A new modular polyketide synthase in the erythromycin producer Saccharopolyspora erythraea

A previously unidentified set of genes encoding a modular polyketide synthase (PKS) has been sequenced in Saccharopolyspora erythraea, producer of the antibiotic erythromycin. This new PKS gene cluster (pke) contains four adjacent large open reading frames (ORFs) encoding eight extension modules, flanked by a number of other ORFs which can be plausibly assigned roles in polyketide biosynthesis. Disruption of the pke PKS genes gave S. erythraea mutant JC2::pSBKS6, whose growth characteristics and pattern of secondary metabolite production did not apparently differ from the parent strain under any of the growth conditions tested. However, the pke PKS loading module and individual pke acyltransferase domains were shown to be active when used in engineered hybrid PKSs, making it highly likely that under appropriate conditions these biosynthetic genes are indeed expressed and active, and synthesize a novel polyketide product.     

Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.

The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675-679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.     

Characterization of a gene cluster responsible for the biosynthesis of anticancer agent FK228 in Chromobacterium violaceum No. 968

A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates.     

Characterization of the biosynthetic gene cluster for the antifungal polyketide soraphen A from Sorangium cellulosum So ce26

A genomic DNA region of over 80 kb that contains the complete biosynthetic gene cluster for the synthesis of the antifungal polyketide metabolite soraphen A was cloned from Sorangium cellulosum So ce26. The nucleotide sequence of the soraphen A gene region, including 67,523 bp was determined. Examination of this sequence led to the identification of two adjacent type I polyketide synthase (PKS) genes that encode the soraphen synthase. One of the soraphen A PKS genes includes three biosynthetic modules and the second contains five additional modules for a total of eight. The predicted substrate specificities of the acyltransferase (AT) domains, as well as the reductive loop domains identified within each module, are consistent with expectations from the structure of soraphen A. Genes were identified in the regions flanking the two soraphen synthase genes that are proposed to have roles in the biosynthesis of soraphen A. Downstream of the soraphen PKS genes is an O-methyltransferase (OMT) gene. Upstream of the soraphen PKS genes there is a gene encoding a reductase and a group of genes that are postulated to have roles in the synthesis of methoxymalonyl-acyl carrier protein (ACP). This unusual extender unit is proposed to be incorporated in two positions of the soraphen polyketide chain. One of the genes in this group contains distinct domains for an AT, an ACP, and an OMT.     

The gene encoding Escherichia coli acyl carrier protein lies within a cluster of fatty acid biosynthetic genes.

The gene encoding Escherichia coli acyl carrier protein (ACP) has been isolated and sequenced. The ACP gene (called acpP) was located on the genetic map between fabF and fabD which encode two fatty acid biosynthetic enzymes, 3-ketoacyl-ACP synthase II and malonyl CoA-ACP transacylase, respectively. An open reading frame between acpP and fabD encodes a 26.5-kDa protein that has significant sequence identity (greater than 40%) with two acetoacetyl-CoA reductases and thus is believed to encode a 3-ketoacyl-ACP reductase. This gene (called fabG) is cotranscribed with acpP. Thus, the gene encoding ACP, the key carrier protein of fatty acid synthesis, is located within a cluster of fatty acid biosynthetic genes.     

Characterization of a Gene Cluster Responsible for the Biosynthesis of Anticancer Agent FK228 in Chromobacterium violaceum No. 968

A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates.     

The putative elaiophylin biosynthetic gene cluster in Streptomyces sp. DSM4137 is adjacent to genes encoding adenosylcobalamin-dependent methylmalonyl CoA mutase and to genes for synthesis of cobalamin

A type I PKS gene probe obtained from RAPB of the rapamycin producer Streptomyces hygroscopicus, strongly hybridised to 92 out of 1120 cosmids from a genomic library of the elaiophylin-producing strain Streptomyces sp. DSM4137. Partial cosmid sequencing suggested the presence of 10 separate sequences encoding type I PKS genes. One entire DNA sequence was obtained and found exactly to match the gene organisation expected for the biosynthesis of the unusual macrodiolide polyketide elaiophylin. The putative elaiophylin gene cluster contains five large open-reading frames encoding typical modular polyketide synthases, which together catalyse the synthesis of the octaketide monomer of elaiophylin. Other genes were identified that would be required for provision of the ethylmalonate extender unit, for the synthesis and attachment of 2-deoxy-L-fucose and in regulation, or in export of the product. Immediately adjacent to the putative elaiophylin biosynthetic gene cluster is a 30-kbp region containing the gene for adenosylcobalamin-dependent methylmalonyl CoA mutase and also genes involved in the biosynthesis of the cobalamin cofactor. Analysis of the latter gene set confirms the view that cbiD of the anaerobic pathway and cobF in the aerobic pathway catalyse the same methylation of precorrin-5. The proximity of these genes to the putative elaiophylin gene cluster can best be rationalised if in this organism succinyl-CoA is a significant source of the methylmalonate units for complex polyketide biosynthesis.     

