Polyketide Synthase Type Research Articles

Bioactivity Assessment of Indian Origin-Mangrove Actinobacteria against Candida albicans.

Actinobacteria is found to have a potent metabolic activity against pathogens. The present study reveals the assessment of potent antifungal secondary metabolites from actinobacteria isolated from Indian marine mangrove sediments. The samples were collected from the coastal regions of Muthupet, Andaman and the Nicobar Islands. Identification was carried out using 16S rRNA analysis and biosynthetic genes (Polyketide synthase type I/II and Non-ribosomal peptide synthase) were screened. Actinobacteria were assayed for their antifungal activity against 16 clinical Candida albicans and the compound analysis was performed using gas chromatography-mass spectrometry GC-MS. The 31 actinobacterial strains were isolated and 16S rRNA gene sequencing revealed that this ecosystem is rich on actinobacteria, with Streptomyces as the predominant genus. The PCR based screening of biosynthetic genes revealed the presence of PKS-I in six strains, PKS-II in four strains and NRPS in 11 strains. The isolated actinobacteria VITGAP240 and VITGAP241 (two isolates) were found to have a potential antifungal activity against all the tested C. albicans. GC-MS results revealed that the actinobacterial compounds were belonging to heterocyclic, polyketides and peptides. Overall, the strains possess a wide spectrum of antifungal properties which affords the production of significant bioactive metabolites as potential antibiotics.

Complete genome sequence of Streptomyces peucetius ATCC 27952, the producer of anticancer anthracyclines and diverse secondary metabolites

Streptomyces peucetius ATCC 27952 is a filamentous soil bacterium with potential to produce anthracyclines such as doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for the treatment of cancer. Here we present the complete genome sequence of S. peucetius ATCC 27952, which consists of 8,023,114 bp with a linear chromosome, 7187 protein-coding genes, 18 rRNA operons and 66 tRNAs. Bioinformatic analysis of the genome sequence revealed ∼68 putative gene clusters involved in the biosynthesis of secondary metabolites, including diverse classes of natural products. Diverse secondary metabolites of PKS (polyketide synthase) type II (doxorubicin and daunorubicin), NRPS (non-ribosomal peptide synthase) (T1-pks), terpene (hopene) etc. have already been reported for this strain. In addition, in silico analysis suggests the potential to produce diverse compound classes such as lantipeptides, lassopeptides, NRPS and polyketides. Furthermore, many catalytically-efficient enzymes involved in hydroxylation, methylation etc. have been characterized in this strain. The availability of genomic information provides valuable insight for devising rational strategies for the production and isolation of diverse bioactive compounds as well as for the industrial application of efficient enzymes.

Diversity and Bioactivity of Marine Bacteria Associated with the Sponges Candidaspongia flabellata and Rhopaloeides odorabile from the Great Barrier Reef in Australia

