth aspects, the modification preferably provides for increased expression of nucleic acid encoding more than one of AccB, AccE, AccAl and AccA2, more preferably at least AccB and AccE or at least AccB and either AccAl or AccA2, more preferably AccB, AccE and either AccAl or AccA2. Of AccAl and AccA2, AccA2 is preferred. Increased expression of nucleic acid encoding both AccAl and AccA2 (usually in combination with AccB and optionally AccE) is also contemplated.
The methods of the fifth and sixth aspects preferably include a step of transforming a Streptomyces ce~.l with a said nucleic acid under the control of a constitutive or inducible promoter, preferably a strong promoter. However, the expression of existing said nucleic acid could be increased, e.g. by placing them under the control of a stronger promoter sequence or sequences.
Exogenous said nucleic acid can replace existing said nucleic acid in the cell, or can be added without removing or functionally deleting existing said nucleic acid.
Acc polypeptides and acc genes In the definitions herein of the invention, and of the scope of protection (but not, except where the context requires otherwise, in the experimental sections), the term AccB is intended to include not only a polypeptide having the deduced amino acid sequence encoded by the nucleic acid sequence of Fig. 12 (though this is a preferred embodiment), but also a polypeptide which is a variant (e.g. an allelic or isoallelic variant) or a derivative of said polypeptide, having at least about 60% amino acid identity with said polypeptide, preferably at least about 65%, 70% or 75%, especially preferably (in view of the similarity of AccB as disclosed herein to another protein of unconfirmed function) at least about 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% identity. Such a variant or derivative may possess any one or more of the biological properties of the wild-type AccB protein, as disclosed herein, e.g. complex formation with AccAl, .AccA2 and/or AccE (or allosteric regulation by AccE), ACCase and/or PCCase activity when AccB is co-expressed with AccAl, AccA2 and/or AccE, or increased secondary metabolite production when AccB is overexpressed in Streptomyces species (preferably in conjuction with AccAl, AccA2 and/or AccE).
Similarly, the term AccE is intended to include not only a polypeptide having the deduced amino acid sequence encoded by the nucleic acid sequence of Fig. 13 (though this is a preferred embodiment), but also a polypeptide which is a variant (e.g. an allelic or isoallelic variant) or a derivative of said polypeptide, having at least about 40%
amino acid identity with said polypeptide, preferably at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99%
identity. Such a variant or derivative may possess any one or more of the biological properties of the wild-type AccE
protein, as demonstrated herein, e.g. complex formation with AccAl, AccA2 and/or AccB (or allosteric regulation of AccB), ACCase and/or PCCase activity when AccE is co-expressed with AccB, or increased secondary metabolite production when AccE is overexpressed in Streptomyces species (preferably in conjuction with AccB).
Similarly, the terms AccAl and AccA2 are intended to include not only the polypeptides having the amino acid sequences shown in Fig. 11 (though these are respective preferred embodiments), but also polypeptides which are variants (e.g. allelic or isoallelic variants) or are derivatives of said polypeptides, having at least about 75%
amino acid identity with said polypeptide, preferably at least about 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99%
identity. Such variants or derivatives may possess any one or more of the biological properties of the wild-type AccAl or AccA2 polypeptides, as disclosed herein, e.g. complex formation with AccB and/or AccE, ACCase and/or PCCase activity when AccAl or AccA2 is co-expressed with AccB
and/or AccE, or increased secondary metabolite production when AccB is overexpressed in Streptomyces species (preferably in conjuction with AccB and/or AccE).
A variant or a derivative of a given peptide may have one or more of internal deletions, internal insertions, terminal truncations, terminal additions, or substitutions of one or more amino acids, compared to the given peptide.
References to nucleic acid encoding AccAl, AccA2, AccB
and/or AccE (or to accAl, accA2, accB and/or accE genes) should be interpreted accordingly.
In relation to the first aspect, preferred nucleic acids comprise a nucleic acid sequence having at least about 50%, preferably at least about 60%, 70%, 80%, 85%, 90%, 95%, 98%
or 99% nucleic acid sequence identity with the accB nucleic acid sequence shown in Fig. 12. Other preferred nucleic acids comprise a nucleic acid sequence having at least about 40%, preferably at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% nucleic acid sequence identity with the accE nucleic acid sequence shown in Fig. 13.
Similarly, in relation to the second aspect, preferred nucleic acids comprise a nucleic acid sequence having at least about 50%, preferably at least about 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% nucleic acid sequence identity with the accAl or accA2 nucleic acid sequence shown in Fig.
11.
Secondary metabolites and Streptomyces species While the experimental disclosure herein relates to the production of Act (actinomycin) and Red (undecylprodigiosin) in S. coelicolor A3(2) (strain M145), it is thought that the teaching is applicable to other strains of Streptomyces in particular, it is thought that overexpession of all three Acc polypeptides (i.e. AccB, AccE and AccAl and/or AccA2) will lead to increased malonyl-CoA production in substantially any Streptomyces species or even in other actinomycetes or in fungi (which also produce polyketide compounds). Since malonyl-CoA is an essential metabolic substrate, it is thought that this will lead to greater yield of desired secondary metabolites (for which see page 1), e.g. polyketides (including antibiotic polyketidss) and fatty acids.
Preferred secondary metabolites are, however, antibiotics, especially Act and Red.
Preferred Streptomyces species are the closely related species S. coelicolor, S. violaceoruber, S. lividans and S.
parvulus, especially S. coelicolor. Strains of such species are commonly available, e.g. from the ATCC, for example under ATCC deposit numbers 12434 for S. parvulus and 19832 for S. violaceoruber. S. coelicolor A3(2) and S.
lividans 66 are available from the John Innes Culture Collection (Norwich, UK) under JICC deposit numbers 1147 and 1326, respectively. However, the invention is not limited to such particular strains.
Acetyl-CoA
In preferred embodiments, present invention further provides for the increased production in Streptomyces of acetyl-CoA, since it is thought that when ACCase activity is increased by the methods and means of the present invention, production of malonyl-CoA may become limited by the availability of the substrate acetyl-CoA. It is proposed that increased acetyl-CoA production could then lead to a further increased rate of malonyl-CoA production and hence secondary metabolite production. For example, oils or fatty acids could be used as the carbon ~urce (together with glucose); fatty acids are degraded by b-oxidation giving high levels of acetyl-CoA.
Sequence identity "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.
The work underlying the invention will now be described in detail, by way of example only, with reference to the accompanying figures.
Figures Fig. 1 Organization of the genomic region of S.
coelicolor M145 chromosome harbouring accB and accE
genes. A. Genetic and physical map of the 6.2 kb insert in pRM08. The secondary structure downstream accE represents a rho-independent transcriptional terminator. Fragments I and II were amplified by PCR
with the pair of oligos accBup-accBdown and accBEup-accBEdown respectively, uniquely labelled at the 5'-end (*) and used as probes in transcriptional analysis of the accBE operon. B. Physical map of the DNA
fragments cloned in pET22b(+) and used for the heterologous expression of accB and/or accE. Only the most relevant restriction sites are shown: B, BamHI;

Bc, BclI; E, EcoRI; K, KpnI; Nd, NdeI; N, NotI; S, SpHI .
Fig. 2 Attempted d:i.sruption of accB. A. Diagram showing the integration of pTR124 through one of the accBE
flanking regions and the resolution of the cointegrate by a second event of homologous recombination. The crossed out arrow indicates the impossibility of obtaining the reF>lacement of the wild-type accB by the HygR mutant allele. B. The integration of a second copy of the accB~; genes in the ~C31 att site of T'124 (to yield strain T149) allowed the replacement of the wild-type accB by a mutant allele containing the Hyg resistance cassette.
Fig. 3 Growth~phasF~ dependent expression and transcription start site of the accBE operon. A. S1 nuclease mappin:.~ of accB, actII-ORF4 and hrdB, using RNA isolated from a liquid time course of S.
coelicolor M145. Exp, Trans and Stat indicate the exponential, transition and stationary phase of growth, respective:Ly.B.rsEQ.ID.NO~s.:l8-2o) The nucleotide sequences of both strands from the accB promoter region are shown.
The arrow indicates the mcst likely transcription.
start point for the accBE promoter, as determined. by Sl nuclease mapp:i.ng. The potential -10 and -35 regions for the accBEp ar-a underlined. C. S1 nuclease mapping of the accB accE intergenic region using a 563 nt probe. FLP repre~~ents the full-length RNA-protected fragment that is ~.3 nt shorter than the probe.
Fig. 4 Growth-phase dependent expression of accA2 and accA2. S1 nuclease mapping of accA2 (A) and accAl. (B), using RNA isolated from a liquid time course of S.
coelicolor M145.
Fig. S Mapping of t:he accA2 and accAl transcription start point. A. H:i.gh resolution S1 nuclease mapping of the Send of the accA2 transcript. S1, RNA-protected products of the S1 nuclease protection assay. Lanes labelled A, C, G and T indi;~ate a dideoxy sequencing ladder using the same oligonucleotide that was used to make the S1 probe (accA2down). B. High resolution S1 nuclease mapping of the S' end of the accA1 transcript. S1, RNA-protected products of the S1 nuclease protect::ion assay. Lanes labelled T, G, C and A indicate a dideoxy sequencing ladder using the same oligonucleotide that was used to make the S1 probe (accAldown).C.(sEQ.I~.NGS.:21-23) Sequence of the accA2 and accAl upstream regions, indicating the most likely transcript;~on st::art points for the promoters of each of the accAl and accA2 genes (bent arrows). The potential -10 arid. -35 sequences for the accAl and accA2 promoters are underlined. The potential ribosomal bindi::ca sites (rbs) are highlighted with bold letters. The 16 nt direct repeats (DR) found upstream o~ the t:a~anscription start point of accAlpl are indicated with straight arrows.
Fig. 6 Construct.ioh. and analysis of the accBE
conditional mutant:. A. Diagram showing the integration of ~>IJ8600 in strain M86 and the expected organisation of t:3ne Campbell integration of pTR94 in M94. Restriction sites: B. BamHI; N, NotI; Nd, NdeI;
S, SacI; Sp, Sphl; Xb, XbaI. B. Hybridisation analysis of Southern blot. of SacI-digested DNAs from M145, M86 and M94. The prcbe was the internal NdeI-XbaI
fragment of accB shown in A (see Fig. 10).
Fig. 7 Expression of the acyl-CoA components in M86 and M94. A. SDS-PAGE of cell-free extracts of S.
coelicolor M86 and. M94 strains grown in YEME medium containing 10 ~g/m.l Am with or without the addition of S ~g/ml Th. B. A duplicate of t:he SDS-PAGE gel shown in A was subjected to Western blotting and stained for biotinylated prouteins by using alkaline phosphatase-streptavidin conjugate.
Fig. 8 Growth curves of M145, M86 and M94 strains. 108 spores of strain's M86 and M94 were inoculated in YEME
medium containing 10 ~g of Am or 10 ~tg/ml Am and 5 ~g/ml of Th'. 108 ,pores of M145 were inoculated in YEME. The growth was followed by measuring OD4sown.
Fig. 9 Morphologic:a.l and physi-ological differentiation of M86 and M94 i.n the presence of Th. Spores of M86 and M94 were spread in R2 or R~s medium containing 10~g/ml Am. A drop containing 1 ~g of Th was spotted in the centre of each plate. The picture shows the results obtained after the incubation of the plate at 30°C for 48h.
Flg. 10 (sEQ.ID.NO.:24) 'he sequence of the amplification product obtained from accB using primers TC16 and TC17. NdeI
(CATATG) and XbaI (TCTAGA) :sites introduced into the accB by the primers are shown in bold. The 1 kb NdeI-XbaI fragment was cloned into pIJ8600.
Fig. 11 A. (SEQ.ID.NOS.:25--26) Amino acid sequences and B. iSEQ.ID.N05.:27-2ii Nucleic acid sequences of accA1 and accA2.