Cloning, nucleotide sequence, and expression of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase.

The Escherichia coli fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase (MCT) was cloned by complementation of a thermosensitive E. coli fabD mutant (fabD89). Expression of the fabD gene in an appropriate E. coli expression vector resulted in an accumulation of the MCT protein of up to 10% of total soluble protein, which was accompanied by an approximately 1,000-fold increase in the MCT activity. DNA sequence analysis and expression studies revealed that the fabD gene is part of an operon consisting of at least three genes involved in fatty acid biosynthesis. Comparison with available DNA and protein data bases suggest that a 3-ketoacyl-acyl carrier protein synthase and a ketoacyl-acyl carrier protein reductase gene are located immediately upstream and downstream, respectively, of fabD within this fab operon. Western immunoblot analysis with antiserum raised against wild-type E. coli MCT showed that the fabD89 allele encodes a polypeptide with an apparent molecular weight of 27,000 in addition to the normal MCT protein of 32,000. The nature of the temperature-sensitive fabD89 gene product is discussed.     

Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis.

The genes encoding the polyketide synthase (PKS) portion of the niddamycin biosynthetic pathway were isolated from a library of Streptomyces caelestis NRRL-2821 chromosomal DNA. Analysis of 40 kb of DNA revealed the presence of five large open reading frames (ORFs) encoding the seven modular sets of enzymatic activities required for the synthesis of a 16-membered lactone ring. The enzymatic motifs identified within each module were consistent with those predicted from the structure of niddamycin. Disruption of the second ORF of the PKS coding region eliminated niddamycin production, demonstrating that the cloned genes are involved in the biosynthesis of this compound.     

The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units

FK520 (ascomycin) is a macrolide produced by Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) that has immunosuppressive, neurotrophic and antifungal activities. To further elucidate the biosynthesis of this and related macrolides, we cloned and sequenced an 80kb region encompassing the FK520 gene cluster. Genes encoding the three polyketide synthase (PKS) subunits (fkbB, fkbC and fkbA), the peptide synthetase (fkbP), the 31-O-methyltransferase (fkbM), the C-9 hydroxylase (fkbD) and the 9-hydroxyl oxidase (fkbO) had the same organization as the genes reported in the FK506 gene cluster of Streptomyces sp. MA6548 (Motamedi, H., Shafiee, A., 1998. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur. J. Biochem. 256, 528-534). Disruption of a PKS gene in the cluster using the φC31 phage vector, KC515, led to antibiotic non-producing strains, proving the identity of the cluster. Previous labeling data have indicated that FK520 biosynthesis uses novel polyketide extender units (Byrne, K.M., Shafiee, A., Nielson, J., Arison, B., Monaghan, R.L., Kaplan, L., 1993. The biosynthesis and enzymology of an immunosuppressant, immunomycin, produced by Streptomyces hygroscopicus var, ascomyceticus. Dev. Ind. Microbiol. 32, 29-45). Genes in the flanking regions of the FK520 cluster were identified that appear to be involved in synthesis of these extender units. All but two of these genes were homologous to genes with known function. In addition to a crotonyl-CoA reductase gene (fkbS), at least two other genes are proposed to be involved in biosynthesis of the atypical PKS extender unit ethylmalonyl-CoA, which accounts for the ethyl side chain on C-21 of FK520. A set of five contiguous genes (fkbGHIJK) is proposed to be involved in biosynthesis of an unusual PKS extender unit bearing an oxygen on the alpha-carbon, and leading to the 13- and 15-methoxy side chains. These putative precursor synthesis genes in the flanking regions of the FK520 cluster are not found in the flanking regions of the rapamycin cluster (Molnár, I., Aparicio, J.F., Haydock, S.F., Khaw, L.E., Schwecke, T., K?nig, A., Staunton, J., Leadlay, P.F., 1996. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. Gene 169, 1-7), consistent with labeling data showing that rapamycin biosynthesis uses only malonyl and methylmalonyl extender units.     