Sponges and their associated microbial communities have sparked much interest in recent decades due on the abundant production of chemically diverse metabolites that in nature serve as functional compounds required by the marine sponge host. These compounds were found to carry therapeutic importance for medicinal applications. In the presented study, 123 bacterial isolates from the culture collection of the Australian Institute of Marine Science (AIMS) previously isolated from two different sponge species, namely Candidaspongia flabellata and Rhopaloeides odorabile, originating from different locations on the Great Barrier Reef in Queensland, Australia, were thus studied for their bioactivity. The symbiotic bacterial isolates were first identified using 16S rRNA gene analysis and they were found to belong to five different dominating classes of Domain Bacteria, namely Alphaproteobacteria, Gammaproteobacteria, Flavobacteria, Bacilli and Actinobacteria. Following their taxonomical categorization, the isolates were screened for their antimicrobial activity against human pathogenic microbial reference strains: Escherichia coli (ATCC® BAA-196™), E. coli (ATCC® 13706™), E. coli (ATCC® 25922™), Klebsiella pneumoniae (ATCC® BAA-1705™), Enterococcus faecalis (ATCC® 51575™), Bacillus subtilis (ATCC® 19659™), Staphylococcus aureus (ATCC® 29247™), Candida albicans (ATCC® 10231™) and Aspergillus niger (ATCC® 16888™). Over 50% of the isolates displayed antimicrobial activity against one or more of the reference strains tested. The subset of these bioactive bacterial isolates was further investigated to identify their biosynthetic genes such as polyketide synthase (PKS) type I and non-ribosomal peptide synthetase (NRPS) genes. This was done using polymerase chain reaction (PCR) with degenerate primers that have been previously used to amplify PKS-I and NRPS genes. These specific genes have been reported to be possibly involved in bacterial secondary metabolite production. In 47% of the bacterial isolates investigated, the PKS and NRPS genes were located. Some of the bacterial isolates were found to possess both gene types, which agrees with the previous reported biosynthetic ability of certain sponge-symbiotic bacteria such as the Actinobacteria or Gammaproteobacteria to produce secondary metabolites with antimicrobial activity. All these reported activities further confirm that sponge-symbiotic bacteria hold significant bioactivity with medicinal and biotechnological importance.

Production of Potent Antimicrobial Compounds from Streptomyces cyaneofuscatus Associated with Fresh Water Sediment.

The genus Streptomyces under phylum actinobacteria has been recognized as a prolific source for the production of bioactive secondary metabolites. An actinobacterial strain designated as DST103 isolated from a wetland fresh water sediment of Tamdil Lake, Mizoram, Northeast, India was identified as Streptomyces cyaneofuscatus (KY287599) using 16SrRNA gene sequencing which shares 99.87% sequence similarity with Streptomyces cyaneofuscatus NRRL B-2570T. The strain showed broad spectrum antimicrobial activities against Gram negative bacteria (Escherichia coli MTCC 739 and Pseudomonas aeruginosa MTCC 2453), Gram positive bacteria (Micrococcus luteus NCIM 2170 and Staphylococcus aureus MTCC 96) and yeast pathogen Candida albicans MTCC 3017). The methanolic extract of the strain DST103 exhibited highest antimicrobial activity against E. coli (IC50 = 2.10 μg/mL) and minimum activity against S. aureus (IC50 = 43.63 μg/mL). Five antibiotics [trimethoprim (18 μg/g), fluconazole (6 μg/g), ketoconazole (18 μg/g), nalidixic acid (135 μg/g), and rifampicin (56 μg/g)] were detected and quantified using ultra-performance liquid chromatography (UPLC-ESI-MS/MS). Further, biosynthetic potential genes [polyketide synthases type II, non-ribosomal peptide synthetases, and aminodeoxyisochorismate synthase (phzE)] were also detected in strain DST103 which may possibly be responsible for the production of antimicrobial compounds. Additionally, gas chromatography-mass spectrometry analysis showed the presence of four volatile compounds which might be responsible for their diverse biological activity. The present study revealed the presence of bioactive compounds in strain DST103, which may be a promising resource for the discovery of novel bioactive metabolites against wide range of pathogens.

A new strategy for strain improvement of Aurantiochytrium sp. based on heavy-ions mutagenesis and synergistic effects of cold stress and inhibitors of enoyl-ACP reductase.