Fig. 12 (SEQ.ID.NOS.:29 and 31) Nucleic acid sequence of accB.
Fig. 13 (SEQ.ID.NOS.:30 and 32) Nucleic acid sequence of accE.
Fig. 14 Plasmid map for the construction of an expression vector for accA, accB and accE.
Example 1: Cloning of accBE genes pccB of S. coelicolor (Rodriguez and Gramajo, 1999) was used as an heterologaus probe in Southern blot experiments.
When a BamHI digest af: S. coelicalor DNA was probed with pccB and washed under low stringent conditions, a second, low hybridising, band was readily detected (data not shown). The target sequence was cloned from a size-enriched library as a 2.5 kb BamHl fragment and sequenced as described in Experimental Procedures (below). The sequence revealed the presence of an in'omplete ORF with high homology to pccB. The complete gene was finally cloned as a 6 kb SstI fragment yielding pR~I0~3 (:Fig. 1). Sequencing and analysis of this DNA 1_z:agment revealed the presence of an ORF that exhibited enca.-tc-end similarity with a putative decarboxylase (though the real functicn is unknown) of S.
cyanogenous (Westri~:t,. et al . , 1999) , with the S.
coelicolor PccB (Rodriguez and Gramajc, 1999) and with the (3-subunit (PccB! of tree Sac. erythraea PCCase (Donadio, et al., 1996). The leve~~~~r of identity were 76%, 57% and 56%, respectively. The gene encoding this new putative carboxyl transferase was callec_~ accB.
Surprisingly, the sequence also revealed the presence of a small ORF, designated accE, whose start codon is only 17 by downstream of the termination codon of acc8. A 17 nt inverted repeat, which could function as a factor-independent bidirectional transcriptional terminator (reviewed in Lewin, 1994), separates accE from three convergent ORFs with homology to putative proteins of M.
tuberculosis with unknown functions. The putative AccE
polypeptide has a deduced molecular mass of 7.07 kDa and no significant homology to this polypeptide was found in a search of sequences deposited in the GenBank database.
Upstream of accB there is an ORF highly homologous to several known hialuronidases.
Example 2: accB is essential for S. coelicolor viability An accB mutant was constructed by gene replacement (Fig.
2A). A Hyg-resistant cassette was cloned in the unique BamHI site present in the coding sequence of accB, contained in pTR80. After an intermediate construction in pIJ2925, a BglII fragment containing the mutated allele was finally cloned in the conjugative vector pSETl5l. The resulting plasmid, pTR124, was cloned into the E. coli donor strain ET12567/pUZ8002 and transferred by conjugation into M145. Exconjugants were selected for ThR HygR for a simple crossover event. One of the exconjugants, named T124, was taken through four rounds of non-selective growth (SFM Hyg) to promote homologous recombination for the second crossover. Spores were plated to give single colonies and several thousands screened for Th sensitivity (which would have reflected successful gene replacement), but no Ths isolates were obtained. This result suggested that accB is essential for S. coelicolor viability.

The inventors proposed, however, that if a second copy of accB were present in the chromosome of T124, a second crossover event (leading to the replacement of the wild type gene by the HygR mutant allele) would then be allowed.
To confirm this hypothesis, pTR149, which contains a copy of the accBE genes under its own promoter (see Experimental procedures, Fig. 2B), was first integrated in the ~C31 attB
site of T124. (The introduction of a second copy of both genes into the chromosome was prompted by the probability of a polar effect on accE taking place after the gene replacement event and because AccE is important for the recovery of a fully active acyl-CoA carboxylase complex -see in vitro reconstitution experiments below). The resultant strain T149 (HygR, ThR, AmR) was passed through three rounds of sporulation on SFM Hyg Am and after the screening of approximately 500 colonies, 20 were found to be AmR HygR Ths. The final chromosomal organization of accB
in each of the strains constructed (T124, T149 and T149A), was analyzed by Southern blots using an internal fragment of accB as a probe.
Example 3: Heterologous expression of accB, accE and in vitro reconstitution of an acyl-CoA carboxylase complex.
Since accB proved to be essential for S. coelicolor viability, we could not clearly evaluate in vivo the physiological function of this gene product.
In order to study if AccB and AccE were components of an acyl-CoA carboxylase complex, we attempted in vitro reconstitution of the enzyme activity by mixing E. coli cell-free extracts containing the AccB and AccE with cell-free extracts containing the biotinylated sub-units AccAl or AccA2. E. coli does not contain an ACCase enzyme, so ACCase activity cannot be assayed directly by carboxylation of acetyl-CoA (Polakis et al., 1972); therefore, the acyl-CoA carboxylase activity measured in these crude extracts exclusively represents the activity of the heterologous complexes reconstituted in vitro.
Heterologous expression of accB and accE was att~npted by introducing a NdeI site at the ATG start codon of accB;
after an intermediate construction (see Experimental procedures), accBE was cloned as a NdeI-SacI fragment into pET22(b), yielding pTR88 (Fig. 1). Transformation of E.
coli BL21(DE3) with this plasmid yielded strain RG8 (Table 1). Crude extracts of RG8, prepared from IPTG-induced cultures, showed a clear over-expression of a 64 kDa protein in a 15 s SDS-PAGE, corresponding to AccB; by contrast, AccE was not clearly visualised by Coomassie blue staining of the same gel (data not shown). In vitro reconstitution of an acyl-CoA carboxylase was then attempted my mixing crude extracts prepared from IPTG-induced cultures of RG8 with cell-free extracts of the E.
coli strains RG7, which overproduces the biotinylated protein AccAl. After incubation for 1 h at 4 °C, the mixture was assayed for ACCase and PCCase activity. As shown in Table 2 an enzyme complex showing high levels of both ACCase and PCCase activities was successfully reconstituted To study if cell-free extracts containing AccB but not AccE
were capable of reconstituting an active acyl-CoA
carboxylase complex when mixed with cell-free extracts containing AccAl, we constructed a new pET22(b) derivative that only expresses accB. For this we took advantage of the NotI site present approximately in the middle of the coding sequence of accE and cloned the NdeI-NotI fragment from pTR88 into the expression vector, yielding pTR90 (Fig. 1).
Cell-free extracts of RG9, obtained by transformation of BL21(DE3) with pTR90, showed high levels of soluble AccB
after IPTG induction. However, the acyl-CoA carboxylase complex reconstituted in vitro, after mixing cell-free extracts of RG9 (AccB) and RG7 (AccAl), showed much lower levels (approximately 10~) of ACCase and PCCase activities than the acyl-CoA carboxylase previously obtained by mixing RG8 with RG7 cell-free extracts (Table 2). Since the levels of AccB in cell-free extracts of RG8 and RG9 were essentially the same, we inferred from these experiments that AccE was necessary in order to obtain a fully active acyl-CoA carboxylase complex.
To confirm that the absence of AccE was the responsible of the lower acyl-CoA carboxylase activities, we studied the effect that the addition of cell-free extract containing AccE, had on the crude extracts containing AccB and AccAl proteins. For this we first constructed strain RG10 (BL21(DE3) containing pTR107) that expresses high levels of soluble AccE (data not shown).
When cell-free extracts of RG10 where mixed with those of RG9 (AccB) and RG7 (AccAl) and incubated for 1h on ice, the levels of enzyme activity where at least five times higher than in the control experiment, without the addition of AccE (Table 2). Although the results presented in this section clearly show that AccE is a functional part of the acyl-CoA carboxylase, enzyme kinetics studies with purified components will be necessary to understand more precisely the role of this protein in the enzyme complex activity.

Similar results were obtained in all the reconstitution experiments mentioned above when AccAl was replaced by AccA2 as the biotinylated component of the acyl-CoA
carboxylase, indicating that either AccAl or AccA2 can be efficiently used as the a-subunit of this enzyme complex.
Example 4: Transcriptional analysis of accBF~,-accA1 and accA2 At least four combinations that resulted in active carboxylase complexes have been reconstituted by mixing the ~-subunits PccB (Rodriguez and Gramajo, 1999) or AccB (this work) with either of the two almost identical a-subunits, AccAl or AccA2. In any of these complexes the carboxyl transferase subunit seems to dictate the substrate specificity; thus, PccB seems to recognize only propionyl-CoA, while AccB has a broader substrate specificity, which allows the enzyme to recognize either acetyl- or propionyl-CoA. Moreover, a third.complex with PCCase activity has also being described in S. coelicolor (Bramwell, et al., 1996). These findings show a remarkable overlapping of gene function in Streptomyces species. We followed two different approaches to gain more information on this; one was the generation of mutants and the second the study of the mRNA
levels of some of these four genes throughout the different growth stages by using S1 nuclease protection.
S. coelicolor A3(2) strain M145 was grown in SMM medium and RNA extracted at exponential, transition and stationary phase. S1 nuclease protection of accB was performed by using a 483 by PCR product, uniquely labelled at the 5'end of the downstream oligo. Transcription of accB occurs primarily during active growth (exponential and transition phases), while its level of expression decayed significantly after entering into stationary phase (Fig 3A). The transcripts of the major essential sigma factor hrdB and of the pathway-specific activator gene for acitnorhodin biosynthesis, actII-ORF4, were also studied as positive controls for the RNAs used in these experiments.
As expected from previous results, hrdB was expressed constantly throughout growth (Buttner, M.J., 1990), while actII-ORF4 had a peak of expression during transition phase that shut off in stationary phase (Gramajo, et al., 1993).
The RNA-protected fragments found for accB corresponded to a transcription start site 1 by upstream, or in the adenine, of the most likely translation start site of accB.
Upstream of the transcription initiation site we found a putative -10 and -35 promoter regions with a high consensus sequences of promoters recognised by the vegetative okras (Strohl, 1991) (Fig. 3B).
In order to find out if accB and accE were co-transcribed as a unique bi-cistronic mRNA, a new 563 by probe was obtained by PCR. For this we used a 5'oligo corresponding to a sequence within the coding region of accB and a 3'oligo corresponding to a sequence within accE. The full-length RNA-protected fragment was easily differentiated from the probe-probe re-annealing due to the addition of a 13 nt tail to the 5'oligonucleotide (Experimental Procedures). The results obtained in this experiment clearly showed that accB and accE were part of the same transcript, confirming that these two genes form a single-copy operon (Fig. 3C). Moreover, the expression of accBE
during the different growth phases as detected with this new probe followed the same profile as the expression observed with the probe used for accB.

The levels of accA2 and accA1 mRNA present throughout growth were also studied by S1 protection experiments (Fig.
4). The probe used for accA2 was a 766 by DNA fragment generated by PCR and uniquely labelled on the 5'end of the oligo corresponding to the sequence within accA2. This experiment showed the existence of three mRNA-protected fragments. The growth phase-dependent expression of two of them, accA2p1 and accA2p2, resemble very much that of the accBE operon. Thus, a constant and high level of expression occurs during exponential and transition phase (TP), while the transcription shuts down when the cultures reach.
stationary phase (Fig. 4A).
Considering that the nucleotide sequences of accAl and accA2 are identical from the first two nucleotides upstream of the most probable GTG translation start sites down to the end of the probe (Rodriguez and Gramajo, 1999), it is important to note that a fragment of 185 by of the accA2 probe could also be protected by the accA1 mRNA. Since the lowest RNA-protected fragment observed in Fig. 4A shows a different pattern of expression with respect to accA2p1 and p2, and considering that the size of the band corresponds to a 185 by fragment, we believe that this band might represent the level of expression of accA1 (although we cannot rule out the existence of a third promoter for accA2, regulated in a different manner).
S1 nuclease protection of accAl mRNA was performed by using a 563 by PCR product, uniquely labelled at the 5'end of the downstream oligo, corresponding to a sequence within accAl.
As shown in Fig. 4B, the expression of this gene occurs from at least three different putative promoters, and all of them showed a clear burst of expression during the first hours of the TP, which rapidly shut down during late TP.

