Streptomyces Galilaeus Research Articles

Insecticidal potential of Streptomyces sp. dichloromethane extracts against the cactus cochineal Dactylopius opuntiae (Cockerell)

Destructive damages caused by Dactylopius opuntiae (Cockerell) to cactus worldwide require an ecofriendly IPM approach. Streptomyces sp. produce wide range of biologically active secondary metabolites that can be an interesting alternative to chemical insecticides for pest control, as they are less toxic and easily biodegradable. The efficacy of dichloromethane extracts of four Moroccan Streptomyces sp. strains: Streptomyces bellus- E23-2, Streptomyces galilaeus- E23-9, Streptomyces africanus- E23-3, and Streptomyces bellus- E25-12 (applied at 11, 13, 15, 17 and 20 mg mL-1) against D. opuntiae nymphs and adult females was evaluated under laboratory and greenhouse conditions. Results showed that Streptomyces bellus- E23-2 and Streptomyces galilaeus- E23-9 dichloromethane extracts applied at 20 mg mL-1 were more effective, causing higher mortality against nymphs (92% and 91%, respectively) and adult females (90% and 95%, respectively) after 8 days of exposure, resulting in an LT50 value of 3.0 days (nymph), and 3.0 and 6.0 days (adult female), respectively. Streptomyces bellus- E25-12 extract had the lowest mortalities [88% (nymph) and 68% (adult female)]. In greenhouse experiment, the highest first instar nymph mortality was achieved by Streptomyces bellus- E23-2 (55.5%) and Streptomyces galilaeus- E23-9 (50.5%) dichloromethane extracts at 20 mg mL-1. The metabolites found in dichloromethane extracts of Streptomyces bellus- E23-2 and Streptomyces galilaeus- E23-9 show considerable potential to be used in the development of new biopesticide formulations for use in integrated pest management programs against D. opuntiae.

Stress-Resistance and Growth-Promoting Characteristics and Effects on Vegetable Seed Germination of Streptomyces sp. Strains Isolated from Wetland Plant Rhizospheres

Wetlands are the most biologically diverse ecosystems on Earth. The isolation of Streptomyces strains from wetlands is helpful to study their diversity and functions in such habitats. In this study, six strains of Streptomyces were isolated from the rhizosphere soil of three plant species in the Huaxi Wetland at Guiyang and were identified as Streptomyces galilaeus, S. avidinii, S. albogriseolus, S. albidoflavus, S. spororaveus, and S. cellulosae, respectively. The six strains all solubilized phosphate, fixed nitrogen, and produced ACC deaminase and siderophores, and four strains also secreted indole-3-acetic acid. The six strains had the ability to resist to certain degrees of salinity, drought, and acidic/alkaline pH stress. In addition, the S. avidinii WL3 and S. cellulosae WL9 strains significantly promoted seed germination of mung bean, pepper, and cucumber, especially the WL3 strain. A pot experiment further showed that WL3 significantly promoted the growth of cucumber seedlings. Thus, strains of six species of Streptomyces with multiple plant growth-promoting characteristics were isolated from the wetland. These results lay a foundation for their potential use as microbial agents for seed-coating treatments.

Digital livestock systems and probiotic mixtures can improve the growth performance of swine by enhancing immune function, cecal bacteria, short-chain fatty acid, and nutrient digestibility.