Developing a strain with high docosahexaenoic acid (DHA) yield and stable fermenting-performance is an imperative way to improve DHA production using Aurantiochytrium sp., a microorganism with two fatty acid synthesis pathways: polyketide synthase (PKS) pathway and Type I fatty acid synthase (FAS) pathway. This study investigated the growth and metabolism response of Aurantiochytrium sp. CGMCC 6208 to two inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan), and proposed a method of screening high DHA yield Aurantiochytrium sp. strains with heavy ion mutagenesis and pre-selection by synergistic usage of cold stress (4°C) and FAS inhibitors (triclosan and isoniazid). Results showed that (1) isoniazid and triclosan have positive effects on improving DHA level of cells; (2) mutants from irradiation dosage of 120Gy yielded more DHA compared with cells from 40Gy, 80Gy treatment and wild type; (3) DHA contents of mutants pre-selected by inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan)at 4°C, were significantly higher than that of wild type; (4) compared to the wild type, the DHA productivity and yield of a mutant (T-99) obtained from Aurantiochytrium sp. CGMCC 6208 by the proposed method increased by 50% from 0.18 to 0.27g/Lh and 30% from 21 to 27g/L, respectively. In conclusion, this study developed a feasible method to screen Aurantiochytrium sp. with high DHA yield by a combination of heavy-ion mutagenesis and mutant-preselection by FAS inhibitors and cold stress.

Ketide Synthase (KS) Domain Prediction and Analysis of Iterative Type II PKS Gene in Marine Sponge-Associated Actinobacteria Producing Biosurfactants and Antimicrobial Agents.

The important biological macromolecules, such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS) genes was explored. A unique feature of type II PKS genes is their high amino acid (AA) sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of various domains. Therefore, deciphering the order of AA sequence encoded by PKS domains tailored the chemical structure of polyketide analogs still remains a great challenge. The present work deals with an in vitro and in silico analysis of PKS type II genes from five actinobacterial species to correlate KS domain architecture and structural features. Our present analysis reveals the unique protein domain organization of iterative type II PKS and KS domain of marine actinobacteria. The findings of this study would have implications in metabolic pathway reconstruction and design of semi-synthetic genomes to achieve rational design of novel natural products.

Diversity of <em>Micromonospora</em> strains from the deep Mediterranean Sea and their potential to produce bioactive compounds

During studies on bacteria from the Eastern Mediterranean deep-sea, incubation under in situ conditions (salinity, temperature and pressure) and heat treatment were used to selectively enrich representatives of Micromonospora. From sediments of the Ierapetra Basin (4400 m depth) and the Herodotos Plain (2800 m depth), 21 isolates were identified as members of the genus Micromonospora. According to phylogenetic analysis of 16S rRNA gene sequences, the Micromonospora isolates could be assigned to 14 different phylotypes with an exclusion limit of ≥ 99.5% sequence similarity. They formed 7 phylogenetic clusters. Two of these clusters, which contain isolates obtained after enrichment under pressure incubation and phylogenetically are distinct from representative reference organism, could represent bacteria specifically adapted to the conditions in situ and to life in these deep-sea sediments. The majority of the Micromonospora isolates (90%) contained at least one gene cluster for biosynthesis of secondary metabolites for non-ribosomal polypeptides and polyketides (polyketide synthases type I and type II). The determination of biological activities of culture extracts revealed that almost half of the strains produced substances inhibitory to the growth of Gram-positive bacteria. Chemical analyses of culture extracts demonstrated the presence of different metabolite profiles also in closely related strains. Therefore, deep-sea Micromonospora isolates are considered to have a large potential for the production of new antibiotic compounds.

Distribution and Identification of Endophytic Streptomyces Species from Schima wallichii as Potential Biocontrol Agents against Fungal Plant Pathogens.