This pattern of transcription resembled very much the one observed for the third RNA-protected band found for accA2.
The transcription starts sites for the accA2p1 and p2 were mapped by high resolution S1 mapping (Fig. 5A and B). The transcription start points and the putative -10 and -35 promoter regions of these two promoters are shown in Fig.
5C. A certain degree of homology was found between the -10 consensus sequence of accA2p1 and p2 and the promoters recognised by the vegetative ahras (Strohl, 1992). High resolution S1 mapping of accAl revealed that the transcription start point of the most abundant mRNA species starts 88 by upstream of the GTG initiation codon of AccAl and the putative -10 regions resemble, in some extent, the consensus sequences of promoters recognised by ahr~.
Interestingly, two direct repeat (DR) sequences of 16 bp, containing only two mismatches, were found flanking the putative -35 region of accAlpl and the transcription start point of accAlp2 (Fig. 5C). These DRs could represent DNA
binding sites recognised by a putative regulator. A third putative promoter, accAlp3, was also detected in longer exposures and the most probable nucleotide start sites are also indicated in Fig. 5C.
Example 5: accBE genes are essential in the presence of malonate The presence of MatC and MatB homologues in S. coelicolor suggested that this micro-organism was potentially capable of transporting malonate within the cell through the MatC
transporter, and then activating malonate to malonyl-CoA
with the putative malonyl-CoA synthetase MatB. To test whether S. coelicolor was able to utilize malonate as a sole carbon and energy source, we grew S. coelicolor in a modified SMM medium with no casamino-acids and containing 0.4 % malonate instead of glucose as a sole carbon source.
In this medium S. coelicolor M145 was able to grow, indicating that MatC and MatB could be the proteins involved in the transport and activation of malonate to malonyl-CoA, and suggesting that a decarboxylase that could convert malonyl- into acetyl-CoA should also be present in this bacterium, to allow the use of malonate as a carbon and energy source.
This result encouraged us to test whether this route could also be an alternative pathway to provide malonyl-CoA to the cell. To prove this hypothesis we tried to obtain an acyl-CoA carboxylase minus mutant in the presence of malonate. For this we took spores of strain T124 and grew them in liquid MM containing 0.4 % of malonate instead of glucose. After 36 h of growth we sonicated the mycelia and spread them in SFM medium containing 0.4 % of malonate and incubated until sporulation. Spores were collected and treated in the same way one more time. Finally, spores harvested after the second round of sporulation were diluted out, inoculated in SFM malonate to give aprox. 500 colonies per plate and replica plated in SFM medium with or without Th. After analyzing approximately 5000 isolated colonies, no Ths were obtained. This result indicates that although malonate can be efficiently used as a sole carbon and energy source, the pathway involved in its catabolism can not fulfill the malonyl-CoA requirements of the cell.
Example 6: Construction of a strain with the accBE
operon under the control of a tipA promoter As shown above, the accBE operon, which encodes the carboxyl-transferase and a previously unidentified ~ sub-unit of an acyl-CoA carboxylase, is essential for the viability of S. coelicolor A3(2). In order to regulate the expression of this operon and study its effect on the physiology of this microorganism, we constructed a conditional mutant strain where the expression of the accBE
operon was under the control of the thiostrepton-inducible tipA promoter (Murakami, et al., 1989).
A 947 by fragment containing a modified 5'end of the accB
gene was cloned under the tipA promoter in pIJ8600 (Sun et al (1999) supra) to yield pTR93. After removal of the ~C31 integration components (att and int) present in pTR93 we obtained pTR94, which was transformed into the E. coli strain ET12567/pUZ8002 (MacNeil et al (1992)/Paget et al (1999)). Conjugation of pTR94 into the S. coelicolor strain M145 gave several exconjugants ThR. One of these exconjugants, designated M94, was purified in SFM medium for further analysis. Integration of pTR94 could only take place by Campbell recombination through the accBE
homologous sequences, and this event should leave a complete copy of the accBE operon under the tipA promoter (Fig. 6A). To confirm that this event had occurred in M94, we performed Southern blot experiments of DNA samples prepared from strains M145, M94 and M86. The last strain (M86) was obtained by integration of pIJ8600 in the ~C31 att site of the chromosome and used as the best isogenic control for M94 (Fig. 6A). As shown in Fig. 6B, a Sacl digested DNA from M145 and M86 lights up a unique hybridisation band of 5.94 kb that contains the accBE
operon. DNA from M94, instead, lights up two hybridising bands corresponding to the expected sizes for the integration of pTR94 in the accBE operon (Fig. 6A and B).
Example 7: Acyl-CoA carboxylase levels in M94 and M86 Cultures of the conditional accBE mutant M94 grew normally in YEME medium containing 5 ~g of Th. Interestingly, in the absence of the antibiotic, the cultures were still able to grow, although at much lower rate. This experiment re-confirms the leakiness of the tipA promoter (M. J. Bibb, personal communication). In order to determine the levels of the acyl-CoA carboxylase in conditions of induction or non-induction we carried out the following protocol. YEME
medium containing 10 ~g of Am was inoculated with spores of M94 (or M86) to give and initial OD4so= 0.1. Cultures were grown for 12 h at 30 °C and after that time 5 ~g of Th was added to a half of each culture, keeping the other half as a control. Both flasks were then incubated for additional 24 h at 30 °C. The harvested mycelia were disrupted by sonication and cell debris removed by centrifugation. Cell-free extracts were finally analysed by SDS-PAGE and used for enzyme assays. Fig. 7A shows a 60 kDa protein that is only induced in cultures of M94 grown in the presence of Th; the size of this protein corresponded to the molecular mass of AccB. We were not able to detect an inducible band corresponding to AccE. The levels of the biotinylated components (AccAl or AccA2) of the acyl-CoA carboxylase, in each of the cell-free extracts, were analysed by a modified Western Blotting procedure (Fig. 7B). As shown in this figure the levels of AccAl and/or AccA2 were not modified by presence of Th. However, cell free-extracts of M94 do contain a slightly higher amount of the 65 kDa protein compared to M86.
ACCase and PCCase activities were assayed in cell-free extracts of M94 and M86. The levels of both enzyme activities were similar in cell-free extracts prepared from cultures of M86 grown in the presence or in the absence of Th (Table 3). Cell-free extracts prepared from induced cultures of M94 show instead a remarkable increase in both ACCase (11.5 fold) and PCCase (3.5 fold) activities, compared with the levels found in non-induced cultures of the same strain or in M86. Moreover, if the enzyme levels found in the wild type strain M145 (Rodriguez and Gramajo, 1999) are compared with those found for M94, the increase in ACCase and PCCase levels were still 4- and 2-fold, respectively (Table 3). These results indicate that by overproducing only two (~ and e) of the three sub-units that form the acyl-CoA carboxylase of S. coelicolor we can increase significantly the levels of this enzyme activity.
Example 8: Influence of the acyl-CoA carboxylase levels in the physiological properties of M94 Growth curves were determined for the conditional mutant M94 and for M86 by inoculating a spore suspension in YEME
medium supplemented with 10 ~g of Am, with or without the addition of 5 ~g of Th. For M145, YEME medium without the addition of any antibiotic was used. M94 supplemented with the inducer (Th) showed a growth rate during exponential phase very similar to M145, judged from the slope of the curves. However, the initiation of growth for M94 seems to occur sooner than in M145, reaching the stationary phase earlier than the wild type strain. When the cultures were not supplemented with Th, M94 grew considerably slower, reaching stationary phase several hours latter than in the presence of Th. Also, the final OD reached by M94 in the presence of Th and by M145 were very similar (OD4so= 3) after 60 h of growth. Cultures of M86 grew very slowly compared with M94 and M145, independently of the presence or not of Th. However, these cultures levelled off at the final OD reached by M145 and M94 after 50 h of growth.

Actinorhodin and undecylprodigiosin were also quantitated throughout growth. Table 4 shows that antibiotic production was only detected in cultures of M94 grown in the presence of 1 or 5 ug of Th. No antibiotic production was observed in cultures of M145 or M94 without Th, at least until after 60 h of growth. No antibiotic production was detected in M86.
To determine the effect of Th induction in M86 and M94, 1 ~g of the antibiotic was spotted to a confluent lawn of these strains in R2 and R5 medium supplemented with 10 ~g of Am. A striking stimulatory effect in both sporulation and antibiotic production was observed in M94 after 48 h.
No stimulation of growth or antibiotic production was observed in M86.
Example 9: Co-expression of accA, accB and accE in S.
coelicolor The NdeI-XbaI fragment of pTR154 (Fig. 14) is introduced into pIJ8600 and then transformed into S. coelicolor M145 (Fig. 14). Transformants are selected with apramycin and thiostrepton. Overexpression of the three components accA2, accB and accE results in increased ACCase activity and antibiotic production compared to the wild type M145 strain.

Discussion The use of pccB (Rodriguez and Gramajo, 1999) as an heterologous probe, allowed the successful isolation of a chromosomal DNA fragment containing accB, a gene encoding for a putative new carboxyl transferase of S. coelicolor.
This predicted function was based on the high percentage of identity that AccB showed not only to the S. coelicolor PccB, but to several others biochemical and/or genetically characterized carboxyl transferases reported for actinomycetes, such as the PccB of Sac. erythraea (Donadio, et al., 1996) and to a less extent to the AccD5 of M.
tuberculosis (Cole, et al., 1998) and PccB of M. leprae (Doukhan, 1995). An interesting finding from the analysis of the cloned sequence was the presence of a very small ORF, named accE, immediately downstream of accB.
The successful expression of accB, accE and the BC-BCCP-(biotin carboxylase- and biotin carboxylase carrier protein-)encoding genes accAl and accA2 in E. coli allowed in vitro studies to be performed in order to understand the role of the corresponding encoded proteins as components of a previously uncharacterized acyl-CoA carboxylase. The reconstitution, by mixing cell-free extracts of E. coli containing AccB and AccAl (or AccA2), of an active enzyme with the ability to carboxylate either acetyl- or propionyl-CoA clearly established that AccB was the carboxyl transferase component of an acyl-CoA carboxylase complex. Interestingly, the small polypeptide, AccE, also showed to play an important role in the reconstitution of a fully active enzyme complex (Table 2). It remains to be elucidated whether this protein plays a role as an allosteric regulator of the enzyme or whether it is a structural component of the complex. Thus, our results represent the first characterization, at both the genetic and biochemical levels, of a prokaryotic acyl-CoA
carboxylase.
All the acyl-CoA carboxylases studied so far contain the three functional domains in two individual polypeptides (for a review see Brownsey et al., 1997 ), and none of the purified complexes have shown the presence of a small component equivalent to AccE. Therefore, this might be a distinctive feature for Streptomyces sp. In addition, no AccE homologues have been found in any of the bacteria genomes sequenced so far, an observation that could also support this hypothesis.
Malonyl-CoA is an essential component of all living organisms, since it is the main elongation unit for fatty acid biosynthesis (Brownsey et al., 1997). This primary metabolite is synthesised in most species through the carboxylation of acetyl-CoA by an ACCase (Bloch and Vance, 1977). If this was also the case for S. coelicolor and, if AccB was the component of an essential acyl-CoA
carboxylase, mutation of this gene should be lethal for the micro-organism. Replacement of the wild-type accB for the HygR mutant allele prove to be unsuccessful, and it only occurred when a second copy of the accBE genes was present in the chromosome (Fig. 2B).
These experiments clearly indicated that at least accB was essential for S. coelicolor viability. The fact that both AccA2 (Rodriguez and Gramajo, 1999) and AccB have proved to be essential, along with the fact that acyl-CoA carboxylase reconstituted in vitro with these two sub-units has the ability to recognise either acetyl- or propionyl-CoA as substrates, strongly suggests that AccA2 and AccB are the a and ~ components of an essential acyl-CoA carboxylase, whose main physiological role should be the biosynthesis of malonyl-CoA. The transcriptional levels of accB and accA2 throughout growth (Fig.3A and 4A) also support this interpretation, since both genes are principally transcribed during exponential and transition phase.
Moreover, ACCase and PCCase activities also showed the highest and constant levels of activities during exponential and transition phase while in stationary phase the activities were low but readily measurable.
In S. coelicolor, besides the obvious need for malonyl-CoA
biosynthesis during vegetative growth, there is also a requirement for this metabolite during transition and stationary phase, since at least two secondary metabolites (undecylprodigiosin and actinorhodin) are synthesised during these growth-phases and they both require malonyl-CoA for their biosynthesis. Hence, if the ACCase is the only enzyme that synthesises malonyl-CoA in this bacterium, its presence will be also required during the idiophase.
According to the proposed composition of this enzyme complex and based on the transcriptional studies, we propose that the low level of expression of accA2 and accBE
during stationary phase is sufficient to produce enough of the a and ~ components for an active acyl-CoA carboxylase.
From the observation that accA1 mRNA peaks during transition phase, we propose that enough AccAl might be present in the cytoplasm to compete with AccA2 as the main a sub-unit of this enzyme complex in the stationary phase.
However, no difference in antibiotic production has been found between M145 and the isogenic accA1 mutant MA4 (Rodriguez and Gramajo, 1999).

