Self cloning in Micromonospora olivasterospora of fms genes for fortimicin A (astromicin) biosynthesis.

Summary We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employs the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pM0116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.     

Cloning and identification of saprolmycin biosynthetic gene cluster from Streptomyces sp. TK08046

Saprolmycins A–E are anti-Saprolegnia parasitica antibiotics. To identify the gene cluster for saprolmycin biosynthesis in Streptomyces sp. TK08046, polymerase chain reaction using aromatase and cyclase gene-specific primers was performed; the spr gene cluster, which codes for angucycline biosynthesis, was obtained from the strain. The cluster consists of 36 open reading frames, including minimal polyketide synthase, ketoreductase, aromatase, cyclase, oxygenase, and deoxy sugar biosynthetic genes, as defined by homology to the corresponding genes of the urdamycin, Sch-47554, and grincamycin biosynthetic gene clusters in Streptomyces fradiae, Streptomyces sp. SCC-2136, and Streptomyces lusitanus, respectively. To establish the function of the gene cluster, an expression cosmid vector containing all 36 open reading frames was introduced into Streptomyces lividans TK23. The transformant was confirmed to express the biosynthetic genes and produce saprolmycins by liquid chromatography–mass spectrometry analysis of the extract.     

Genetic organization of the biosynthetic gene cluster for the indolocarbazole K-252a in <Emphasis Type="Italic">Nonomuraea longicatena</Emphasis> JCM 11136

Indolocarbazole metabolite K-252a is a natural product that was previously reported as a potent protein kinase C inhibitor with in vitro and in vivo potency. From a biosynthetic viewpoint, this compound possesses structurally interesting features such as an unusual furanosyl sugar moiety, which are absent in the well-studied staurosporine and rebeccamycin. A cosmid library from genomic DNA of Nonomuraea longicatena JCM 11136 was constructed and screened for the presence of genes to be involved in the biosynthesis of indolocarbazole K-252a. Using as a probe an internal fragment of vioB, a Chromobacterium violaceum gene encoding a multifunctional enzyme that catalyzes tryptophan decarboxylation and condensation reaction in violacein biosynthesis, we isolated a DNA region that directed the biosynthesis of K-252a when introduced into the heterologous expression host Streptomyces albus. Sequence analysis of 45 kb revealed genes for indolocarbazole core formation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes. The cloned genes should help to elucidate the molecular basis for indolocarbazole biosynthesis and generate new indolocarbazole analogues by genetic engineering.     

A novel, highly efficient gene-cloning system in Micromonospora applied to the genetic analysis of fortimicin biosynthesis.

We have developed a gene-cloning system in Micromonospora olivasterospora, a fortimicin A (astromicin) producer. Plasmids of Micromonospora from two strains of M. olivasterospora were used for construction of the vectors. Two antibiotic-resistance genes, nmrA and nmrB, cloned from a neomycin-producing Micromonospora, were introduced into these plasmids for the selection of transformants. In a new protoplasting protocol for lysozyme-resistant bacteria, protoplasts of M. olivasterospora were found in short-time incubation with lysozyme and transformed efficiently, indicating that the method was suitable to shotgun cloning. Using this system, seven biosynthetic genes for fortimicin A were cloned. Their physical maps revealed that at least four of these genes were clustered. Analysis of a cosmid library of M. olivasterospora showed that eleven biosynthetic genes and a self-defense gene existed in a region of approx. 25 kb of DNA.     

Identification of AHBA Biosynthetic Genes Related to Geldanamycin Biosynthesis in Streptomyces hygroscopicus 17997

To clone and study the geldanamycin biosynthetic gene cluster in Streptomyces hygroscopicus 17997, we designed degenerate primers based on the conserved sequence of the ansamycin 3-amino-5-hydroxybenzoic acid (AHBA) synthase gene. A 755-bp polymerase chain reaction product was obtained from S. hygroscopicus 17997 genomic DNA, which showed high similarity to ansamycin AHBA synthase genes. Through screening the cosmid library of S. hygroscopicus 17997, two loci of separated AHBA biosynthetic gene clusters were discovered. Comparisons of sequence homology and gene organization indicated that the two AHBA biosynthetic gene clusters could be divided into a benzenic and a naphthalenic subgroup. Gene disruption demonstrated that the benzenic AHBA gene cluster is involved in the biosynthesis of geldanamycin. However, the naphthalenic AHBA genes in the genome of Streptomyces hygroscopicus 17997 could not complement the deficiency of the benzenic AHBA genes. This is the first report on the AHBA biosynthetic gene cluster in a geldanamycin-producing strain. W. He and L. Wu contributed equally to this work.     