In response to climate change, the use of digital livestock systems and probiotic mixtures as technological strategies to improve animal health and production is driving new innovations in the farm animal industry. However, there is little information available regarding the effects of digital livestock systems and probiotic mixtures (consisting of Bacillus subtillus, Streptomyces galilaeus, and Sphingobacteriaceae) on the growth performance of the growth-finishing swine. Thus, the objective of this study was to investigate the effects of digital livestock systems and probiotic mixtures on the immune function, cecal bacteria, short-chain fatty acids, nutrient digestibility, and growth performance of growth-finishing swine. A total of 64 crossbred male swine (Duroc × Landrace × Yorkshire, average body weight: 60.17 ± 1.25 kg) were randomly assigned to four treatment groups: CON (control group with a conventional livestock system without a probiotic mixture), CON0.4 (a conventional livestock system with a 0.4% probiotic mixture), DLSC (a digital livestock system without a probiotic mixture), and DLS0.4 (a digital livestock system with a 0.4% probiotic mixture). The swine were reared under standard environmental conditions until their average body weight reached 110 kg. The results indicated that the growth performance of the swine improved with an increase in nutrient digestibility and immune function via modulation of blood immune markers in the group with a digital livestock system compared to the CON group, although the growth performance of the swine was similar between the DLSC and CON0.4 groups. Moreover, the application of the digital livestock system and the probiotic mixture maintained higher levels of Lactobacillus and balanced short-chain fatty acid profiles compared to the CON group. These results suggest that a digital livestock system and a probiotic mixture can improve the growth performance of swine by enhancing their nutrient digestibility, improving their immune function, and maintaining balanced cecal bacteria and short-chain fatty acids. Therefore, this study provides insights into the application of digital livestock systems and probiotic mixtures as a climate change response strategy to improve swine production.

Characterization of Bacterial Flora from Afşin–Elbistan Lignite Mine for Potential Microbial Biotech Applications

The energy demand is enhancing day by day all over the world with the increasing of huge population. Lignite, a fossil fuel with low energy efficiency, is used in many different fields. However, lignite is primarily an energy source and its use without treatment causes environmental pollution as well as low energy efficiency. Advantages are known for the use of microorganisms in lignite processing technologies, and research is underway to improve this process. In this research designed to meet this need, isolation and characterization of bacteria with potential for use in biotechnological applications resistant to extreme conditions were performed from lignite mine. Turkey’s largest lignite mine bacterial flora have been identified for the first time in this study. In addition to lignite processing technologies, a bacterial strain collection has been created to be used in other biotechnological studies. This collection of bacteria includes Methylorubrum zatmanii, Streptomyces argenteolus, Streptomyces galilaeus, Pseudomonas mandelii, Paenarthrobacter aurescens, Paeniglutamicibacter sulfureus, Arthrobacter humicola, Bacillus pumilus, Bacillus subtilis, Bacillus safensis, Kocuria polaris, Arthrobacter sp., Rhodococcus sp. This specific bacteria collection is open to sharing with other researchers who want to do field applications.

A Novel Streptomyces sp. Strain PBSH9 for Controlling Potato Common Scab Caused by Streptomyces galilaeus.

Potato common scab is an important soilborne disease worldwide that can significantly reduce the quality and economic values of potato. The disease is caused by multiple species of Streptomyces, which are not well controlled due to lack of effective strategies. Streptomyces galilaeus has been recently identified as a dominant species causing potato common scab in Inner Mongolia, China. This study was focused on screening and characterizing antagonists for biological control against pathogenic S. galilaeus. Bacterial strain PBSH9 was isolated from a potato tuber. PBSH9 was identified as a Streptomyces sp. on the basis of morphological, physiological, and biochemical characteristics, as well as DNA sequence analysis. PBSH9 inhibited S. galilaeus with a diameter of inhibitory zone of 19.8 mm on agar plates. The extracellular filtrate of PBSH9 also inhibited S. galilaeus growth with a diameter of inhibition zone of 10.0 mm. Furthermore, PBSH9 promoted potato sprouting and emergence. Disease control was up to 81.88% in greenhouse trials, and from 47.64 to 73.97% in 3-year field trials. Among the tested inoculation methods, seed treatment was more effective than soil drenching for PBSH9 application. PBSH9 not only effectively controlled potato common scab but also increased potato growth. Thus, it can be a potential candidate for biocontrol agent.