The prospective of endophytic microorganisms allied with medicinal plants is disproportionally large compared to those in other biomes. The use of antagonistic microorganisms to control devastating fungal pathogens is an attractive and eco-friendly substitute for chemical pesticides. Many species of actinomycetes, especially the genus Streptomyces, are well known as biocontrol agents. We investigated the culturable community composition and biological control ability of endophytic Streptomyces sp. associated with an ethanobotanical plant Schima wallichi. A total of 22 actinobacterial strains were isolated from different organs of selected medicinal plants and screened for their biocontrol ability against seven fungal phytopathogens. Seven isolates showed significant inhibition activity against most of the selected pathogens. Their identification based on 16S rRNA gene sequence analysis, strongly indicated that all strains belonged to the genus Streptomyces. An endophytic strain BPSAC70 isolated from root tissues showed highest percentage of inhibition (98.3 %) against Fusarium culmorum with significant activity against other tested fungal pathogens. Phylogenetic analysis based on 16S rRNA gene sequences revealed that all seven strains shared 100 % similarity with the genus Streptomyces. In addition, the isolates were subjected to the amplification of antimicrobial genes encoding polyketide synthase type I (PKS-I) and nonribosomal peptide synthetase (NRPS) and found to be present in most of the potent strains. Our results identified some potential endophytic Streptomyces species having antagonistic activity against multiple fungal phytopathogens that could be used as an effective biocontrol agent against pathogenic fungi.

Transcriptomic characterisation and genomic glimps into the toxigenic dinoflagellate Azadinium spinosum, with emphasis on polykeitde synthase genes

BackgroundUnicellular dinoflagellates are an important group of primary producers within the marine plankton community. Many of these species are capable of forming harmful algae blooms (HABs) and of producing potent phycotoxins, thereby causing deleterious impacts on their environment and posing a threat to human health. The recently discovered toxigenic dinoflagellate Azadinium spinosum is known to produce azaspiracid toxins. These toxins are most likely produced by polyketide synthases (PKS). Recently, PKS I-like transcripts have been identified in a number of dinoflagellate species. Despite the global distribution of A. spinosum, little is known about molecular features. In this study, we investigate the genomic and transcriptomic features of A. spinosum with a focus on polyketide synthesis and PKS evolution.ResultsWe identify orphan and homologous genes by comparing the transcriptome data of A. spinosum with a diverse set of 18 other dinoflagellates, five further species out of the Rhizaria Alveolate Stramelopile (RAS)-group, and one representative from the Plantae. The number of orphan genes in the analysed dinoflagellate species averaged 27%. In contrast, within the A. spinosum transcriptome, we discovered 12,661 orphan transcripts (18%). The dinoflagellates toxins known as azaspiracids (AZAs) are structurally polyethers; we therefore analyse the transcriptome of A. spinosum with respect to polyketide synthases (PKSs), the primary biosynthetic enzymes in polyketide synthesis. We find all the genes thought to be potentially essential for polyketide toxin synthesis to be expressed in A. spinosum, whose PKS transcripts fall into the dinoflagellate sub-clade in PKS evolution.ConclusionsOverall, we demonstrate that the number of orphan genes in the A. spinosum genome is relatively small compared to other dinoflagellate species. In addition, all PKS domains needed to produce the azaspiracid carbon backbone are present in A. spinosum. Our study underscores the extraordinary evolution of such gene clusters and, in particular, supports the proposed structural and functional paradigm for PKS Type I genes in dinoflagellates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1205-6) contains supplementary material, which is available to authorized users.

Quantification of polyketide synthase genes in tropical urban soils using real-time PCR

Polyketide synthases (PKSs) catalyze the biosynthesis of polyketides and may contribute to the natural production of antibiotics and pose selective pressure for the development of antibiotic resistant bacteria in the environment. Although conventional PCR have been developed to detect the presence of PKS genes, no previous studies have been done to quantify the abundance of PKS genes in environmental samples. In this study, two sets of degenerate real-time PCR (qPCR) primers (PKS1-F/PKS1-R, PKS2-F/PKS2-R) with high specificity and sensitivity were developed to quantify PKS type I and type II genes. These primers were subsequently used to quantify PKS genes in tropical urban soils, and both PKS genes were widely detected in all soil samples. The absolute abundance of PKS type I ranged from 1.7×106 to 4.7×106 copies per gram of soil and the absolute abundance of PKS type II genes ranged from 2.4×105 to 1.5×106 per gram of soil, and the abundance of PKS type I gene was consistently higher than that of PKS type II gene. The relative abundance of PKS type I gene was positively correlated with that of PKS type II gene (p<0.01). Regression analyses indicate that PKS gene abundance was negatively correlated with environmental factors, such as selected antibiotics, sulfate, and metals (p<0.05), but was not correlated with land use type. The studies on the correlation between environmental factors and PKS genes could provide useful information to understand natural production of antibiotics and its associated environmental risks.