PKSI-AT结构域及在组合生物合成研究中的应用

I型聚酮合酶（PKSI）的模块型分子结构组织方式非常适合于组合生物合成研究.结构域和模块通过二级组织方式构成了PKSI的催化单元,其它结构多肽则作为“支架”.在“支架”上对结构域和模块两个水平进行突变、替换、插入、缺失等基因操作形成重组PKS,可以理性设计并获得复杂多样的新活性或高活性的聚酮化合物.利用PKSI进行组合生物合成以期获得新聚酮化合物的研究迄今已有约25年,但是目前仍不能够对PKS进行完美的理性设计,快速合成目标活性的新聚酮化合物.PKS中的酰基转移酶结构域的研究在PKS的组合生物合成研究中一直发挥着重要作用.本文结合本课题组的研究基础,对AT结构域的结构、功能及在组合生物合成研究中的最新研究成果作以分析总结.     

Characterization of biosynthetic gene cluster for the production of virginiamycin M, a streptogramin type A antibiotic, in Streptomyces virginiae

Virginiamycin M (VM) of Streptomyces virginiae is a hybrid polyketide-peptide antibiotic with peptide antibiotic virginiamycin S (VS) as its synergistic counterpart. VM and VS belong to the Streptogramin family, which is characterized by strong synergistic antibacterial activity, and their water-soluble derivatives are a new therapeutic option for combating vancomycin-resistant Gram-positive bacteria. Here, the VM biosynthetic gene cluster was isolated from S. virginiae in the 62-kb region located in the vicinity of the regulatory island for virginiamycin production. Sequence analysis revealed that the region consists of 19 complete open reading frames (ORFs) and one C-terminally truncated ORF, encoding hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS), typical PKS, enzymes synthesizing precursors for VM, transporters for resistance, regulatory proteins, and auxiliary enzymes. The involvement of the cloned gene cluster in VM biosynthesis was confirmed by gene disruption of virA encoding a hybrid PKS-NRPS megasynthetase, which resulted in complete loss of VM production without any effect on VS production. To assemble the VM core structure, VirA, VirF, VirG, and VirH consisting, as a whole, of 24 domains in 8 PKS modules and 7 domains in 2 NRPS modules were predicted to act as an acyltransferase (AT)-less hybrid PKS-NRPS, whereas VirB, VirC, VirD, and VirE are likely to be essential for the incorporation of the methyl group into the VM framework by a HMG-CoA synthase-based reaction. Among several uncommon features of gene organization in the VM gene cluster, the lack of AT domain in every PKS module and the presence of a discrete AT encoded by virI are notable. AT-overexpression by an additional copy of virI driven by ermEp() resulted in 1.5-fold increase of VM production, suggesting that the amount of VirI is partly limiting VM biosynthesis.     

Biosynthesis of the anti-parasitic agent megalomicin: transformation of erythromycin to megalomicin in Saccharopolyspora erythraea

Megalomicin is a therapeutically diverse compound which possesses antiparasitic, antiviral and antibacterial properties. It is produced by Micromonospora megalomicea and differs from the well-known macrolide antibiotic erythromycin by the addition of a unique deoxyamino sugar, megosamine, to the C-6 hydroxyl. We have cloned and sequenced a 48 kb segment of the megalomicin (meg) biosynthetic gene cluster which contains the modular polyketide synthase (PKS) and the complete pathway for megosamine biosynthesis. The similarities and distinctions between the related megalomicin and erythromycin gene clusters are discussed. Heterologous expression of the megalomicin PKS in Streptomyces lividans led to production of 6-deoxyerythronolide B, the same macrolactone intermediate for erythromycin. A 12 kb fragment harbouring the putative megosamine pathway was expressed in Saccharopolyspora erythraea, resulting in the conversion of erythromycin to megalomicin. Considering the extensive knowledge surrounding the genetic engineering of the erythromycin PKS and the familiarity with genetic manipulation and fermentation of S. erythraea, the ability to produce megalomicin in this strain should allow the engineering of novel megalomicin analogues with potentially improved therapeutic activities.     

The tallysomycin biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.     