Monitoring the efficiency of Streptomyces galilaeus strain KPS-C004 against root knot disease and the promotion of plant growth in the plant-parasitic nematode infested soils

Streptomyces galilaeus strain KPS-C004 obtained from the plant-parasitic nematode infested soil suppressed up to 58% of root knot disease of chili caused by Meloidogyne incognita in a greenhouse experiment. The strain promoted plant growth by increasing biomass, shoot and root length by 81%, 46% and 100%, respectively. The same trend was observed with plant nutrients, increases of nitrogen, phosphorus, potassium, calcium, magnesium and iron in chili in the treatments inoculated with spores of the strain KPS-C004 were up to 27%, 27%, 40%, 18%, 37% and 137%, respectively. The highest control efficiency of the strain was recorded when inoculated its spores in the vicinity of chili roots before nematode invasion. In addition, S. galilaeus strain KPS-C004 was capable of surviving and proliferating in the nematode infested soils throughout the entire 45days of a cultivation period. However, reinoculation at every 15days is recommended in order to achieve better control potential of the strain. Besides its biological control potential and plant growth-promoting attributes, the strain did not affect soil bacterial community. Altogether, its beneficial characteristics suggested that S. galilaeus strain KPS-C004 could be a potential biocontrol agent for being integrated in the root knot disease management program.

Streptomyces rhizosphaerihabitans sp. nov. and Streptomyces adustus sp. nov., isolated from bamboo forest soil.

Three novel isolates belonging to the genus Streptomyces, designated JR-35T, JR-46 and WH-9T, were isolated from bamboo forest soil in Damyang, Korea. The 16S rRNA gene sequences of strains JR-35T and JR-46 showed highest similarities with Streptomyces olivochromogenes NBRC 3178T (99.1 %), Streptomyces siamensis KC-038T (98.9 %), Streptomyces chartreusis NBRC 12753T (98.9 %), Streptomyces resistomycificus NRRL ISP-5133T (98.9 %) and Streptomyces bobili JCM 4627T (98.8 %), and strain WH-9Tshowed highest sequence similarities with Streptomyces. bobili JCM 4627T (99.2 %), Streptomyces phaeoluteigriseus NRRL ISP-5182T (99.2 %), Streptomyces alboniger NBRC 12738T (99.2 %), Streptomyces galilaeus JCM 4757T (99.1 %) and Streptomyces pseudovenezuelae NBRC 12904T (99.1 %). The predominant menaquinones were MK-9 (H6) and MK-9 (H8). The major fatty acids were anteiso-C15 : 0, iso-C16 : 0, iso-C14 : 0 and iso-C15 : 0 for strains JR-35T and JR-46 and anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0 for strain WH-9T. The G+C content of the genomic DNA of strains JR-35T, JR-46 and WH-9T were 69.4, 74.4 and 74.1 mol%, respectively. Based on the phenotypic and genotypic data, the three strains are assigned to two novel species of the genus Streptomyces, for which the names Streptomyces rhizosphaerihabitans sp. nov. (type stain JR-35T=KACC 17181T=NBRC 109807T) and Streptomyces adustus sp. nov. (type strain WH-9T=KACC 17197T=NBRC 109810T) are proposed.

Antifungal performance of extracellular chitinases and culture supernatants of Streptomyces galilaeus CFFSUR-B12 against Mycosphaerella fijiensis Morelet.

The tropical and mycoparasite strain Streptomyces galilaeus CFFSUR-B12 was evaluated as an antagonist of Mycosphaerella fijiensis Morelet, causal agent of the Black Sigatoka Disease (BSD) of banana. On zymograms of CFFSUR-B12 culture supernatants, we detected four chitinases of approximately 32 kDa (Chi32), 20 kDa (Chi20), and two with masses well over 170 kDa (ChiU) that showed little migration during denaturing electrophoresis at different concentrations of polyacrylamide. The thymol-sulphuric acid assay showed that the ChiU were glycosylated chitinases. Moreover, matrix assisted laser desorption ionization time-of-flight MS analysis revealed that the ChiU are the same protein and identical to a family 18 chitinase from Streptomyces sp. S4 (gi|498328075). Chi32 was similar to an extracellular protein from Streptomyces albus J1074 (gi|478687481) and Chi20 was non-significantly similar to chitinases from five different strains of Streptomyces (P > 0.05). Subsequently, Chi32 and Chi20 were partially purified by anion exchange and hydrophobic interaction chromatography and tested against M. fijiensis. Chitinases failed to inhibit ascospore germination, but inhibited up to 35 and 62% of germ tube elongation and mycelial growth, respectively. We found that crude culture supernatant and living cells of S. galilaeus CFFSUR-B12 were the most effective in inhibiting M. fijiensis and are potential biocontrol agents of BSD.