Bäckerhefe Saccharomyces cerevisiae als universelle chemische Mikrofabrik

Abstract One of the applications of the emerging synthetic biology field is the enzymatic production of chemicals, either by individual enzymes or by cascades of whole metabolic pathways. Here, the discovery of novel metabolites from baker’s yeast transformed with yeast artificial chromosomes is described. One particular class of compounds found repeatedly are structurally diverse novel polyketides obtained by expression of mixes of PKSIII (polyketide synthase type III).

Three New Non-reducing Polyketide Synthase Genes from the Lichen-Forming Fungus Usnea longissima

Usnea longissima has a long history of use as a traditional medicine. Several bioactive compounds, primarily belonging to the polyketide family, have been isolated from U. longissima. However, the genes for the biosynthesis of these compounds are yet to be identified. In the present study, three different types of non-reducing polyketide synthases (UlPKS2, UlPKS4, and UlPKS6) were identified from a cultured lichen-forming fungus of U. longissima. Phylogenetic analysis of product template domains showed that UlPKS2 and UlPKS4 belong to group IV, which includes the non-reducing polyketide synthases with an methyltransferase (MeT) domain that are involved in methylorcinol-based compound synthesis; UlPKS6 was found to belong to group I, which includes the non-reducing polyketide synthases that synthesize single aromatic ring polyketides, such as orsellinic acid. Reverse transcriptase-PCR analysis demonstrated that UlPKS2 and UlPKS4 were upregulated by sucrose; UlPKS6 was downregulated by asparagine, glycine, and alanine.

Biosynthetic mechanism for sunscreens of the biocontrol agent Lysobacter enzymogenes.

Lysobacter are ubiquitous environmental bacteria emerging as novel biocontrol agents and new sources of anti-infectives. So far, very little effort has been invested in the study of the biology of these Gram-negative gliding bacteria. Many Lysobacter species are characterized by their yellow-orange appearance. Using transposon mutagenesis, we identified a stand-alone polyketide synthase (PKS) gene cluster required for the pigment production in L. enzymogenes OH11. The yellow pigments were abolished in the “white” mutants generated by target-specific deletions of ketosynthase (KS), acyl carrier protein, or ketoreductase. Spectroscopic data suggested that the pigments belong to xanthomonadin-like aryl polyenes. Polyene-type polyketides are known to be biosynthesized by modular PKS (Type I), not by stand-alone PKS (Type II) which always contain the heterodimer KS-CLF (chain-length factor) as the key catalytic component. Remarkably, this aryl polyene PKS complex only contains the KS (ORF17), but not the CLF. Instead, a hypothetical protein (ORF16) is located immediately next to ORF17. ORF16–17 homologs are widespread in numerous uncharacterized microbial genomes, in which an ORF17 homolog is always accompanied by an ORF16 homolog. The deletion of ORF16 eliminated pigment production, and homology modeling suggested that ORF16 shares a structural similarity to the N-terminal half of CLF. A point-mutation of glutamine (Q166A) that is the conserved active site of known CLF abolished pigment production. The “white” mutants are significantly more sensitive to UV/visible light radiation or H2O2 treatment than the wild type. These results unveil the first example of Type II PKS-synthesized polyene pigments and show that the metabolites serve as Lysobacter “sunscreens” that are important for the survival of these ubiquitous environmental organisms.

