Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring

A 6-kb region from the chromosome of Streptomyces antibioticus, an oleandomycin producer, was cloned and sequenced. This region was located between the 3′ end of the gene encoding the third subunit of the oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P₄₅₀ monooxygenase and an oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P₄₅₀-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

Cloning, Molecular Characterization, and mRNA Expression of the Thermostable Family 3 β-Glucosidase from the Rare Fungus Stachybotrys microspora

The filamentous fungus Stachybotrys microspora possess a rich β-glucosidase system composed of five β-glucosidases. Three of them were already purified to homogeneity and characterized. In order to isolate the β-glucosidase genes from S. microspora and study their regulation, a PCR strategy using consensus primers was used as a first step. This approach enabled the isolation of three different fragments of family 3 β-glucosidase gene. A representative genomic library was constructed and probed with one amplified fragment gene belonging to family 3 of β-glucosidase. After two rounds of hybridization, seven clones were obtained and the analysis of DNA plasmids leads to the isolation of one clone (CF3) with the largest insert of 7 kb. The regulatory region shows multiple TC-rich elements characteristic of constitutive promoter, explaining the expression of this gene under glucose condition, as shown by zymogram and RT-PCR analysis. The tertiary structure of the deduced amino acid sequence of Smbgl3 was predicted and has shown three conserved domains: an (α/β)₈ triose phosphate isomerase (TIM) barrel, (α/β)₅ sandwich, and fibronectin type III domain involved in protein thermostability. Zymogram analysis highlighted such thermostable character of this novel β-glucosidase.     

Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase,ketoacyl reductase,acyl carrier protein,and biotin carboxyl carrier protein

The actI gene, encoding a component of the actinorhodin polyketide synthase of Streptomyces coelicolor, was used to identify and clone a homologous 11.7 kb BamHI DNA fragment from Saccharopolyspora hirsuta 367. The cloned fragment complemented actinorhodin production in a strain of Streptomyces coelicolor bearing a mutant actI gene. The DNA sequence of a 5.1 kb fragment revealed 6 open reading frames (ORF). ORF1 does not resemble any known DNA or deduced protein sequence, while the deduced protein sequence of ORF2 resembles that of biotin carboxyl carrier proteins. Based on the similarity to deduced protein sequences from cloned genes of polyketide producers, ORF3 would code for a ketoreductase, ORF4 and ORF5 for the putative heterodimeric β-ketoacyl synthase, and ORF6 for an acyl carrier protein.     

Distinctive features of the microbial diversity and the polyketide synthase genes spectrum in the community of the endemic Baikal sponge <Emphasis Type="Italic">Swartschewskia papyracea</Emphasis>

The diversity of the symbiotic community of the endemic Baikal sponge Swartschewskia papyracea was studied, and an analysis of the polyketide synthases genes spectrum in sponge-associated microorganisms was carried out. Six bacterial phyla were detected in the S. papyracea microbiome: Verrucomicrobia, Cyanobacteria, Actinobacteria, Bacteroidetes, Proteobacteria, and Planctomycetes. Unlike the microbial associations of other freshwater sponges, the community under study was dominated by the phylaVerrucomicrobia (42.1%) and Cyanobacteria (17.5%), while the proportion of the Proteobacteria was unusually low (9.7%). In the S. papyracea community metagenome, there were identified 18 polyketide synthases genes fragments, the closest homologues of which included the polyketide synthases of the microorganisms belonging to the bacterial phyla Cyanobacteria, Proteobacteria (classes Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria), and Acidobacteria as well as the eukaryotic algae of the phylum Heterokonta (class Eustigmatophyceae). Polyketide synthase sequences from S. papyracea formed three groups on the phylogenetic tree: a group of hybrid NRPS/PKS complexes, a group of cyanobacterial polyketide synthases, and a group of homologues of the eukaryotic alga Nannochloropsis gaditana. Notably, the identified polyketide synthase genes fragments showed only a 57–88% similarity to the sequences from the databases, which implies the presence of genes controlling the synthesis of the novel, still unstudied, polyketide compounds in the S. papyracea community. It was proposed that the habitat conditions of S. papyracea affect the taxonomic composition of the microorganisms associated with the sponge, including the diversity of the producers of secondary metabolites.     

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis

Summary Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 by fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1–5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS : a heterodimeric -ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a -ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORFl-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM 160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.     

Composition of essential oils from turkish Salvia species

The chemical composition of the essential oils from five Salvia species from Turkey was determined by GC. The species were S. candidissima, S. cryptantha, S. fruticosa, S. officinalis and S. tomentosa. 24, 22, 20, 19, and 22 components were identified, respectively, the major ones being β-pinene (candidissima), borneol (cryptantha), 1,8-cineole (fruticosa), camphor (officinalis) and β-pinene (tomentosa). α-Pinene in candidissima, camphor in cryptantha, α-thujone in officinalis and 1,8-cineole in tomentosa were the other important components.     