We have clearly demonstrated the ability of S. coelicolor to efficiently utilize malonate as a sole carbon and energy source. A putative pathway for the utilization of this substrate could involve the R. trifolii MatC and MatB
homologues which are found in the genome of S. coelicolor.
The biochemical characterization of MatB in R. trifolii demonstrated that this protein is a malonyl-CoA
synthetase, which catalyzes the formation of malonyl-CoA
directly from malonate and CoA. MatC, instead, has not been characterized biochemically but computer analysis indicate that it is a transmembrane protein that could function as a dicarboxylate (malonate for example) carrier (An and Kim, 1998). If these enzymes were part of the pathway that allows S. coelicolor to utilize malonate as a sole carbon source, one could also presume that the malonyl-CoA
synthesized by MatB should fulfill the malonyl-CoA
requirements of Ithe micro-organism. However, we could not show that under these conditions the essential acyl-CoA
carboxylase becomes dispensable.
Interestingly, the addition of 0.4% malonate to SFM and glucose-MM media produced a clear stimulation of actinorhodin production (data not shown). From this we propose that higher levels of malonyl-CoA were probably available under this growth conditions. From this, and the observation that even the limited levels of the ACCase activity found during the stationary phase of growth of this bacterium are sufficient to allowed regular levels of antibiotic production, the inventors propose that increasing the expression of the ACCase components will probably lead to an improved production of antibiotics.
Experimental Procedures Bacterial strains, cultures and transformation conditions S. coelicolor A3 (2) strain M145 (SCP1- SCP2-) was manipulated as described by Hopwood et al. (1985). The strain was grown on various agar media - SFM (Rodriguez and Gramajo, 1999), R2 and R5 - or in 50 ml SMM or YEME liquid media (Hopwood et a1 (1985) supra). Escherichia coli strain DHSa (Hanahan 1983) was used for routine subcloning and was transformed according to Sambrook et a1. (1989).
Transformants were selected on media supplemented with the appropiate antibiotics . ampicillin (Ap) 100 ~tg/ml;
apramaycin (Am) 100 ~tg/ml; chloramphenicol (Cm) 25 ~g/ml or kanamycin (Km) 30 ~,g/ml. Strain BL21(DE3) is an E. coli B
strain [F- ompT (r$- m$-) (DE3) ] lysogenized with 1DE3, a prophage that expresses the T7 RNA polymerase downstream of the IPTG-inducible lacUVS promoter (Studier & Moffat, 1986). ET12567/pUZ8002 (MacNeil et al (1992)/Paget et al (1999)) was used for E. coli - S. coelicolor conjugation experiments (Bierman, 1992). For selection of Streptomyces transformants and exconjugants, media were overlayed with thiostrepton (Th) (300 ~g per plate), hygromycin (Hyg) (1 mg per plate) or apramycin (Am) (1 mg per plate). Strains and recombinant plasmids are listed in Table 1.
Growth conditions, protein expression and preparation of cell-free extracts S. coelicolor M145 was grown at 30°C in shake flasks in YEME medium for 24-48 h. When necessary, 10 mg Am ml-1 or 5 mg Th ml-1 were added to the medium. Mycelia were harvested by centrifugation at 5000 x g for 10 min at 4 °C, washed in 100 mM potassium phosphate buffer pH 8 containing 0.1 mM
DTT, 1 mM EDTA, 1 mM PMSF and 10% glycerol (buffer A) and resuspended in 1 ml of the same buffer. The cells were disrupted by sonic treatment (4 or 5 s bursts) using a VibraCell Ultrasonic Processor (Sonics & Materials, Inc.).

Cell debris was removed by centrifugation and the supernatant used as cell-free extract. For the expression of heterologous proteins, E. coli strain BL21(DE3) harbouring the appropriate plasmids were grown at 37°C in shake flasks in LB medium in the presence of 25 ~g Cm ml-1 or 100 ~g Ap ml-1 for plasmid maintenance. For the expression of biotinylated proteins, 10 ~M d-biotin was supplemented to the medium. Overnight cultures were diluted 1:10 in fresh medium and grown to A6oo 0.4-0.5 before the addition of IPTG to a final concentration of 0.1 mM.
Induction was allowed to proceed for 4 h. The cells were then harvested, washed and resuspended in 1 ml buffer A.
Cell-free extracts were prepared as described above.
Protein methods Cell-free extracts were analysed by denaturing (SDS)-PAGE
(Laemmli, 1970) using the Bio Rad mini-gel apparatus. The final acrylamide monomer concentration was 12% (w/v) for the separating gel and 5% for the stacking gel. Coomassie brilliant blue was used to stain protein bands. The biotinylated proteins were detected by a modification of the Western blotting procedure described by Nikolau et a1.
(1985). After electrophoretic separation, proteins were electro-blotted onto nitrocellulose membranes (Bio-Rad) and probed with alkaline phosphatase-streptavidin conjugate (Bio-Rad) diluted 1:10000. Protein content was determined by the method of Bradford (1976) with BSA as standard.
In vitro reconstitution and assay of the acyl-CoA
carboxylase complex In vitro reconstitution of the enzyme complex was carried out by mixing 100 ~g of each of the cell-free extracts shown in Table 2 in a final volume of 300 ~1. When AccE was not included in the incubation mix, 100 ~tg of BSA were added instead. The mixes were incubated for 1 h at 4 °C and 100 ~.g of each used for enzyme assay.
ACCase and PCCase activities in cell-free extracts were measured following the incorporation of H14C03- into acid non-volatile material (Huanaiti & Kolattukudy, 1982;
Bramwell et al., 1996). The reaction mixture contained 100 mM potassium phosphate pH 8.0, 300 ~g BSA, 3 mM ATP, 5 mM
MgCl2, 50 mM NaH14C03 [specific activity 200 ~tCi mmol-1 (740 kBq mmol-1)], 1 mM substrate (acetyl-CoA or propionyl-CoA) and 100 ~g cell-free protein extract in a total reaction volume of 100 ~1. The reaction was initiated by the addition of NaH14C03, allowed to proceed at 30 °C for 15 min and stopped with 200 ~.1 6 M HC1. The contents of the tubes were then evaporated to dryness at 95 °C. The residue was resuspended in 100 ~l water, 1 ml of Optiphase liquid scintillation (Wallac Oy) was added and 14C radioactivity determined in a Beckman scintillation liquid counter. Non-specific C02 fixation by crude extracts was assayed in the absence of substrate. One unit of enzyme activity catalysed the incorporation of 1 ~mol 14C into acid-stable products per min.
DNA manipulations Isolation of chromosomal and plasmid DNA, restriction enzyme digestion and agarose gel electrophoresis were carried out by conventional methods (Sambrook et al., 1989;
Hopwood et al., 1985). Southern analyses were performed by using P-labelled probes made by random oligonucleotide priming (Prime-a-gene kit; Promega).

Gene cloning and plasmid construction The synthetic oligonucleotides TC1, 5'-CAGAATTCAAGCAGCACGCCAA(3GGC AAG ( :3EQ . :ID . N0 . : 1 ) , and TC2 , 5 ' -CAGAATTCGATGCCGTCGTGCTCCTGGTC,(sEQ.ID.NO.:2) were used to amplify an internal fragment of the S. coelicolor pccB gene. The reaction mixture contained 10 mM Tris-HCl pH 8.3, 50 mM
KCl, 1 mM MgCl2 , 6% glycerol, 25 ~M of each of tile four dNTPs, 2.5 U Taq DNApo_Lymerase, 20 pmol of each primer and 50 ng of S. coelicolor chromosomal DNA in a final volume of 100 ~l. Samples were subjected to 30 cycles of denaturation (95°C, 30 s), annealing (65°C, 30 s) and extension (72°C, min) . A 1 kb PCR fragmE=nt was used as a 32P-labelled probe to screen a size-enriched library. A 2.7 kb BamHI fragment containing an incomplete accB gene was cloned in BamHI-cleaved pBluescript SK(+), yielding pTR62.
The synthetic oligonuc.Leotide TC16 (5' TATTCTAGACATATGACCGTTT'rGGATGAGG ( sEQ . ID. No . : 3 ) used to introduce an Ndel site at the translational start codon of the S. coelicolor accB gene ) and TC17 ( 5 ' -.~1CCTCTAGACAACGCTCGTGGACC , ( SEQ . ID . NO . : 4 ) used to introduce an Xbal site in the accB coding sequence) were used to amplify an internal fragment of S. coelicolor accB
gene, having the sequence shown in Fig. 10. The reaction mixture was the same as the one indicated above. Samples were subjected to 30 o:r 35 cycles of: denaturation (95°C, 30 s) , annealing (65°C, 30 s) and extension ('72°C, 1 min) . The 1 kb PCR product was digested with NdeI and XbaI (these sites were introduced in the 5~ ends of the oligos TC16 and TC17 and are shown in bold in Fig. 10) and cloned in XbaI-cleaved pBluescript SK.(+) in E. coli DHSa, yielding pTR82.
This plasmid was digested with BstEII and SacI, ligated with a BstEII-SacI fragment cleaved from pRM08 and introduced by transformation into E. coli DHSa, yielding pTR87.