Aromatic Polyketides Produced by Bacterial Iterative Type I Polyketide Synthases

Polyketides comprise a large and highly diverse group of natural products produced by polyketide synthases (PKSs), and many of these compounds display remarkable biological activities. Although PKSs share a common mechanism in the assembly of polyketides from short carboxylic acid precursors, different types of PKSs have been classified according to their structures and modes of action. This review discusses a growing group of bacterial PKSs that are structurally type I but act in an iterative manner to produce aromatic polyketides. We summarize the genetic and biochemical features of these enzymes and compare them with other types of PKSs with an emphasis on the evolutionary relationship. We also discuss the different mechanisms for polyketide off-loading and the diverse post-PKS modifications on the resulting aromatic rings. Insights into bacterial iterative type I PKSs for aromatic polyketide formation and the relevant tailoring enzymes may guide rational bioengineering efforts to produce novel natural...

New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction

Bacillus strains produce non-ribosomal lipopeptides that can be grouped into three families: surfactins or lichenysins, iturins and fengycins or plispastatins. These biosurfactants show a broad spectrum of biological activities. To detect strains able to produce these lipopeptides, a new polymerase chain reaction screening approach was developed using degenerated primers based on the intraoperon alignment of adenylation and thiolation nucleic acid domains of all enzymes implicated in the biosynthesis of each lipopeptide family. The comparative bioinformatics analyses of each operon led to the design of four primer pairs for the three families taking into account the differences between open reading frames of each synthetase gene. Tested on different Bacillus sp. strains, this technique was used successfully to detect not only the expected genes in the lipopeptide producing strains but also the presence of a plispastatin gene in Bacillus subtilis ATCC 21332 and a gene showing a high similarity with the polyketide synthase type I gene in the B. subtilis ATCC 6633 genome. It also led to the discovery of the presence of non-ribosomal peptide synthetase genes in Bacillus thuringiensis serovar berliner 1915 and in Bacillus cereus LMG 2098. In addition, this work highlighted the differences between the fengycin and plipastatin operon at DNA level.

Development of an Analysis Program of Type I Polyketide Synthase Gene Clusters Using Homology Search and Profile Hidden Markov Model

MAPSI (Management and Analysis for Polyketide Synthase Type I) has been developed to offer computational analysis methods to detect type I PKS (polyketide synthase) gene clusters in genome sequences. MAPSI provides a genome analysis component, which detects PKS gene clusters by identifying domains in proteins of a genome. MAPSI also contains databases on polyketides and genome annotation data, as well as analytic components such as new PKS assembly and domain analysis. The polyketide data and analysis component are accessible through Web interfaces and are displayed with diverse information. MAPSI, which was developed to aid researchers studying type I polyketides, provides diverse components to access and analyze polyketide information and should become a very powerful computational tool for polyketide research. The system can be extended through further studies of factors related to the biological activities of polyketides.

Combination Methods for Screening Marine Actinomycetes Producing Potential Compounds as Anticancer

Marine actinomycetes is a robust source of secondary metabolites including anticancer compounds . The objective of this research was to select marine actinomycetes producing potential compounds as anticancer used combination methods that consist of amplification PKS I (polyketide synthases type I) and NRPS (non ribosomal peptide synthetases) genes, analysis the diversity of secondary metabolites and genetic. Selected isolates were used for cytotoxicity assay. PKS I and NRPS genes were amplified using sets of degenerate primers. K1F and M6R were used for amplify ketosynthase and methyl-malonyl-CoA transferase modules of PKS I gene which targeted sequences 1200-1400 bp. A3F and A7R were used for amplify adenilation domains of NRPS gene which targeted sequences 700-800 bp. The diversity of secondary metabolites was analized by TLC and densitometry of ethyl acetate extracts. Genetic diversity was analized by repetitive DNA fingerprinting using BOXA1R primers. The cytotoxicity of secondary metabolites on T47D and MCF7 breast cell lines cancer was measured by MTT assay method. Fifty two marine actinomycetes isolates were screened using combination methods. Ten isolates were detected encoding both PKS I and NRPS genes, whereas 11 isolates were detected encoding the NRPS gene. The screening by analysis of secondary metabolites and genetic diversity methods were obtained 6 selected isolates for cytotoxicity assay, which consist of 3 isolates encoding both PKS I and NRPS genes and 3 isolates encoding NRPS gene.Isolate 1 had high cytotoxicity with the IC50 on T47D cell was 19 mg/ml and the IC50 on MCF7 cell was 7 g/ml. This findings suggests that combination methods were effective and efficient way to select marine actinomycetes producing potential compounds as anticancer.