Characterization of a key aminoglycoside phosphotransferase in gentamicin biosynthesis

Gentamicin is an aminoglycoside antibiotic obtained from cultures of Micromonospora as the important anti-infective agents. Gentamicin which lacks 3′-hydroxyl group can avoid the attack from the modification enzymes of antibiotic-resistant bacteria in clinic. Consequently, C-3′ dehydroxylation is the key step in gentamicins biosynthesis. We suppose that there are some enzymes responsible for converting intermediate JI-20A to 3′,4′-bisdehydroxylated final product gentamicin C_1a, while phosphorylation of 3′-OH is possibly the first step for C-3′ dehydroxylation. The gentamicin biosynthetic gene gntI, encoding an aminoglycoside phosphotransferase, was cloned from Micromonospora echinospora ATCC15835 and overexpressed in Escherichia coli. The resulting phosphotransferase was purified, and the kinetic parameters for Kanamycin A, Kanamycin B, Neomycin B and Amikacin were determined. Elucidation of NMR data of phosphorylated kanamycin B has unambiguously demonstrated a regiospecific phosphorylation of 3′-hydroxyl of the 6-aminohexose ring. The results described here partly confirm that the 3′-dehydroxylation step is preceded by a 3′ phosphorylation step. It is predicted that GntI belongs to a new aminoglycoside phosphotransferase group involved with aminoglycoside antibiotics biosynthesis pathway.     

Characterization of CalS9 in the biosynthesis of UDP-xylose and the production of xylosyl-attached hybrid compound

The gene cluster of calicheamicin contains calS9, which encodes UDP-GlcA decarboxylase that converts UDP-GlcA to UDP-xylose. calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21 (DE3) to characterize its putative function. The reaction product was analyzed by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by gene replacement to generate Streptomyces sp. GerSM2 mutant, which was unable to produce dihydrochalcomycin. calS7, calS8, and calS9 UDP-xylose biosynthetic genes were cloned in an integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in Streptomyces sp. GerSM2. Finally, novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was isolated and analyzed by HPLC and liquid chromatography–mass spectrometry.     

Identification of functionally clustered nystatin-like biosynthetic genes in a rare actinomycetes, <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

The polyene antibiotics, including nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very valuable antifungal polyketide compounds, and they are typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain genes potentially encoding polyene biosynthesis. Here, sequence information of an approximately 125.7-kb contiguous DNA region in five overlapping cosmids isolated from the P. autotrophica KCTC9441 genomic library revealed a total of 23 open reading frames, which are presumably involved in the biosynthesis of a nystatin-like compound tentatively named NPP. The deduced roles for six multi-modular polyketide synthase (PKS) catalytic domains were found to be highly homologous to those of previously identified nystatin biosynthetic genes. Low NPP productivity suggests that the functionally clustered NPP biosynthetic pathway genes are tightly regulated in P. autotrophica. Disruption of a NPP PKS gene completely abolished both NPP biosynthesis and antifungal activity against Candida albicans, suggesting that polyene-specific genome screening may constitute an efficient method for isolation of potentially valuable previously identified polyene genes and compounds from various rare actinomycetes widespread in nature.     

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.     

Cloning and analysis of geldanamycin partial biosynthetic gene cluster of streptomyces hygroscopicus 17997

A geldanamycin (GDM)-producing strain, Streptomyces hygroscopicus 17997, was isolated from the soil of Yunnan, China, by the researchers of the Institute of Medicinal Biotechnology, CAMS & PUMC. GDM is an ansamycin antibiotic, which has the ability to bind with Hsp90 (heat shock protein 90) and alter its function. Hsp90 plays a key role in regulating the physiology of cells exposed to environmental stress and in maintaining the malignant phenotype of tumor cells. As an inhibitor of Hsp90, GDM possesses potent antitumoral and antiviral bioactivity, but the hypatotoxicity and poor solubility in water limit its clinical use. To accomplish the structural modification of GDM by genetic means, an attempt to obtain the biosynthetic gene cluster of GDM from S. hygroscopicus 17997 was made. In this study, a pair of primers was designed according to a conserved sequence of one of the possible post-PKS (polyketides synthase) modification genes, the carbamoyltransferase (CT) gene (gdmN) in GDM biosynthesis. The 732-bp PCR product was obtained from the S. hygroscopicus 17997 genomic DNA. Through the colony-PCR Binary Search Method, using the CT gene primers, six positive cosmid clones, CT1-6, were identified from the S. hygroscopicus 17997 cosmid genomic library. The CT-4 positive cosmid was then sub-cloned and sequenced. Approximately 28.356 kb of foreign gene sequence from CT-4 cosmid and by further PCR extension reaction was obtained. According to BLAST analysis, this sequence contains 13 possible ORFs, and they are believed to be involved in GDM production. The obtained possible GDM biosynthetic gene cluster in S. hygroscopicus 17997 will facilitate the further functional analysis of the genes and the modification of the structure of GDM through combinatorial biosynthesis.     