Diversity of Polyketide Synthase (PKS) genes in metagenomic community of freshwater sponge Lubomirskia baicalensis

The screening of metagenomic DNA of the microbial community associated with the Baikalian sponge Lubomirskia baicalensis was performed in order to investigate the presence of polyketide synthase (PKS) genes. PKS enzyme systems take part in the synthesis of a great number of biologically active substances. The cloning and sequencing of amplified products of the ketosynthase domain section of the PKS gene cluster revealed 15 fragments of PKS genes with amino acid sequences differing from each other by 35?C65%. A BLASTX analysis showed that all of these sequences belong to KS domains identified in various groups of microorganisms, i.e., Alpha-, Beta-, and Deltaproteobacteria; Verrucomicrobia; Cyanobacteria; and Chlorophyta. Some sequences were related to genes that participate in the biosynthesis of curacin A (CurI, CurJ), stigmatellin (StiC, StiG), nostophycin (NpnB), and cryptophycin (CrpB). The homology of the found sequences with those of the EMBL database lies in the range of 50?C82%, which indicates that the freshwater sponge community contains genes that encode new, not yet studied polyketide substances of potential biotechnological significance.     

Isolation and characterization of the naphthocyclinone gene cluster from Streptomyces arenae DSM 40737 and heterologous expression of the polyketide synthase genes

Streptomyces arenae produces the aromatic polyketide naphthocyclinone, which exhibits activity against Gram-positive bacteria. A cosmid clone containing the putative naphthocyclinone gene cluster was isolated from a genomic library of S. arenae by hybridization with a conserved region from the actinorhodin PKS of S. coelicolor. Sequence analysis of a 5.5-kb DNA fragment, which hybridizes with the actI probe, revealed three open reading frames coding for the minimal polyketide synthase. A strong sequence similarity was found to several previously described ketosynthases, chain length factors and acyl carrier proteins from other polyketide gene clusters. An additional open reading frame downstream of the PKS genes of S. arenae showed 53% identity to act VII probably encoding an aromatase. Another open reading frame was identified in a region of 1.436 bp upstream of the PKS genes, which, however, had no similarity to known genes in the database. Approximately 8 kb upstream of the PKS genes, a DNA fragment was identified that hybridizes to an actVII--actIV specific probe coding for a cyclase and a putative regulatory protein, respectively. Disruption of the proposed naphthocyclinone gene cluster by insertion of a thiostrepton resistance gene completely abolished production of naphthocyclinones in the mutant strain, showing that indeed the naphthocyclinone gene cluster had been isolated. Heterologous expression of the minimal PKS genes in S. coelicolor CH999 in the presence of the act ketoreductase led to the production of mutactin and dehydromutactin, indicating that the S. arenae polyketide synthase forms a C-16 backbone that is subsequently dimerized to build naphthocyclinone. The functions of the proposed cyclase and aromatase were examined by coexpression with genes from different polyketide core producers.     

Diversity of bacteria and polyketide synthase associated with marine sponge Haliclona sp.

The microbial community associated with a marine sponge (Haliclona sp.) collected from Tateyama city, Japan was studied using 16S rRNA gene clone libraries. Two DNA templates were prepared using methods recommended for Gram-positive and Gram-negative bacteria in the Qiagen kit manual. From each DNA template, two 16S rRNA genes were PCR amplified, using the combination of universal bacterial primer 27f and primers 1385r and 1492r, respectively. A total of 347 clones were sequenced and compared with those available in DNA data banks. These sequences were members of ten bacterial phyla. Interestingly, more than 30 % of the clones represent novel sequences. A comparison of these sequences with sequences in a library prepared from DNA extracted from the surrounding water shows minimum DNA contamination. Taxonomically, the highest diversity was detected in the clone library prepared using a combination of primers 27f and 1492r and DNA isolated using the Gram-positive bacteria protocol. The potential of Haliclona sp.-associated bacteria to produce secondary metabolites was studied by cloning and sequencing the polyketide synthase (PKS, type 1) gene using the same DNA samples. Analysis of partial sequences derived from the sponge metagenome revealed 27 unique ketosynthase domains of PKS type I. This study suggests strongly that this Haliclona sp. plays host to diverse novel bacteria with a potential to produce novel polyketides.     

Bacterial Populations Colonizing and Degrading Rice Straw in Anoxic Paddy Soil

Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the α, β, and γ Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa.     

Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei

In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.     