Diversity of actinomycetes isolated from Challenger Deep sediment (10,898 m) from the Mariana Trench

Thirty-eight actinomycetes were isolated from sediment collected from the Mariana Trench (10,898 m) using marine agar and media selective for actinomycetes, notably raffinose-histidine agar. The isolates were assigned to the class Actinobacteria using primers specific for members of this taxon. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Dermacoccus, Kocuria, Micromonospora, Streptomyces, Tsukamurella and Williamsia. All of the isolates were screened for genes encoding nonribosomal peptide and polyketide synthetases. Nonribosomal peptide synthetase sequences were detected in more than half of the isolates and polyketide synthases type I (PKS-I) were identified in five out of 38 strains. The Streptomyces isolates produced several unusual secondary metabolites, including a PKS-I associated product. In initial testing for piezotolerance, the Dermacoccus strain MT1.1 grew at elevated hydrostatic pressures.

Functional Analysis of Type II Thioesterase of Streptomyces lydicus AS 4.2501

Constructing a mutant strain of single gene disruption is the basis for the study of gene function and metabolomics. Systematic and complete genome sequencing is the basis of genetic manipulation. In the case of a little knowledge about the Streptomyces lydicus genome and the speculation that polyketide synthases (type I) might be responsible for the polyketide side chain biosynthesis of streptolydigin, a 588-bp fragment was amplified by polymerase chain reaction (PCR) according to the homology existing in the same functional genes among Streptomyces. A mutant strain of this gene was constructed by single crossover homologous recombination. The results of sequence analysis as well as the metabolite analysis of the mutant and the original strain by liquid chromatography/mass spectroscopy indicated that this fragment was part of type II thioesterase (TE) gene, which was required for streptolydigin biosynthesis like other type II TEs function in related antibiotics biosynthesis. Furthermore, targeted gene manipulation based on PCR was a powerful tool for studying gene function and metabolomics, especially when little was known about the genomic sequence of streptomyces.

Tandemly Duplicated Acyl Carrier Proteins, Which Increase Polyketide Antibiotic Production, Can Apparently Function Either in Parallel or in Series

Polyketide biosynthesis involves the addition of subunits commonly derived from malonate or methylmalonate to a starter unit such as acetate. Type I polyketide synthases are multifunctional polypeptides that contain one or more modules, each of which normally contains all the enzymatic domains for a single round of extension and modification of the polyketide backbone. Acyl carrier proteins (ACP(s)) hold the extender unit to which the starter or growing chain is added. Normally there is one ACP for each ketosynthase module. However, there are an increasing number of known examples of tandemly repeated ACP domains, whose function is as yet unknown. For the doublet and triplet ACP domains in the biosynthetic pathway for the antibiotic mupirocin from Pseudomonas fluorescens NCIMB10586 we have inactivated ACP domains by inframe deletion and amino acid substitution of the active site serine. By deletion analysis each individual ACP from a cluster can provide a basic but reduced activity for the pathway. In the doublet cluster, substitution analysis indicates that the pathway may follow two parallel routes, one via each of the ACPs, thus increasing overall pathway flow. In the triplet cluster, substitution in ACP5 blocked the pathway. Thus ACP5 appears to be arranged "in series" to ACP6 and ACP7. Thus although both the doublet and triplet clusters increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved. The function of some ACPs may be determined by their location in the protein rather than absolute enzymic activity.