First Report of Streptomyces galilaeus Associated with Common Scab in China.

During a survey of potato scab pathogens in China from 2003 to 2012, a new pathogen was found in Shanxi and Neimenggu provinces. The incidence was approximately 20% of all recovered strains. The lesions caused by the pathogen were slightly raised and similar to those caused by Streptomyces scabies (3). Lesions were excised (approximately 10 mm3) from 40 infected tubers, surface-disinfested with 0.3% NaOCl for 30 s, rinsed in sterile water three times, cut into 5 mm3, then sliced into 1-mm pieces, and plated on water agar amended with ampicillin (50 μg/ml). Plates were incubated at 28°C in the dark for 4 days. The spores of Streptomyces sp. strains growing from the tuber pieces were collected from single bacterial colonies and cultured on oatmeal agar. To fulfill Koch's postulates, one strain, CPS-2, was grown at 28°C for 10 days and the spores were washed from the plates as inoculum. One hundred milliliters of inoculum (1 × 105 CFU/ml) was mixed with autoclaved soil and vermiculite (1:1) in each pot (15 cm in diameter). Cut tubers were planted in the pots (potato cv. Favorita, one plant per pot, five replicates) and grown under greenhouse conditions (22 ± 5°C). Typical common scab symptoms consisting of small, brown, raised lesions developed on potato tubers 12 weeks after planting. The same strain was re-isolated from the lesions of the new scabby tubers. Non-inoculated plants, treated as described above, but without strain CPS-2, remained healthy. The CPS-2 strain was identified based on morphological and physiological characterization and 16S rDNA sequence. On yeast-malt extract agar, the test strain produced grayish-white aerial hypha, reddish brown substrate mycelium and pigments, and loose spiral spore chains. Spores were smooth and were 0.8 to 0.9 × 1.1 to 1.2 μm in size (diameter and length). The ability of the strain to use single sources of carbon and nitrogen was verified according to the International Streptomyces project (4). The strain grew in media supplemented with L-arabinose, D-fructose, D-glucose, rhamnose, raffinose, meso-inositol, sucrose, and D-xylose, but not D-mannitol. It used L-hydroxyproline, L-methionine, and L-histidine, and produced melanin on tyrosine and peptone yeast extract agar. The strain did not grow at a pH less than 5.0 and was sensitive to streptomycin (20 μg/ml), phenol (0.1%), and crystal violet (0.5 μg/mL), but not to penicillin (10 IU/ml). The strain also produced hydrogen sulfide. The biological characteristics of strain CPS-2 were in accord with Streptomyces galilaeus. CPS-2 produced thaxtomin A in oatmeal liquid medium and the txt AB gene fragment was successfully amplified using specific primers (2). The 16S rDNA sequence of CPS-2 was amplified by PCR with primers 16S1-F: 5'-CATTCACGGAGAGTTTGATCC-3' and 16S1-R: 5'-AGAAAGGAGGTGATCCAGCC-3' (1) and sequenced. A BLAST search of the 16S rDNA sequence for CPS-2 was conducted using the NCBI GenBank database, resulting in 99.8% similarity to S. galilaeus (NR_040857). The 16S rDNA sequence for CPS-2 (1,388 bp) was deposited in GenBank (AY621378). To our knowledge, this is the first report of S. galilaeus causing common scab of potato in China.

ChemInform Abstract: Hybrid Anthracyclines from a Genetically Engineered Streptomyces galilaeus Mutant.