An NdeI-XbaI fragment :From the plasmid pTR82 was cloned in NdeI-XbaI-cleaved pIJ8f;00 (Sun et al (1999)), yielding pTR93. In order to place the chromosomal copy of accBE
operon under the tipA promoter we removed from pTR93 a HindIII fragment containing the int gene and att of ~C31, yielding pTR94. Plasmid pTR94 was transformed into strain ET12567/pUZ8002 and transferred by conjugation to S.
coelicolor M145 (Hopwoc~d et al (1985)).
A NdeI-SacI fragment f:rom the plasmid pTR87 was cloned in NdeI-Sacl-cleaved pET22b(+) (Novagen) (pTR88), thus placing the accBE operon under the control of the powerful T7 promoter and ribosome-binding sequences. The synthetic 011.goriuCleotldeS NaCCE, 5'-TTATCTAGACATATGTCCCCTGCCGAC, (SEQ.ID.N0.:5) used to introduce an NcleI site at the translational start codon of the S. coelicc~lor accE gene, and CaccE, 5'-ATGAATTCTATGCATCGGGTCAGCGCC.~GCTG,(SEQ.ID.N0.:6) were used to amplify the accE gene of S. coelicc~lor. The reaction mixture was the same as the one indicated above. Samples were subjected to 35 cycles of denaturat:ion (95°C, 30 s), annealing (65°C, 30 s) and extension (72°C, 30 s). The PCR product was cloned using pGEM-T easy vector (Promega) in E. coli DHSa, yielding pTR106. A Ndel-EcoRI fragment from the plasmid pTR106 was cloned in NdeI-EcoRI-cleaved pET22(b) (Novagen) yielding the plasmid p'rR107, thus placing the accE gene under the control of the powerful T7 promoter and ribosome-binding sequences.
Plasmid pIJ8600 was digested with. BglII and EcoRI and the fragment containing or:i~' RK2, on pUClB, attP site, int ~C31 and aac (3) IV (AmR cassette) genes was ligated with a linker containing the following enzymes (Mike Butler personal comunication): BglII, AseI, EcoRI, BglII, NdeI, KpnI, XbaI, PstI, HindIII, BamHI, SstI, NotI and EcoRI, yielding pTR141. A 4.0 kb KpnI fragment containing the complete accBE operon from pRM08 was cloned into KpnI-cleaved pTR141, yieldi:c~g pTR149.
For an efficient over-expression in S. coelicolor of the three components of the acyl-CoA carboxylase complex of this micro-organism, we carried out the construction of pTR156 through the fol=Lowing steps. First we did a PCR.
amplification of the chromosomal accBE operon using the:
O Z 1g0 TC 16 ( 5 ' -TATTCTAGAC:ATATGACCGTTTTGGATGAGG 3 ' ) ( SEQ . ID . NO .
: 3 ) to introduce a NdeI site at the translation start codon c>f accB, and the oligo C--accE (5'ATG AAT TCT ATG CAT CGG GTC
AGC GCC AGC 3')(SEQ.ID.N0.:7) to introduce a Nsil restriction site at the 3' end of accE. The amp:Lified DNA, was then cloned into pGEM-T (Promega), to g_Lve pTR99. To introduce a NsiI site upstream of the RBS of accA2 we amplified this gene using the oligo N-accA2 (5' ATG AAT TCA TGC ATG AGG GAG CCT CAA
TCG 3' ) , (SEQ.ID.NO. :8> , for ~.I-;e 5' end and the oligo C-accA2 (5' AGA
TCT
AGA TCA GTC CTT GAT CTC GC 3')(SEQ.ID.N0.:9) containing a Xbal and a EcoRI site, for the 3' end of the gene. The amplified DNA
was cloned in pGEM-T to give pTR112. The NdeI-NsiI DNA
fragment from pTR99 and the NsiI-EcoRI fragment isolated from pTR112 were final=Ly cloned into pET22(b) (Stratagene), previously digested with NdeI and EcoRI, to yield pTR154.
In order to introduce these genes in S. coelicolor we sub-cloned the NdeI-XbaI fragment, containing aceBE and accA2, from pTR154 to pIJ8600 digested with the same enzymes to give pTRl56. See Fig. 14 for plasmid constructions.
Nucleotide sequencing The sequence of the SphI original fragment was performed from plasmids DNA constructed by subcloning ApaI DNA
fragments from pRM08 into pSKBluescri.be SK(+). Synthetic oligonucleotides were ~zsed to complete the sequence. The nucleotide sequence of the accBE region was determined by dideoxy sequencing (Sanger et al., 1977) using the Promega TaqTrack sequencing kit. and double-stranded DNA templates.
The complete sequence <.~f the 1C2 cosmid, that includes the SphI fragment harbouring accBE, is available from the S.
coelicolor genome sequE~ncing project.
S1 nuclease mapping For each S1 nuclease rE~action, 30 ~g of RNA were hybridized in NaTCA buffer (Murray, 1986); solid NaTCA (Aldrich) was dissolved to 3M in 50mM PIPES (pH 7.0), 5mM EDTA, to about 0.002 pmol (approximatf=_:ly 104 cpm) of the following probes.
For accA2 the synthet:Lc: oligonucleotide 5'-GCTTTGAGGACCTTGGCGATG (SEQ.I:D.NO.:10) (AccA2down), corresponding to the sequence within the coding region of accA2, was uniquely labelled at the 5' end of the oligonucleotide with [32P]-ATP using T4 polynucleotide kinase. The labelled oligo was then used in the PCR :reaction with the unlabelled OllgOnuCleOtlde (aCCA2~Llp) 5'-GAAGTAC.'AGGCCGAAGACCAC (SEQ.ID.N0.:11), which corresponds to a regioxi upstream of the accA2 promoter region, to generate a '766 by probe. For accAl the synthetic oligonucleotide (accAldown) 5'-GCGATTTCGCCACGATTGGCG, (SEQ.ID.NO.: 12), corresponding to the region within the coding region of accAl, was uniquely labelled with [3~P]-ATP using T4 polynucleotide kinase at the 5' end of the oligonucleotide.
The accAldown oligo was later used in the PCR reaction with the unlabelled oligonuc;leotide (accAl.up) 5'-CCGATATCAGCCCCTGATGAC,(SEQ.ID..N0.:13), which corresponds to a region upstream of the accAl promoter to generate a 563 by probe.
For accB the synthetic oligonucleotide (accBdown) 5'-CGTCAGCTTGCCCTTGGCGTG,(SEQ.ID.N0.:14), corresponding to the region within the coding region of accB, was uniquely labelled with [32P]-ATP using T4 polynucleotide kinase at the 5' end of the oligonucleotide. accBdown waa, then used in the PCR
reaction with the unlabelled oligonucleotide (accBup) 5'-CTACGCTCCGGGTGAGCGAAC,(SEQ.ID.t~0.:15), which corresponds to a region upstream of the accB promoter, to generate a 483 by probe.
For accBE the synthetic oligonucleotide (accBEdown) 5'-GGAGGGCCGTGATGGCGGCGACTTCCTCGGG,(SEQ.ID.N0.:16), corresponding to the region within the coding region of accE was uniquely labelled with [32P]-ATP using T4 polynucleotide kinase at the 5' end of the oligonucleotide. The accBEdown oligo was then used in the PCR reaction with the unlabelled oligonucleotide (accBEup) 5'-GAGGAACTGGTACGCGCGGGCG (GTACAe~.GCAAGCT),(SEQ.ID.N0.:17), which corresponds to a region within the coding region of accB Cbracketed oligonucleotides are a tail added to the probe to differentiate probe re<~nnealing from fully protected DNA-RNA complexes), to genE~rate a 563 by probe. Subsequent steps were as described by Strauch et al. (1991).
Determination oflactinorhodin 1 ml of whole broth wars mixed with 0.5 ml of 3N KOH to give a final concentration of 1N KOH. The solutions were mixed vigorously and centrifuge at 4000 x g for 5 minutes. The supernatant was collected and measured at A64onm.
Actinorhodin concentration was calculated using the molar extinction coefficient (in 1 N KOH) at 640 nm of 25.320 (Bystrykh et al., 1996?.
Determination of undecylprodigiosin This was carried out a~~cording to the procedure of Hobbs et al. (1990) .

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All the above references are hereby incorporated by reference in their entirety, individually and for all purposes.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Plant Bioscience Limited (B) STREET: Norwich Research Park, Colney Lane (C) CITY: Norwich (D) STATE: Norfolk (E) COUNTRY: UK
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(ii) TITLE OF INVENTION: Ant:.ibiotic Production (II) (iii) NUMBER OF SEQUENCES: 32 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Bereskin & Parr (B) STREET: Suite 4000, Scotia P7.aza, Box 401, 40 King Street West (C) CITY: Toronto (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP).- M5H 3Y2 (v) COMPUTER READABLE FORM:
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(C) SOFTWARE: Patent:In Release #1.0, Version #1.25 (EPO) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,322,105 (B) FILING DATE: 23-OC:T-;?000 (viii) PATENT AGENT INFORMATION:
(A) NAME: David W. R. Langton (B) REGISTRATION N0: :?800 (C) REFERENCE NUMBER: 420-360 (2) INFORMATION FOR SEQ ID N0: :L:
( i ) SEQUENCE CHARACTERISTIc_:S
(A) LENGTH: 29 base p<zi.rs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ :CD N0: 2:
(i) SEQUENCE CHARACTERISTI~::S:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic a~~id (C) STRANDEDNESS: single (D) TOPOLOGY: .Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TATTCTAGAC ATATGACCGT TTT(3GATGAG C; 31 (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:

(2) INFORMATION FOR SEQ ID NO: 'i:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: si.nc;~le (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
TTATCTAGAC ATATGTCCCC TGC:CGAC 27 (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTIC'.S:
(A) LENGTH: 32 base p<~irs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ATGAATTCTA TGCATCGGGT CAGCGCCi~GC TG 32 (2) INFORMATION FOR SEQ :CD NO: '7:
( i ) SEQUENCE CHARACTERIS'PIC:>
(A) LENGTH: 30 base pairs (B) TYPE: nucl~=_ic acid (C) STRANDEDNESS: single (D) TOPOLOGY: .Linea:r (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID N0: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: :>EQ ID N0: 8:
ATGAATTCAT GCATGAGGGA GCC'PCAATCC.; 30 (2) INFORMATION FOR SEQ ID N0: ~!:
(i) SEQUENCE CHARACTERISTIC'S:
(A) LENGTH: 26 base p<xirs (B) TYPE: nucleic acid (C) STRANDEDNESS: sinc;~le (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: :>EQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: :LO:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic aci<L
(C) STRANDEDNESS: sinc;~le (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID N0: 'L1.:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base p<~irs (B) TYPE: nucleic acid (C) STRANDEDNESS: s:inc~le (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

(2) INFORMATION FOR SEQ ID NO: 12:
( i ) SEQUENCE CHARACTERIS'rI~::
(A) LENGTH: 21 base pairs (B) TYPE: nucleic aci3 (C) STRANDEDNESS: single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

(2) INFORMATION FOR SEQ ID NO: 13 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: ::EQ ID NO: 13:

(2) INFORMATION FOR SEQ ID N0: 1.4:
(i) SEQUENCE CHARACTERISTIC:'S:
(A) LENGTH: 21 base pair's (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: :3EQ ID N0: 14:

(2) INFORMATION FOR SEQ ID NO: :1.5:
(i) SEQUENCE CHARACTERISTI(:~S:
(A) LENGTH: 21 base pe~irs (B) TYPE: nuclE:ic ac:ic:i (C) STRANDEDNESS: si_n~xle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 15:

(2) INFORMATION FOR SEQ ID N0: 16:
( i ) SEQUENCE CHARACTERIS'CI~S
(A) LENGTH: 31 base p<~irs (B) TYPE: nucleic acir.~
(C) STRANDEDNESS: s:ingl.e (D) TOPOLOGY: .Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGAGGGCCGT GATGGCGGCG ACTTCCT~~GG G 31 (2) INFORMATION FOR SEQ ID N0: 17:
(i) SEQUENCE CHARACTERISTIC:>:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sin gle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GAGGAACTGG TACGCGCGGG CGGTACAAE'~C AAGCT 35 (2) INFORMATION FOR SEQ ID NO: 18 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 120 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (xi) SEQUENCE DESCRIPTION: :~EQ ID N0: 18:
AAACGGGCCG GAGACTGTAC GGAGTCGACC; GCTCGCAATC CTTGCTCGGC TTCGTAGAGT 60 CGCTACATGA CCGTTTTGGA TGAGGCGCCC:~ C~GCGAGCCGA CGGACGCGCG CGGGCGGc3TG 120 (2) INFORMATION FOR SEQ ID NO: 1.9:
(i) SEQUENCE CHARACTERISTI<:'S:
(A) LENGTH: 120 base C:>airs (B) TYPE: nucleic acic:3 (C) STRANDEDNESS: douk:Re (D) TOPOLOGY: circul.axv (xi) SEQUENCE DESCRIPTION: >EQ ID NO: .L9:
CACCCGCCCG CGCGCGTCCG TCGGCTCGCce CGGCGCCTCA TCCAAAACGG TCATGTAGCG 60 ACTCTACGAA GCCGAGCAAG GATTGCGAGC_' CGTCGACTCC GTAC'AGTC'rC CGGCCCGTTT 120 (2 ) INFORMATION FOR SEQ 7:D N0: :?0 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: sin<!le (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 20:
Met Thr Val Leu Asp Glu Ala Pro Gly Glu Pro Thr Asp Ala Arg Gly Arg Val (2) INFORMATION FOR SEQ :CD N0: ',?1.:
( i ) SEQUENCE CHARACTERIS'PI~~S
(A) LENGTH: 240 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 21:

ACCGGCCGAT ACGTTCTGCG CATGACTGTT CCGAACAGGG CC>GCGTGAAT TCCAACCGTT 180 GGCCGTCGGC GAGCCCCGAT CAGTAATCGA. GTGAGTGAGG A(sAATCT'PCG TGCGCAAGGT 240 (2) INFORMATION FOR SEQ ID NO: 22 (i1 SEQUENCE CHARACTERISTIC."S:
(A) LENGTH: 240 base x>airs (B) TYPE: nucleic acid (C) STRANDEDNESS: dout:~le (D) TOPOLOGY: circular:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
CCACAAAGAA ACCGCGTGGC CCGCAGCACG CC'CTTACAGA GACCTTGACC ACACAGGAGG 60 GCTAGGGTTT CCCCCAGGAG TCCTGCGTA<.: CGCGGTACTA CAAGGGCT'PT CGGGGGTCGA 120 GCGAGCCTCG AATCACGCTC CGTGTGGGCh AGCTCACCAT TGGGGACGGG 'PCGAAGTGCC 180 GTGTCGGCAG TCCCTAAACT CGGCTTGTT~I' CAAGGAGGGA GCCTCAATCG TGCGCAAGGT 240 (2) INFORMATION FOR SEQ ID NO: 23 (i) SEQUENCE CHARACTERISTIC=S:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: sincFle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: ;vEQ ID N0: 23:
Val Arg Val Lys (2) INFORMATION FOR SEQ :
ID NO: '..24 (i) SEQUENCE CHARACTERISTIC~~:

(A) LENGTH: 968 base irs .pa (B) TYPE: nucleic aci:l (C) STRANDEDNESS: doubl e (D) TOPOLOGY: circu.Lar (xi) SEQUENCE DESCRIPTION:Q ID
SE: N0:
24:

TATTCTAGAC ATATGACCGT GCGCCGGGCGAGCCGAC'GGA CGCGCGCGGG60 TTTGGATGA~~

CGGGTGGCCG AGCTGCACGG GCGGC;GCTCGCC.'Gt3GCC'GAG 120 GATCCGTGCA TGAGAAGGCG

ACGGCGGCGC AGCACGCCAA ACGG(~ACGTGAGCGCATCGA GCTGCTCCTG180 GGc3CAAGCTG

GGTCGAGCAG

CTGGAGGCCA AGAAGCCGTA GTCATCACCGGC:TGGGGCAC GGTCGAGGGC300 CACCGACGGT

CCACGAC'rTC

CACGCCACGA AGATCCACAA ATGGC_CATCGCGGCCGC>TGC CCCGCTGGTG420 GA'I'CATGGAC

CGCCCGTATC

CACCAAGGCG

CGCGGCCTAC

GGCCCGGACCi GAGGAGATCACGCAGAACGG TCTGGGCGGC' ACcsCCGAGAC GTCCGGCGTG720 GC'CGAC:GTGC'.