Plumbing the depths of PUFA biosynthesis: a novel polyketide synthase-like pathway from marine organisms

Polyunsaturated fatty acids (PUFAs) are involved in determining the biophysical properties of membranes as well as being precursors for signalling molecules. C(20+) PUFA biosynthesis is catalysed by sequential desaturation and fatty acyl elongation reactions. This aerobic biosynthetic pathway was thought to be taxonomically conserved, but an alternative anaerobic pathway for the biosynthesis of polyunsaturated fatty acids is now known to exist that is analogous to polyketide synthases (PKS). These novel PKS genes could be used to direct the synthesis of PUFAs in heterologous hosts, as well as exploiting the combinatorial chemistry of PKSs to make unusual fatty acids.     

Evolutionary implications of bacterial polyketide synthases

Polyketide synthases (PKS) perform a stepwise biosynthesis of diverse carbon skeletons from simple activated carboxylic acid units. The products of the complex pathways possess a wide range of pharmaceutical properties, including antibiotic, antitumor, antifungal, and immunosuppressive activities. We have performed a comprehensive phylogenetic analysis of multimodular and iterative PKS of bacteria and fungi and of the distinct types of fatty acid synthases (FAS) from different groups of organisms based on the highly conserved ketoacyl synthase (KS) domains. Apart from enzymes that meet the classification standards we have included enzymes involved in the biosynthesis of mycolic acids, polyunsaturated fatty acids (PUFA), and glycolipids in bacteria. This study has revealed that PKS and FAS have passed through a long joint evolution process, in which modular PKS have a central position. They appear to have derived from bacterial FAS and primary iterative PKS and, in addition, share a common ancestor with animal FAS and secondary iterative PKS. Furthermore, we have carried out a phylogenomic analysis of all modular PKS that are encoded by the complete eubacterial genomes currently available in the database. The phylogenetic distribution of acyltransferase and KS domain sequences revealed that multiple gene duplications, gene losses, as well as horizontal gene transfer (HGT) have contributed to the evolution of PKS I in bacteria. The impact of these factors seems to vary considerably between the bacterial groups. Whereas in actinobacteria and cyanobacteria the majority of PKS I genes may have evolved from a common ancestor, several lines of evidence indicate that HGT has strongly contributed to the evolution of PKS I in proteobacteria. Discovery of new evolutionary links between PKS and FAS and between the different PKS pathways in bacteria may help us in understanding the selective advantage that has led to the evolution of multiple secondary metabolite biosyntheses within individual bacteria.     

Engineering modular ester fermentative pathways in Escherichia coli

Sensation profiles are observed all around us and are made up of many different molecules, such as esters. These profiles can be mimicked in everyday items for their uses in foods, beverages, cosmetics, perfumes, solvents, and biofuels. Here, we developed a systematic ‘natural’ way to derive these products via fermentative biosynthesis. Each ester fermentative pathway was designed as an exchangeable ester production module for generating two precursors− alcohols and acyl-CoAs that were condensed by an alcohol acyltransferase to produce a combinatorial library of unique esters. As a proof-of-principle, we coupled these ester modules with an engineered, modular, Escherichia coli chassis in a plug-and-play fashion to create microbial cell factories for enhanced anaerobic production of a butyrate ester library. We demonstrated tight coupling between the modular chassis and ester modules for enhanced product biosynthesis, an engineered phenotype useful for directed metabolic pathway evolution. Compared to the wildtype, the engineered cell factories yielded up to 48 fold increase in butyrate ester production from glucose.     

Purification,overproduction, and partial characterization of beta-RFAP synthase,a key enzyme in the methanopterin biosynthesis pathway

Methanopterin is a folate analog involved in the C1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria. Although a pathway for methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway. The enzyme beta-ribofuranosylaminobenzene 5'-phosphate synthase (beta-RFAP synthase) catalyzes the first unique step to be identified in the pathway of methanopterin biosynthesis, namely, the condensation of p-aminobenzoic acid with phosphoribosylpyrophosphate to form beta-RFAP, CO2, and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding beta-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of beta-RFAP synthase. The enzyme was purified from the methane-producing archaeon Methanosarcina thermophila, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus. The putative beta-RFAP synthase gene from A. fulgidus was expressed in Escherichia coli, and the enzymatic activity of the recombinant gene product was verified. A BLAST search using the deduced amino acid sequence of the beta-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C1 metabolism by the bacterium Methylobacterium extorquens. The identification of a gene encoding a potential beta-RFAP synthase in M. extorquens is the first report of a putative methanopterin biosynthetic gene found in the Bacteria and provides evidence that the pathways of methanopterin biosynthesis in Bacteria and Archaea are similar.     

Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization

The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway-specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently-proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin-linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.