ChemInform Abstract: Hybrid Anthracyclines from a Genetically Engineered Streptomyces galilaeus Mutant.

Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA–DNA hybridization, and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens

The Streptomyces albidoflavus 16S rRNA gene clade contains 10 species and subspecies with identical 16S rRNA gene sequences and very similar numerical taxonomic data, including Streptomyces griseus subsp. solvifaciens. Type strains of this clade, as well as three CGMCC strains which were received as Streptomyces galilaeus, Streptomyces sioyaensis and Streptomyces vinaceus, respectively, that shared the same 16S rRNA gene sequences with the clade, were subjected to multilocus sequence analysis (MLSA), DNA–DNA hybridization (DDH) and phenotypic characterization for a comprehensive reevaluation. The 13 strains still formed a distinct, albeit loosely related, clade in the phylogenetic tree based on concatenated sequences of aptD, gyrB, recA, rpoB and trpB genes, supported by a high bootstrap value and different tree-making algorithms, with MLSA evolutionary distances ranging from 0 to 0.003. DDH values among these strains were well above the 70% cut-off point for species delineation. Based on the genotypic data of MLSA and DDH, combined with key phenotypic properties in common, it is proposed that the 10 species and subspecies of the S. albidoflavus clade, namely S. albidoflavus, S. canescens, S. champavatii, S. coelicolor, S. felleus, S. globisporus subsp. caucasicus, S. griseus subsp. solvifaciens, S. limosus, S. odorifer and S. sampsonii, should be merged into a single genomic species, for which the name S. albidoflavus is retained, and that the three strains S. galilaeus CGMCC 4.1320, S. sioyaensis CGMCC 4.1306 and S. vinaceus CGMCC 4.1305 should be assigned to S. albidoflavus as well. The results also indicated that MLSA could be the procedure of choice for distinguishing between species within Streptomyces 16S rRNA gene clades.

Crystal Structure of the Polyketide Cyclase AknH with Bound Substrate and Product Analogue: Implications for Catalytic Mechanism and Product Stereoselectivity

AknH is a small polyketide cyclase that catalyses the closure of the fourth carbon ring in aclacinomycin biosynthesis in Streptomyces galilaeus, converting aklanonic acid methyl ester to aklaviketone. The crystal structure analysis of this enzyme, in complex with substrate and product analogue, showed that it is closely related in fold and mechanism to the polyketide cyclase SnoaL that catalyses the corresponding reaction in the biosynthesis of nogalamycin. Similarity is also apparent at a functional level as AknH can convert nogalonic acid methyl ester, the natural substrate of SnoaL, to auraviketone in vitro and in constructs in vivo. Despite the conserved structural and mechanistic features between these enzymes, the reaction products of AknH and SnoaL are stereochemically distinct. Supplied with the same substrate, AknH yields a C9-R product, like most members of this family of polyketide cyclases, whereas the product of SnoaL has the opposite C9-S stereochemistry. A comparison of high-resolution crystal structures of the two enzymes combined with in vitro mutagenesis studies revealed two critical amino acid substitutions in the active sites, which contribute to product stereoselectivity in AknH. Replacement of residues Tyr15 and Asn51 of AknH, located in the vicinity of the main catalytic residue Asp121, by their SnoaL counter-parts phenylalanine and leucine, respectively, results in a complete loss of product stereoselectivity.

Expression, purification, and characterization of AknX anthrone oxygenase, which is involved in aklavinone biosynthesis in Streptomyces galilaeus.

In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus. In order to identify its function, the aknX gene was expressed in Escherichia coli. The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His(6) tag was efficiently purified to homogeneity by Ni(2+) affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.

Characterization of mutations in aclacinomycin A-non-producing Streptomyces galilaeus strains with altered glycosylation patterns.