TGCCACTTCGCCTACGACGA CGAGGAGACC:' AGGTCCGCTA CCTCCTCTCC780 TGCCTCGCCG

CTCCTCCCGCAGAACAACCG GGAGAACCCG AG'PCCTCCGA CCCCGTGGAC840 C.'CCCGCGCCG

CGCCGCTCGGACACCCTCCT CGACCTGGTC' GCAA.CCGCCC GTACGACATG900 CCGGCGGACG

(2) INFORMATION
FOR
SEQ
ID
NO:
25:

(i) SEQUENCE
CHARACTERISTIC:.'S:

(A) LENGTH: 590 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: si.nc:~le (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Val Arg Lys Val Leu Ile Ala Asn Arg Gly Glu Ile Ala Val Arg V<~1 Ala Arg Ala Cys Arg Asp Ala Gly Ile A:La Sex' Val Ala Val Tyr Ala Asp Pro Asp Arg Asp Ala Le~.i His Val Arg Ala Ala Asp Glu Ala Phe Ala Leu Gly Gly Asp Thr Pro Ala Thr Ser Tyr Leu Asp Ile Ala Lys 50 'i5 60 Val Leu Lys Ala Ala Arg (~lu Ser Gly Ala Asp Ala Ile His Pro Gly Tyr Gly Phe Leu Ser Glu Asn Ala Glu Phe A1<~ Gln Ala Val Leu Asp Ala Gly Leu Ile Trp Ile Gly Pro Pro Pro Hi;> Ala Ile Arg Asp Arg Gly Glu Lys Val Ala Ala Arcfi His 11e Ala Gln Arg Ala G.Ly Ala Pro Leu Val Ala Gly Thr Pro Asp Pro Val Ser Gly Ala Asp Glu Val Val Ala Phe Ala Lys Glu His Gl;y Leu Pro Ile A1<~ Ile Lys Ala Ala Phe Gly Gly Gly Gly Arg Gly :Leu Lys Val Ala Ary Thr Leu Glu Glu Val 165 1.70 175 Pro Glu Leu Tyr Asp Ser Ala Val Arg Glu Ala Va.L Ala Ala Phe Gly Arg Gly Glu Cys Phe Val ~Jlu Arg Tyr Leu Asp Lys Pro Arg His Val Glu Thr Gln Cys Leu Ala Asp Thr His Gly Asn Val Val Val Val Ser 210 215 2,20 Thr Arg Asp Cys Ser Leu Gln Arg Arg His Gln Lys Leu Val Glu Glu Ala Pro Ala Pro Phe Leu Ser Glu Ala Gln Thr G1u Gln Leu Tyr Ser Ser Ser Lys Ala Ile Leu L~y:-, Glu Ala Gly 'ryr Gly Gly Ala Gly Thr Val Glu Phe Leu Val Gly Met: Asp Gly Thr Ile Phe Phe Leu Glu Val Asn Thr Arg Leu Gln Val Glu His Pro Val T.hr Glu Glu Val Ala Gly Ile Asp Leu Val Arg Glu stet: Phe Arg Ile Ala Asp Gly Glu Glu Leu 305 310 315 3'<:0 Gly Tyr Asp Asp Pro Ala Lei, Arg Gly His Ser Phe Glu Phe Arg Ile Asn Gly Glu Asp Pro Gly Arcl Gly Phe Leu Pro Ala Pro Gly Thr Val Thr Leu Phe Asp Ala Pro Th::' Gl.y Pro Gly Val. Arg Leu A:~p Ala Gly Val Glu Ser Gly Ser Val Ile Gly Pro Ala Trp Asp Ser Leu Leu Aia 370 =.75 380 Lys Leu Ile Val Thr Gly Arg Thr Arg Ala Glu Ala Leu Gln Arg A.La Ala Arg Ala Leu Asp Glu Phf=_ Thr Val Glu Gly Met Ala Thr Ala I.Le Pro Phe His Arg Thr Val Va.L Arg Asp Pro Ala Phe Ala Pro G1u Leu Thr Gly Ser Thr Asp Pro Phe Thr Val His Thr Arg Trp Ile Glu Thr Glu Phe Val Asn Glu Ile Ly,s Pro Phe Thr Thr Pro Ala Asp Thr Glu Thr Asp Glu Glu Ser Gly Arg G:Lu Thr Val Va:L Val Glu Val Gly Gly Lys Arg Leu Glu Val Ser Leu Pro Ser Ser Leu Gly Met Ser Leu Ala Arg Thr Gly Leu Ala Ala Gl:y Ala Arg Pro Lys Arg Arg Ala Ala Lys Lys Ser Gly Pro Ala Ala Ser Gly Asp Thr Leu Ala Ser Pro Met Gln Gly Thr Ile Val Lys Ile Ala Val C~lu Glu C~ly Gln Glu Val Gln Glu Gly Asp Leu Ile Val Val Leu Glu Ala Met Lys Met Glu Gln Pro Leu Asn Ala His Arg Ser Gly 'rhr Ile Lys Gly Leu Thr Ala Glu Val Gly Ala Ser Leu Thr Ser Gly Ala Ala I.le Cys Glu I.Le Lys Asp (2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTIC:'S.
(A) LENGTH: 590 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: ;.E(~ ID NO: 26:
Val Arg Lys Val Leu Ile Ala ~~sn Arg Gly Glu Ile Ala Val Arg Val Ala Arg Ala Cys Arg Asp Ala Giy Ile A.La Ser Val Ala Val Tyr Ala Asp Pro Asp Arg Asp Ala Leu liis Val Arg Ala Ala Asp Glu Ala Phe Ala Leu Gly Gly Asp Thr Pro ~~:a Thr Ser Tyr Leu Asp Ile Ala Lys Val Leu Lys Ala Ala Arg Gl:~ 3er Gly Ala Asp Ala Ile His Pro Gly Tyr Gly Phe Leu Ser Glu Asn ,Ala Asp Phe Ala Gln Ala Val Leu Asp Ala Gly Leu Ile Trp Ile G1;~ Pr_o Pro Pro His Ala Ile Arg Asp Arg Gly Glu Lys Val Ala Ala Arg l-Lis I1e Ala Gln Arg Ala Gly Ala Pro Leu Val Ala Gly Thr Pro Asp :Pro Val Ser Gly A1<r Asp Glu Val Val 130 13':. 140 Ala Phe Ala Lys Glu His Gly Leu Pro Ile Ala Ile Lys Ala Ala Phe 145 150 1.55 160 Gly Gly Gly Gly Arg Gly Leu C~ys Val Ala Ard Thr Leu Glu Glu Val Pro Glu Leu Tyr Asp Ser Ala 'Val Arg Glu Ala Val Ala Ala Phe Gly 180 1.85 190 Arg Gly Glu Cys Phe Val Glnr.Arg Tyr Leu Asp Lys Pro Arg His Val Glu Thr Gln Cys Leu Ala Asp 'rhr His Gly Asn Val Val Val Val Ser Thr Arg Asp Cys Ser Leu Gln Arg Arg His Gln Lys Leu Val Glu Glu Ala Pro Ala Pro Phe Leu Ser :,1u Ala Gln Thr Glu Gln Leu Tyr Ser Ser Ser Lys Ala Ile Leu Lys Glu Ala Gly Tyr Va.1 Gly Ala Gly Thr Val Glu Phe Leu Val Gly Met Asp Gly Thr I:le Ser Phe Leu Glu Val 275 2.30 285 Asn Thr Arg Leu Gln Val Glu His Pro Val Thr Glu Glu Val Ala Gly 290 29'> 300 Ile Asp Leu Val Arg Glu Met Phe Arg Ile Ala Asp Gly Glu Glu Leu Gly Tyr Asp Asp Pro Ala Leu Arg Gly His Ser Phe Glu Phe Arg Ile Asn Gly Asp His Pro Gly Arg (31y Phe Leu Pro Ala Pro Gly Thr Val Thr Leu Phe Asp Ala Pro Thr Gly Pro Gly Val Arg Leu Asp Ala Gly 355 :3fi0 365 Val Glu Ser Gly Ser Val Ile tziy Pro Ala Trp Asp Ser Leu Leu Ala Lys Leu Ile Val Thr Gly Arc7 'Phr Arg Ala Glu Ala Leu Gln Arg Ala Ala Arg Ala Leu Asp Glu Phe 'Phr Val Glu Gly Met, Ala Thr Ala Ile Pro Phe His Arg Thr Val Val.:~lrg Asp Pro Ala Phe Ala Pro Glu Leu Thr Gly Ser Thr Asp Pro Phe 'Phr Val His Thr Arc) Trp Ile Glu Thr 435 ~140 445 Glu Phe Val Asn Glu Ile Lys 'Pro Phe Thr Thr Pro Ala Asp Thr Glu 450 45'. 46!) Thr Asp Glu Glu Ser Gly Arc)G1u Thr Val Val Val Glu Val Gly Gly Lys Arg Leu Glu Val Ser Lei:WPro Ser Ser Leu Gly Met Ser Leu Ala Arg Thr Gly Leu Ala Ala G1;~,~11a Arg Pro Lys Arg Arg Ala Ala Lys Lys Ser Gly Pro Ala Ala Ser Gly Asp Thr Leu Ala Ser Pro Met Gln 515 !p20 525 Gly Thr Ile Val Lys Ile Ala 'J<~1 Glu Glu Gly Gln Glu Val Gln Glu 530 53'i 54() Gly Asp Leu Ile Val Val Le~.i G.Lu Ala Met Lys Met= Glu Gln Pro Leu Asn Ala His Arg Ser Gly Thr I.Le Lys Gly Leu Thr Ala Glu Val Gly Ala Ser Leu Thr Ser Gly Alai .~la Ile Cys Glu Ile Lys Asp (2) INFORMATION FOR SEQ TD N0: 27:
(i) SEQUENCE CHARACTERISTIi'S':
(A) LENGTH: 1773 base p~sirs (B) TYPE: nucleic acid (C) STRANDEDNESS: doubl,=_ (D) TOPOLOGY: circular (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
GTGCGCAAGG TGCTCATCGC CAATCGTGGC'.GAAATCGCTG TCCC>CGTGGC CCGGGCCTGC 60 CGGGACGCCG GGATCGCGAG CGTGGCCGTt:'Pi~CGCGGATC CGGACCGGGA CGCGTTGCAC 120 GTCCGTGCCG CTGATGAGGC GTTCGCCCT(>Gt3TGGTGACA CCCt~CGCGAC CAGCTATCTG 180 TACGGATTCCTCTCGGAGAA TCCTGGAC;GC CGGCCTGATC300 CGCCGAGTTC.' GC;GCAGGCGG

TGGATCGGCCCGCCCCCGCACGCCATCCGC'(iACCGTGGCGAAAAGGTCGC CGCCCGCCAC360 ATCGCCCAGCGGGCCGGCGCCCCCCTGGTC.' CCGACCCCGT CTCCGGCGCG420 GCCGGCACCC