A fragmented aflatoxin-like gene cluster in the forest pathogen Dothistroma septosporum

The polyketide toxin dothistromin is very similar in structure to the aflatoxin precursor, versicolorin B. Dothistromin is made by a pine needle pathogen, Dothistroma septosporum, both in culture and in planta. Orthologs of aflatoxin biosynthetic genes have been identified that are required for dothistromin biosynthesis in D. septosporum. In contrast to the situation in aflatoxin-producing fungi where 25 aflatoxin biosynthetic and regulatory genes are tightly clustered in one region of the genome, the dothistromin gene cluster is fragmented. Three mini-clusters of dothistromin genes have been identified, each located on a 1.3-Mb chromosome and each grouped with non-dothistromin genes. There are no obvious patterns of repeated sequences or transposon relics to suggest recent recombination events. Most dothistromin genes within the mini-clusters are co-regulated, suggesting that coordinate control of gene expression is achieved despite this unusual arrangement of secondary metabolite biosynthetic genes.     

Isolation and partial characterization of a cryptic polyene gene cluster in Pseudonocardia autotrophica

The polyene antibiotics, a category that includes nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very promising antifungal polyketide compounds and are typically produced by soil actinomycetes. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly similar cytochrome P450 hydroxylase (CYP) genes. Using polyene CYP-specific PCR screening with several actinomycete genomic DNAs, Pseudonocardia autotrophica was determined to contain a unique polyene-specific CYP gene. Genomic DNA library screening using the polyene-specific CYP gene probe identified a positive cosmid clone, which contained a DNA fragment of approximately 34.5 kb. The complete sequencing of this DNA fragment revealed a total of seven complete and two incomplete open reading frames, which were found to be highly similar, but still unique, when compared to previously known polyene biosynthetic genes. These results suggest that the polyene-specific screening approach may constitute an efficient method for the isolation of potentially valuable cryptic polyene biosynthetic gene clusters from various rare actinomycetes.     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

水杨酸诱导下蝙蝠蛾拟青霉转录组分析及虫草酸代谢途径关键酶基因挖掘

【目的】虫草酸是虫草中重要的活性成分之一,但其低含量极大地限制了其工业应用。水杨酸(salicylic acid, SA)是一种非生物诱导子,可以显著提高蝙蝠蛾拟青霉中虫草酸的合成,但蝙蝠蛾拟青霉虫草酸代谢途径及其对水杨酸的响应尚不明确。本研究旨在获得蝙蝠蛾拟青霉响应SA处理的转录组学信息,挖掘蝙蝠蛾拟青霉中虫草酸代谢途径关键酶基因。【方法】采用SA诱导培养蝙蝠蛾拟青霉,8 h后选取诱导和未诱导的菌丝进行转录组高通量测序分析。【结果】测序最终获得40.37 Gb的clean data,拼接得到20 317条unigene,平均长度为1 357.13 bp,功能注释共获得13 592条unigene。差异基因分析共筛选出差异基因2 574个,其中有1 135个上调,1 439个下调。KEGG富集分析表明,差异基因主要富集于细胞周期、减数分裂、半乳糖代谢、DNA复制、糖醇脂类生物合成、甘油脂类代谢等KEGG通路中。进一步分析得到与虫草酸代谢相关的基因13条,其中参与虫草酸生物合成的基因glk、gpi、gla、mpi、fbp、mtld在SA处理后表达量上调,而涉及虫草酸消耗的基因mdh在SA...     

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.     

Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5-triphosphate (UTP) to cytidine 5-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.     

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of ~1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.     

Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways

Mycobacteria produce two siderophores, mycobactin and exochelin. Mycobacterium smegmatis mutants defective in the production of exochelin were isolated using agar medium containing chrome azural S for the sensitive detection of siderophores. Cosmids of genomic libraries from M. smegmatis and Mycobacterium bovis BCG were screened for complementation of the mutation. Subcloning of the complementing M. smegmatis cosmid identified a 4.3 kb fragment required for restoring exochelin biosynthesis. Sequencing of the DNA revealed four open reading frames whose genes were named fxuA. fxuB, fxuC, and fxbA. FxuA, FxuB, and FxuC share amino acid sequence homology with the iron permeases FepG, FepC. and FepD from Escherichia coli. respectively. Deletion analysis identified fxbA as the gene required to restore exochelin biosynthesis in our mutant. Although fxbA does not share amino acid sequence homology with any of the published sequences for siderophore biosynthetic genes, it does show limited homology with the phosphoribosyl-glycineamide formyltransferases (GAR enzymes) and methionyl-tRNA formyltransferase over a limited region of the sequence, suggesting that fxbA may code for an enzyme which adds a formyl group in the synthesis of exochelin. A fusion of fxbA with the E. coli lacZ gene demonstrated regulation of gene expression by iron. The ability of M. smegmatis mutants to produce mycobactin in the absence of exochelin supports the hypothesis that exochelin is not a precursor of mycobactin and suggests that the siderophores have independent biosynthetic pathways. In addition, complementation of the M.