In this study a set of Streptomyces galilaeus ATCC 31615 mutants was characterized, which are incapable of synthesizing some or all of the deoxyhexose sugars of aclacinomycin A. Complementation experiments with the the mutant strains H026, H038, H039, H054, H063, H065 and H075 were carried out with glycosylation genes previously derived from the wild-type S. galilaeus. Mutations in strains H038, H063 and H075 were complemented with single PCR-amplified genes. Furthermore, amplification and sequencing of the corresponding genes from the mutant strains revealed single point mutations in the sequences. First, in H038 a transition mutation in aknQ, encoding a putative dTDP-hexose 3-ketoreductase, causes an amino acid substitution from glycine to aspartate, suppressing the biosynthesis of both 2-deoxyfucose and rhodinose and thus leading to the accumulation of aclacinomycin T with rhodosamine as its only sugar. Second, in H063, which accumulates aklavinone without a sugar moiety, amino acid substitution occurs, with threonine being substituted by isoleucine in dTDP-glucose synthase, the first enzyme participating in deoxyhexose biosynthesis, encoded by aknY. Third, a nonsense mutation in aknP leads to truncated dTDP-hexose 3-dehydratase in H075, which is incapable of synthesizing rhodinose. In addition, mutants H054 and H065, which accumulate aclacinomycins without aminosugars, were complemented by a gene for an aminotransferase, aknZ. Characterization of the nature of the mutations adds to the usefulness and value of the mutants in the analysis of gene function and in the creation of novel compounds by combinatorial biosynthesis. Furthermore, these results strengthen the assignments of akn gene products and enlighten the biosynthetic pathway for deoxyhexoses.

Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster

We have cloned and sequenced polyketide synthase (PKS) genes from the aclacinomycin producer Streptomyces galilaeus ATCC 31,615. The sequenced 13.5-kb region contained 13 complete genes. Their organization as well as their protein sequences showed high similarity to those of other type II PKS genes. The continuous region included the genes for the minimal PKS, consisting of ketosynthase I (aknB), ketosynthase II (aknC), and acyl carrier protein (aknD). These were followed by the daunomycin dpsC and dpsD homologues (aknE2 and F, respectively), which are rare in type II PKS clusters. They are associated with the unusual starter unit, propionate, used in the biosynthesis of aklavinone, a common precursor of aclacinomycin and daunomycin. Accordingly, when aclacinomycins minimal PKS genes were substituted for those of nogalamycin in the plasmid carrying genes for auramycinone biosynthesis, aklavinone was produced in the heterologous hosts. In addition to the minimal PKS, the cloned region included the PKS genes for polyketide ketoreductase (aknA), aromatase (aknE1) and oxygenase (aknX), as well as genes putatively encoding an aklanonic acid methyl transferase (aknG) and an aklanonic acid methyl ester cyclase (aknH) for post-polyketide steps were found. Moreover, the region carried genes for an activator (aknI), a glycosyl transferase (aknK) and an epimerase (aknL) taking part in deoxysugar biosynthesis.

Modifications of aclacinomycin T by aclacinomycin methyl esterase (RdmC) and aclacinomycin-10-hydroxylase (RdmB) from Streptomyces purpurascens

The genes rdmB and rdmC of Streptomyces purpurascens encoding aclacinomycin modifying enzymes RdmB and RdmC were expressed in Streptomyces lividans TK24. In contrast to the earlier suggestion that RdmC may be an esterase that causes the removal of the carbomethoxy group from the 10 position of aclacinomycins, RdmC functions as an aclacinomycin methyl esterase and catalyzes the removal of the methoxy group from the C-15 position of aclacinomycin T producing 15-demethoxyaclacinomycin T. RdmB acts upon C-10 of 15-demethoxyaclacinomycin T and is able to remove the carboxylic group from the C-10 position. It functions also as an aclacinomycin-10-hydroxylase being able to add a hydroxyl group at the same, C-10 position in vitro. Aclacinomycin methyl esterase was purified to apparent homogeneity from S. lividans carrying the rdmC and aclacinomycin-10-hydroxylase as a glutathione S-transferase fusion construct from Escherichia coli carrying the rdmB gene, respectively. Aclacinomycin methyl esterase functions as a monomer and aclacinomycin-10-hydroxylase as a tetramer. Aclacinomycin methyl esterase has an exceptionally high temperature stability and has an apparent K m for aclacinomycin T of 15.5 μM. The introduction of rdmC and rdmB in a Streptomyces galilaeus mutant HO38 produced the same modifications of aclacinomycin T in vivo as aclacinomycin methyl esterase and aclacinomycin-10-hydroxylase in vitro.