GACGAGGTCGTCGCCTTCGCCAAGGAGCAC GC=CCTGCCCATCGCCATCAA GGCCGCCTTC480 GGCGGCGGCGGGCGCGGCCTCAAGGTCGCC:'(:GCACCCTCGAAGAGGTC;CC GGAGCTGTAC540 GACTCCGCCGTCCGCGAGGCCGTGGCCGCC' 'f7.'CGGCCGCGGGGAGTGC;TT CGTCGAGCGC600 TACCTCGACAAGCCCCGCCACGTGGAGACC:'(:AGTGCCTGGCCGF~CACC.'CA 660 CGGCAACGTG

GTCGTCGTCTCCACCCGCGACTGCTCCCTC C:AGCGCCGCCACCAAAAGCT CGTCGAGGAG720 GCCCCCGCGCCCTTTCTCTCCGAGGCCCAC~~~C:GGAGCAGCTGTACTCATC CTCCAAGGCC;780 ATCCTGAAGGAGGCCGGCTACGGCGGCGCC:'(zC=CACCGTGGAGTTCCTC;GT CGGCATGGAC840 GGCACGATCTTCTTCCTGGAGGTCAACACC" (~CiCCTCCAGGTCGAGCAC;CC GGTCACCGAG900 GAAGTCGCCGGCATCGACTTGGTCCGCGAG i~TGTTCCGCATCGCCGACGG CGAGGAACTC960 GGTTACGACGACCCCGCCCTGCGCGGCCAC"CC:CTTCGAGTTCCG1CATCAA CGGCGAGGAC1020 CCCGGCCGCGGCTTCCTGCCCGCCCCCGGC' ACCGTCACCCTCTTCGACGC GCCCACCGGC1080 CCCGGCGTCCGCCTGGACGCCGGCGTCGAG ',CCCGGCTCCGTCATCGGC;CC CGCCTGGGAC1140 TCCCTCCTCGCCAAACTGATCGTCACCGGC:'(~GCACCCGCGCCGAGGCACT CCAGCGCGCG1200 GCCCGCGCCCTGGACGAGTTCACCGTCGAG t~GCATGGCCACCGC;CATC;CC CTTCCACCGC1260 ACGGTCGTCCGCGACCCGGCCTTCGCCCCC' GAACTCACCGGCTCCACGGA CCCCTTCACC1320 GTCCACACCCGGTGGATCGAGACGGAGTTC' GTCAACGAGATCAAGCCC;TT CACCACGCCC1380 GCCGACACCGAGACGGACGAGGAGTCGGG<:' TCGTCGTC:GA GGTCGGCGGC1440 (:GGGAGACGG

AAGCGCCTGGAAGTCTCCCTCCCCTCCAGC:' CCCi'GGCC;CG CACCGGCCTG1500 t:'l.'GGGCATGT

GCCGCCGGGGCCCGCCCCAAGCGCCGCGCG (3C;CAAGAAGTCCGC~CCCC:GC CGCCTCGGGC1560 GACACCCTCGCCTCCCCGATGCAGGGCACG ~~TCGTCAAGATCGCCGTCGA GGAAGGCCAG1620 GAAGTCCAGGAAGGCGACCTCATCGTCGTA CTCGAGGCGATGAAGATC~GA ACAGCCCCTC1680 AACGCCCACAGGTCCGGCACCATCAAGGGC' CTCACCGCCGAGGTCGGC;GC CTCCCTCACC1740 TCCGGCGCCGCCATCTGCGAGATCAAGGAC:' 1773 'PGA

(2) INFORMATION
FOR
SEQ
ID
NO:
:8:

(i) EQUENCE
S CHARACTERISTIC'S:

(A) LENGTH:1773 base pairs (B) TYPE:cleic aci<a nu (C) STRANDEDNESS:
douk>l~=_ (D) TOPOLOGY:
circular (xi) SEQUENCE DESCRIPTION: S'EQ ID NO: 28:
GTGCGCAAGG TGCTCATCGC CAATCGTGGt:; GAAATCGCTG TCCC>CGTGGC CCGGGCCTGC 60 CGGGACGCCG GGATCGCGAG CGTGGCCGTC''P~1CGCGGATC CGGACCGGGA CGCGTTGCAC 120 GTCCGTGCCG CTGATGAGGC GTTCGCCCT(.~ GGTGGTGACA CCCCCGCGAC CAGCTATCTG 180 GACATCGCCAAGGTCCTCAA CGGACGCC.'AT CCACCCCGGC240 AGCCGCGCGC:'(;~AGTCGGGCG

TACGGATTCCTCTCGGAGAACGCCGAGTTC." TCCTGGAC:GC CGGCCTGATC300 (3C:GCAGGCGG

TGGATCGGCCCGCCCCCGCACGCCATCCGC'(~ACCGTGGCGAAAAGGTC.'GC CGCCCGCCAC360 ATCGCCCAGCGGGCCGGCGCCCCCCTGGTC (3CCGGCACCCCCGACCCC:GT CTCCGGCGCG420 GACGAGGTCGTCGCCTTCGCCAAGGAGCAC' GGCCTGCCCATCGC:CATC'AA GGCC:GCCTTC480 GGCGGCGGCGGGCGCGGCCTCAAGGTCGC<:'(:GCACCCTCGAAGAGGTGCC GGAGCTGTAC:540 GACTCCGCCGTCCGCGAGGCCGTGGCCGCC' TTCGGCCGCGGGGAGTGC:TT CGTCGAGCGC600 TACCTCGACAAGCCCCGCCACGTGGAGACC: CAGTGCCTGGCCGACACC:CA CGGCAACGTG660 GTCGTCGTCTCCACCCGCGACTGCTCCCTC'(:AGCGCCGCCACCAAAAGCT CGTCGAGGAG720 GCCCCCGCGCCCTTTCTCTCCGAGGCCCACI AC:GGAGCAGCTGTACTCATC CTCCAAGGCC780 ATCCTGAAGGAGGCCGGCTACGGCGGCGCC'..'(~GCACCGTGGAGTTCCTC:GT CGGCATGGAC840 GGCACGATCTTCTTCCTGGAGGTCAACACC (:GCCTCCAGGTCGAGCAC:CC GGTCACCGAG900 GAAGTCGCCGGCATCGACTTGGTCCGCGAG i~TGTTCCGCATCGCCGACGG CGAGGAACTC960 GGTTACGACGACCCCGCCCTGCGCGGCCA(:''PCCTTCGAGTTCCC~CATCAA CGGCGAGGAC1020 CCCGGCCGCGGCTTCCTGCCCGCCCCCGGC:' TCTTCGACGC GCCCACCGGC1080 AC:CGTCACCC

CCCGGCGTCCGCCTGGACGCCGGCGTCGAC~ 'L'C:CGGCTCCGTCATCGGC:CC CGCCTGGGAC1140 TCCCTCCTCGCCAAACTGATCGTCACCGGC (:CiCACCCGCGCCGAGGCACT CCAGCGCGCG1200 GCCCGCGCCCTGGACGAGTTCACCGTCGAC~(3GCATGGCCACCGC:CATCCC CTTCCACCGC1260 ACGGTCGTCCGCGACCCGGCCTTCGCCCC<'(>AACTCACCGGCT(:CACGGA CCCCTTCACC:1320 GTCCACACCCGGTGGATCGAGACGGAGTTC:(sTCAACGAGATCAAGCCC:TT CACCACGCCC1380 GCCGACACCGAGACGGACGAGGAGTCGGGC:.' TCG'7.'CGTC:GA 1440 CGGGAGACGG GGTCGGCGGC

AAGCGCCTGGAAGTCTCCCTCCCCTCCAGC'c:TGGGCATGTCCC'TGGCC:CG CACCGGCCTG1500 GCCGCCGGGGCCCGCCCCAAGCGCCGCGCC:; CCGGCCCC:GC CGCCTCGGGC1560 GC:CAAGAAGT

GACACCCTCGCCTCCCCGATGCAGGGCACC; ATCGTCAAGATCGC:CGTC:GA GGAAGGCCAG1620 GAAGTCCAGGAAGGCGACCTCATCGTCGTI, CTCGAGGCGATGAAGATGGA ACAGCCCCTC1680 AACGCCCACAGGTCCGGCACCATCAAGGG(:: AGG7.'CGGC:GC 1740 t:TCACCGCCG CTCCCTCACC

TCCGGCGCCGCCATCTGCGAGATCAAGGA(:: 1773 'Pt:3A

(2) INFORMATION
FOR
SEQ
ID
N0:
29:

(i) EQUENCE
S CHARACTERISTIC'S:

(A) LENGTH:1584 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS:
double (D) TOPOLOGY:
circular (xi) SEQUENCE DESCRIPTION: ~E',t;~ ID N0: 29:
ATGACCGTTT TGGATGAGGC GCCGGGCGAC> CCGACGGACG CGCGCGGGCG GGTGGCCGAG 60 CTGCACGGGA TCCGTGCAGC GGCGCTCGCC: GGGCCGAGTG AGAAGGCGAC GGCGGCGCAG 120 CACGCCAAGGGCAAGCTGACGGCACGTGAG (;GCATCGAGCTGCTCCTGGA CCCCGGCTCC180 TTCCGCGAGGTCGAGCAGCTGCGCCGGCAC'(~GGGCGACCGGGTTCGGCCT GGAGGCCAAG240 AAGCCGTACACCGACGGTGTCATCACCGGC.' TCGAGGGCCG CACGGTCTTC300 'CGGGGCACGG

GTCTACGCCCACGACTTCCGGATCTTCGGC'(~GCGCGCTGC,GCGAGGCC'.CA CGCCACGAAG360 ATCCACAAGATCATGGACATGGCCATCGCG GC:CGGTGCCCCGCTGGTGTC GCTGAACGAC420 GGCGCCGGCGCCCGTATCCAGGAGGGCGTC.'i~GCGCGCTCGCCGC:GTAC.'GG 480 CGGCATCTTC

CAGCGCAACACCAAGGCGTCCGGCGTCATC'(~CGCAGATCAGCGTGATGCT CGGCCCCTGC540 GCGGGCGGCGCGGCCTACAGCCCCGCCCTC' ACCGACTTCGTCTTCATGGT CCGCGACACC600 TCGCAGATGTTCATCACGGGCCCGGACGTC' (~l'CAAGGCGGTCAC'.CGGC'.GA 660 GGAGATCACG

CAGAACGGTCTGGGCGGCGCCGACGTGCAC (s(:.CGAGACGTCCGC>CGTC~TG CCACTTCGCC720 TACGACGACGAGGAGACCTGCCTCGCCGAO (37.'CCGCTACCTCCTCTCCCT CCTCCCGCAG780 AACAACCGGGAGAACCCGCCCCGCGCCGAi'I'CCTCCGACCCCGTGGAC:CG CCGCTCGGAC840 ACCCTCCTCGACCTGGTCCCGGCGGACGG(:' ACGACATGAC CAAGGTCATC900 AACCGCCCGT

GAGGAACTCGTCGACGAGGGCGAGTACCTi:~(3AGGTCCACGAGCC~TTGGGC CCGCAACATC960 ATCTGCGCGCTGGCCCGTCTCGACGGGCGG GTCGTGGGCATCGTCGCC'.AA CCAGCCGCAG1020 GCCCTGGCCGGTGTCCTGGACATCGAGGCi3 'PC'.GGAGAAGGCGGC:CCGCTT CGTCCAGATG1080 TGCGACGCCTTCAACATCCCGATCATCACT CTTCTGGACGTACC'CGGCTT CCTGCCCGGC1140 GTCGACCAGGAGCACGGCGGGATCATCCG<' CACGGCGCCAAGCTGCTCTA CGCGTACTGC1200 AACGCGACCGTGCCCCGGATCTCGCTGATC' CTGCGCAAGGCGTACGGAGG TGCTTACATC1260 GTCATGGACAGCCAGTCCATCGGCGCCGAC'(:TCACCTACGCCTC~GCCGAC CAACGAGATC1320 GCCGTCATGGGCGCGGAAGGTGC'CGCGAAC.'(~TCATCTTCCGCCGGCAGAT CGCCGACGCC1380 GAGGACCCCGAGGCCATGCGGGCGCGCATC~ G7.'CAAGGAGTACAAGTCCGA GCTGATGCAC1440 CCCTACTACGCGGCCGAACGCGGTCTGGTC'(~ACGACGTCATCGACCCC:GC CGAAACCCGC1500 GAGGTGCTGATCACGTCCCTGGCGATGCTC.' ACGCCGACCT GCCCTCCCGC1560 CACACCAAGC