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21–carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

Polymorphism of the KPI-A gene sequence in the potato subgenera Potatoe (Sect. Petota, Esolonifera, and Lycopersicum) and Solanum

Group A Kunitz-type protease inhibitors (KPI-A) are involved in protecting potato plants from microorganisms and pests. While the nucleotide sequence is known for many KPI-A genes of various potato cultivars (Solanum tuberosum subsp. tuberosum) and a few genes of tomato (Solanum lycopersicum), there are no data on their allelic diversity in other species of the genus Solanum. KPI-A fragments were cloned, amplified, sequenced, and analyzed from plants of the subgenera Potatoe sect. Petota (five genes from S. tuberosum ssp. andigenum and two genes from S. stoloniferum) and Solanum (five genes from S. nugrum), and their consensus sequences were established. An identity of 97–100% was observed among these sequences and the KPI-A sequences of the sections Petota (cultivated potato Solanum tuberosum ssp. tuberosum) and Etuberosum (S. palustre) The interspecific variation of KPI-A did not exceed its intraspecific variation for all but one species (S. lycopersicum). The distribution of highly variable and conserved sequences in the mature protein-coding region was the same in all of the above species. The same primers failed to amplify the homologous genes from Solanum dulcamara, S. lycopersicum, and Mandragora officinarum. Phylogenetic analysis of the KPI-A sequences showed that S. lycopersicum clustered separately from all of the other species examined, that S. nigrum clustered together with species of the sections Etuberosum and Petota, and that these species produced no species-specific clusters. Although S. nigrum is resistant to all known races of the oomycete Phytophthora infestans, which causes one of the most economically important diseases of Solanaceae, the amino acid sequences encoded by S. nigrum KPI-A differed slightly, if at all, from their counterparts of cultivated potato, which is susceptible to P. infestans infection.     

Microbial diversity in formation water and enrichment cultures from the Gangxi bed of the Dagang terrigenous oilfield (PRC)

Microbial diversity and biogeochemical processes of the Gangxi bed with low-mineral water and a temperature gradient from 35 to 54°C were studied. The 16S rRNA gene clone libraries (over 800 clones) were obtained from microbial DNA isolated from formation water and from the primary enrichment cultures for fermenting, sulfate-reducing, methanogenic, and aerobic organotrophic prokaryotes. While both sulfate reduction and methanogenesis were registered in formation water by radioisotope techniques, the genes of sulfate-reducing prokaryotes were not revealed in the 16S rRNA gene clone library from formation water. The 16S rRNA genes of Methanobacterium congolense and Methanococcus vannielii predominated among archaeal sequences retrieved from formation water, while the genes of Methanothermobacter thermoautotrophicus, Methanomethylovorans thermophila, and Methanoculleus sp. predominated in the combined library from enrichment cultures. In the library of Bacteria 16S rRNA genes from formation water, the genes of thermophilic fermentative bacteria of the family Thermoanaerobacteriaceae predominated; the remaining sequences belonged to mesophiles (genera Brevundimonas, Sphingomonas, Oxalicibacterium, and Stenotrophomonas), the phylum Chloroflexi, and unidentified bacteria. The combined library from enrichment cultures, contained, apart from the sequences of the family Thermoanaerobacteriaceae, the genes of fermentative bacteria (genera Anaerobaculum, Coprothermobacter, Thermanaerovibrio, Soehngenia, Bacteroides, and Aminobacterium and the order Thermotogales), of aerobic hydrocarbon-oxidizing bacteria (genera Pannonibacter and Pseudomonas), and of sulfate reducers (genera Desulfomicrobium, Thermodesulfovibrio, and Desulfotomaculum). High coverage was shown for bacterial (97.6%) and archaeal (100%) clone libraries, indicating that a significant portion of the microbial diversity in the studied communities was revealed.     

Putative Polyketide Synthase and Laccase Genes for Biosynthesis of Aurofusarin in Gibberella zeae

Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.     

Organization of ribosomal protein genes in Escherichia coli: II. Mapping of ribosomal protein genes by in vitro synthesis of ribosomal proteins using DNA fragments of a transducing phage as templates

The structural genes for six ribosomal proteins (r-proteins) located in the str-spc region around 64 minutes on the Escherichia coli chromosome have been mapped physically with respect to each other and the neighboring genes aroE and trkA. The genes code for the 30 S r-proteins S4 (ram), S5 (spc), S8, S11, S13 and S14. Furthermore, regions coding for unidentified 50 S r-proteins have been indicated.The mapping was performed by biochemical methods employing DNA from the specialized transducing phage λspc1, which carries the aroE-trkA-spc region of the E. coli chromosome. The phage DNA was cleaved by restriction endonucleases, and the generated DNA fragments used as templates for synthesis of r-proteins in a DNA-dependent cell-free system. Since the relative order of the DNA fragments created by the restriction endonucleases is known, a genetic map could be constructed.     

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402. Received 31 May 2001/ Accepted in revised form 09 July 2001