Folding of the polyketide chain is not dictated by minimal polyketide synthase in the biosynthesis of mithramycin and anthracycline.

Mithramycin, nogalamycin and aclacinomycins are aromatic polyketide antibiotics that exhibit antitumour activity. The precursors of these antibiotics are formed via a polyketide biosynthetic pathway in which acetate (for mithramycinone and nogalamycinone) or propionate (for aklavinone) is used as a starter unit and nine acetates are used as extender units. The assembly of building blocks is catalyzed by the minimal polyketide synthase (PKS). Further steps include regiospecific reductions (if any) and cyclization. In the biosynthesis of mithramycin, however, ketoreduction is omitted and the regiospecificity of the first cyclization differs from that of anthracycline antibiotics (e.g. nogalamycin and aclacinomycins). These significant differences provide a convenient means to analyze the determinants for the regiospecificity of the first cyclization step. In order to analyze a possible role of the minimal PKS in the regiospecificity of the first cyclization in polyketide biosynthesis, we expressed the mtm locus, which includes mithramycin minimal PKS genes, in Streptomyces galilaeus, which normally makes aclacinomycins, and the sno locus, which includes nogalamycin minimal PKS genes, in Streptomyces argillaceus, which normally makes mithramycin. The host strains are defective in the minimal PKS, but they express other antibiotic biosynthesis genes. Expression of the sno minimal PKS in the S. argillaceus polyketide-deficient strain generated mithramycin production. Auramycins, instead of aclacinomycins, accumulated in the recombinant S. galilaeus strains, suggesting that the mithramycin minimal PKS is responsible for the choice of starter unit. We also describe structural analysis of the compounds accumulated by a ketoreductase-deficient S. galilaeus mutant; spectroscopic studies on the major polyketide compound that accumulated revealed a first ring closure which is not typical of anthracyclines, suggesting an important role for the ketoreductase in the regiospecificity of the first cyclization. These experiments clearly support the involvement of ketoreductase and a cyclase in the regiospecific cyclization of the biosynthetic pathway for aromatic polyketides.

Hybrid anthracyclines by a genetically engineered Streptomyces galilaeus mutant

A Streptomyces galilaeus mutant carrying genes transfered from S. purpurascens is reported to produce two hybrid anthracyclines. This study demonstrates the possibility of producing hybrid anthracyclines in a combinatorial way by genetic engineering of anthracycline producing strains.

Production of hybrid anthracycline antibiotics by heterologous expression of Streptomyces nogalater nogalamycin biosynthesis genes.

A cluster of anthracycline biosynthetic genes isolated from Streptomyces nogalater was expressed in Streptomyces lividans and in Streptomyces galilaeus. A 12 kb DNA fragment cloned from this cluster in pIJ486 caused the production of a novel compound when introduced into S. lividans. The compound is derived from nogalonic acid methyl ester, an early intermediate in nogalamycin biosynthesis. Complementation with the cloned 12 kb fragment of S. galilaeus mutants blocked in aclacinomycin biosynthesis caused the production of hybrid anthracyclines. Cloning of the nogalamycin gene cluster should make possible a detailed study of the biosynthesis of this interesting antibiotic, as well as the production of novel anthracyclines of potential value as cytostatic drugs.

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

We have analyzed an anthracycline biosynthesis gene cluster from Streptomyces 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 to Streptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant of S. nogalater. The S. galilaeus mutants carrying the S. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product of snoA seems to be needed for expression of at least the genes for the minimal PKS.