(2) INFORMATION
FOR
SEQ
ID
N0:
.?0:

(i) EQUENCE
S CHARACTERISTIC'S:

(A) LENGTH:198 base pairs (B) TYPE:cleic acid nu (C) STRANDEDNESS:
doux:>le (D) TOPOLOGY:
circular (xi) SEQUENCE DESCRIPTION: ~Et~ ID NO: 30:
ATGTCCCCTG CCGACATCCG CGTCGAGAA<> GGCCACGCCG AGCCCGAGGA AGTCGCCGCC 60 ATCACGGCCC TCCTCCTGGC CCGCGCCGC(:'GC:CCGCCCCG CCGAGATCGC GCCGACCCAC 120 GGCGGCGGCC GCGCCCGCGC CGGCTGGCGC' CGCCTGGAAC GCGAGCCGGG CTTCCGCGCC 180 (2) INFORMATION FOR SEQ ID NO: ~1:
(i) SEQUENCE CHARACTERISTI(:S:
(A) LENGTH: 527 amino ac:Lds (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: 3.inear (xi) SEQUENCE DESCRIPTION: SE~~ ID NO: 31:
Met Thr Val Leu Asp Glu Ala Pro Gly Glu Pro Thr Asp Ala Arg Gly Arg Val Ala Glu Leu His Gly I.Le Arg Ala Ala Ala Leu Ala Gly Pro Ser Glu Lys Ala Thr Ala Ala Gln His Ala Lys Gly Lys Leu Thr Ala Arg Glu Arg Ile Glu Leu Le~.x Leu Asp Pro Gly Ser Phe Arg Glu Val Glu Gln Leu Arg Arg His Arr3 .Ala Thr Gly Phe Gl.y Leu Glu Ala Lys Lys Pro Tyr Thr Asp Gly Va'~ I.Le Thr Gly Trp Gly Thr Val Glu Gly Arg Thr Val Phe Val Tyr Al<i:H:is Asp Phe Arch Ile Phe Gly Gly Ala Leu Gly Glu Ala His Ala Th:r Lys Ile His Lys Il.e Met Asp Met Ala Ile Ala Al.a Gly Ala Pro Le~.z Val Ser Leu Asn Asp Gly Ala Gly Ala Arg Ile Gln Glu Gly Val Sea- A=La Leu Ala Gly Tyr Gly Gly Ile Phe 145 150 15'> 160 Gln Arg Asn Thr Lys Ala Ser 31y Val Ile Pro Gln Ile Ser Val Met Leu Gly Pro Cys Ala Gly Gly Ala Ala Tyr Sex Pro Ala Leu Thr Asp Phe Val Phe Met Val Arg Asp 'rhr Ser Gln Met Phe Ile Thr Gly Pro Asp Val Val Lys Ala Val Thr G.Ly Glu Glu Ile Thr Gln Asn Gly Leu Gly Gly Ala Asp Val His Ala 31u Thr Ser Gly Va.L Cys His Phe Ala Tyr Asp Asp Glu Glu Thr Cys Leu Ala Glu Va1 Arg Tyr Leu Leu Ser Leu Leu Pro Gln Asn Asn Arg Glu Asn Pro Pro Arg Ala Glu Ser Ser Asp Pro Val Asp Arg Arg Se:r Asp Thr Leu Leu Asp Leu Val Pro Ala Asp Gly Asn Arg Pro Tyr Asp Met Thr Lys Va.L Ile Glu Glu Leu Val Asp Glu Gly Glu Tyr Leu Glu 'J<~l His Glu Arg Trp Ala Arg Asn Ile Ile Cys Ala Leu Ala Arg Le~.i.~sp Gly Arg Val Val Gly Ile Val Ala Asn Gln Pro Gln Ala Leu Ala G.Ly Val Leu Asp Ile Glu A1a Ser Glu Lys Ala Ala Arg Phe Val Gln Met Cys Asp Al<~ Phe Asn Ile Pro Ile Ile Thr Leu Leu Asp Val Pro ~3.Ly Phe Leu Pro Gly Val Asp Gln Glu His Gly Gly Ile Ile Arg Hi:~ Gly Ala Lys Leu Leu Tyr Ala Tyr Cys 385 390 39'_i 400 Asn Ala Thr Val Pro Arg Ile Ser Leu Ile Leu Arg Lys Ala Tyr Gly Gly Ala Tyr Ile Val Met Asp Ser Gln Ser Ile Gly Ala Asp Leu Thr Tyr Ala Trp Pro Thr Asn Glu I.Le Ala Val Met Gly Ala Glu Gly Ala Ala Asn Val Ile Phe Arg Arc) G.Ln Ile Ala Asp Ala Glu Asp Pro Glu Ala Met Arg Ala Arg Met Va.I. Lys Glu Tyr Ly:~ Ser Glu Leu Met His Pro Tyr Tyr Ala Ala Glu Arg G.Ly Leu Val Asp As,p Val Ile Asp Pro Ala Glu Thr Arg Glu Val Leu I.Le Thr Ser Leu Ala Met Leu His Thr Lys His Ala Asp Leu Pro Se:r.Arg Lys His Gly Asn Pro Pro Gln (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino <acids (B) TYPE: amino acid (C) STRANDEDNESS: sin:)1.2 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: :>E?~ ID N0: 32:
Met Ser Pro Ala Asp Ile Arch Val Glu Lys G'ly Hi.s Ala Glu Pro Glu Glu Val Ala Ala Ile Thr Ala Leu Leu Leu A1<~ Arg Ala Ala Ala Arg Pro Ala Glu Ile Ala Pro Th:v His Gly Gly Gly Arg Ala Arg Ala Gly Trp Arg Arg Leu Glu Arg Glu Pro Gly Phe Arg Ala Pro His Ser Trp Arg

Claims (31)

1. A nucleic acid comprising a nucleic acid sequence which encodes AccB polypeptide, or a nucleic acid sequence complementary thereto, wherein said AccB polypeptide:
(a) has the amino acid sequence set out in Fig. 12A
and/or the amino acid sequence encoded by the nucleic acid sequence set out in Fig. 12B; or (b) has an amino acid sequence which is at least about 80% identical with the amino acid sequence of (a), and has any one or more of the biological properties cf the polypeptide having the amino acid sequence of (a).

2. The nucleic acid of claim 1, further comprising a nucleic acid sequence which encodes AccE polypeptide, or a nucleic acid sequence complementary thereto, wherein said AccE
polypeptide:
(a) has the amino acid sequence set out in Fig. 13A
and/or the amino acid sequence encoded by the nucleic acid sequence set out in Fig. 13B; or (b) has an amine acid sequence which at least about 80% identical with the amino acid sequence of (a), and has any one or pore of the biological properties of the polypeptide having the amino acid sequence of (a).

3. The nucleic acid of claim 1 or claim 2, further comprising a nucleic acid sequence which encodes AccA1 polypeptide, or a nucleic acid sequence complementary thereto, wherein said AccA1 polypetide:
(a) has the amino acid sequence set out in Fig. 11A
and/or the amino acid sequence encoded by the nucleic acid sequence set out in Fig. 11B; or (b) has an amino acid sequence which is at least about 80% identical with the amino acid sequence of (a), and has any one or more of the biological properties of the polypeptide having the amino acid sequence of (a).

4. The nucleic acid of any preceding claim, further comprising a nucleic acid sequence which encodes AccA2 polypeptide, or a nucleic acid sequence complementary thereto, wherein said AccA2 polypeptide:
(a) has the amino acid sequence set out in Fig. 11A
and/or the amino acid sequence encoded by the nucleic acid sequence set out in Fig. 11B; or (b) has an amino acid sequence which is at least about 80% identical with the amino acid sequence of (a), and has any one or more of the biological properties of the polypeptide having the amino acid sequence of (a).

5. The nucleic acid of any preceding claim, wherein the level of identity is at least about 95%.

6. The nucleic acid of any preceding claim, wherein the level of identity is at least about 90%.

7. the nucleic acid of any preceding claim, wherein:
(a) the nucleic acid sequence which encodes the AccB
polypeptide is at least about 80% identical with the nucleic acid of Fig. 12B; and/or (b) the nucleic acid sequence which encodes the AccE
polypeptide is at least about 80% identical with the nucleic acid of Fig. 13B; and/or (c) the nucleic and sequence which encodes the AccB
polypeptide is at least about 80% identical with the nucleic acid of Fig. 11B; and/or (d) the nucleic acid sequence which encodes the AccB
polypeptide is at least about 80% identical with the nucleic acid of Fig. 11B.

8. The nucleic acid cf whim 7, wherein the level of identity is at least about 90%.

9. The nucleic acid of claim 8, wherein the level of identity is at least abort 95%.

10. The nucleic acid of claim 9, wherein the level of identity is at least about 99%.

11. The nucleic acid of any preceding claim wherein said nucleic acid sequence which encodes AccB polypeptide is in operative association with a regulatory sequence for constitutive or inducible expression of said AccB polypeptide in Streptomyces species.

12. The nucleic acid cf claim 11 wherein each said nucleic acid sequence which encodes AccB, AccE, AccA1 and/or AccA2 polypeptide is in operative association with a regulatory sequence for constitutive or inducible expression of said AccB
polypeptide in Streptomyces species.

13. The nucleic acid of claim 11 or claim 12 wherein said regulatory sequence comprises the tipA inducible promoter, the ermE constitutive promoter, or the ermE* constitutive promoter.

14. A vector comprising the nucleic acid sequence of any one of claims 11 to 13, whereby said vector is capable, after incorporation into a Streptomyces species, of causing or increasing expression of AccB polypeptide as defined in claim 1, and optionally also AccE and/or AccA1 and/or AccA2 polypeptide as defined in claims 2 to 4, respectively.

15. A cell of a Streptomyces species, into which cell the vector of claim 14 has been introduced.

16. A method of producing a polyketide, the method comprising:
providing a cell of a polyketide-producing strain of a Streptomyces species into which cell the vector of claim 14 has been introduced;
culturing said cell under conditions suitable for polyketide synthesis; and extracting said polyketide from the cell culture medium.

17. A method of modifying a polyketide-producing strain of a Streptomyces species to increase production of said polyketide, the method comprising modifying said strain to express, or to increase expression of, nucleic acid according to any one of claims 1 to 13.

18. A modified strain of a Streptomyces species, produced according to the method of claim 17.

19. A method of producing a polyketide, the method comprising:
providing a cell of the modified Streptomyces strain of claim 18;
culturing said cell under conditions suitable for polyketide synthesis; and extracting said polyketide from the cell culture medium.

20. A method of increasing acetyl-CoA carboxylase (ACCase) activity in a strain of a Streptomyces species, the method comprising modifying said strain to express, or to increase expression of, nucleic ac.id according to any one of claims 1 to 13.

21. A method of increasing production of a polyketide in a Streptomyces species, the method comprising culturing cells of said species in the presence of exogenous malonate.

22. The method of claim 21, wherein the malonate is present at a concentration of at least about 0.1%.

23. The method of claim 22, wherein the malonate is present at a concentration of at least about 0.5%.

24. The method of claim 23, wherein the malonate is present at a concentration of at least about 1%.

25. The method of any one of claims 16, 19 or 21-24, further comprising the step of purifying said polyketide.

26. The method of claim 25, further comprising the step of formulating said polyketide as a pharmaceutical.

27. The method or strain, of any one of claims 16 to 19 or 21-26 wherein said polyketide is an antibiotic.

28. The vector, cell, method, or strain of any one of claims 14 to 27, wherein said Streptomyces species is selected from the group consisting of S. coelicolor, S. violaceoruber, S.
lividans and S. parvulus.

29. The vector, cell, method, or strain of claim 28, wherein said Streptomyces species is of a strain selected from the group consisting of ATCC: 12434, ATCC 19832, S. coelicolor A3(2) and S. lividans 66.

30. The vector, cell, method, or strain of claim 29, wherein said species is S. coelicolor.

31. The vector, cell, method, or strain of claim 30, wherein said strain is S. coelicolor A3(2).