Nucleotide sequence of a carboxyphosphonoenolpyruvate phosphonomutase gene isolated from a bialaphos-producing organism, Streptomyces hygroscopicus, and its expression in Streptomyces lividans.

Summary The carboxyphosphonoenolpyruvate (CPEP) phosphonomutase gene of bialaphos-producing Streptomyces hygroscopicus, which encodes a C-P bond forming enzyme was cloned into Streptomyces lividans and sequenced. The amino acid composition of the protein coded in an open reading frame of 295 codons and its calculated molecular mass, 32,800 Da, coincided well with those of the purified enzyme. Introduction of the CPEP phosphonomutase gene, the expression of which is controlled by the promoter of the aph gene, into S. lividans resulted in the production of this enzyme at a level almost equivalent to that in the parent strain.     

Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis

A gene library constructed from large (20 kb) fragments of total DNA from the geldananmycin-producing strain Streptomyces hygroscopicus 3602 cloned in the plasmid vector pIJ61 were used to transform S. lividans TK24. Three transformants of about 800 tested were found to have acquired the ability to produce an antibiotic lethal to a geldanamycin-sensitive strain of Bacillus subtilis. The plasmids isolated from these transformants, pIA101, pIA102 and pIA103, each contained an insert of 15 kb. A 4.5 kb DNA fragment from the insert in pIA102 hybridised to DNA from S. hygroscopicus 3602 and to DNA encoding part of the erythromycin polyketide synthase but not to S. lividans TK24 DNA. The integration-defective phage vector C31 KC515 containing this 4.5 kb fragment was able to lysogenise S. hygroscopicus 3602 to produce lysogens defective in geldanamycin production. Loss of the prophage restored the ability to produce geldanamycin. Extracts of fermentation broth cultures of S. lividans containing pIA101, pIA102 and pIA102 and pIA103 analysed by thin-layer chromatography (TLC) contained compounds identical or very similar to purified geldanamycin, which were not present in S. lividans. These compounds showed a mass spectrum indistinguishable from geldanamycin. The evidence suggests that the clones contain DNA sequences encoding functions required for geldanamycin biosynthesis including components of the polyketide synthase.     

Cloning, overexpression, and purification of novobiocic acid synthetase from Streptomyces spheroides NCIMB 11891

Novobiocic acid synthetase, a key enzyme in the biosynthesis of the antibiotic novobiocin, was cloned from the novobiocin producer Streptomyces spheroides NCIMB 11891. The enzyme is encoded by the gene novL, which codes for a protein of 527 amino acids with a calculated mass of 56,885 Da. The protein was overexpressed as a His(6) fusion protein in Escherichia coli and purified to apparent homogeneity by affinity chromatography and gel chromatography. The purified enzyme catalyzed the formation of an amide bond between 3-dimethylallyl-4-hydroxybenzoic acid (ring A of novobiocin) and 3-amino-4,7-dihydroxy-8-methyl coumarin (ring B of novobiocin) in an ATP-dependent reaction. NovL shows homology to the superfamily of adenylate-forming enzymes, and indeed the formation of an acyl adenylate from ring A and ATP was demonstrated by an ATP-PP(i) exchange assay. The purified enzyme exhibited both activation and transferase activity, i.e. it catalyzed both the activation of ring A as acyl adenylate and the subsequent transfer of the acyl group to the amino group of ring B. It is active as a monomer as determined by gel filtration chromatography. The reaction was specific for ATP as nucleotide triphosphate and dependent on the presence of Mg(2+) or Mn(2+). Apparent K(m) values for ring A and ring B were determined as 19 and 131 micrometer respectively. Of several analogues of ring A, only 3-geranyl-4-hydroxybenzoate and to a lesser extent 3-methyl-4-aminobenzoate were accepted as substrates.     

Cloning, analysis, and overexpression of the gene encoding isobutylamine N-hydroxylase from the valanimycin producer, Streptomyces viridifaciens.

The flavoprotein isobutylamine N-hydroxylase (IBAH) catalyzes the oxidation of isobutylamine to isobutylhydroxylamine, a key step in the biosynthesis of the azoxy antibiotic valanimycin. By using oligonucleotide primers designed from peptide sequence information derived from native IBAH, a fragment of the gene (vlmH) encoding IBAH was amplified by PCR from a genomic library of the valanimycin-producing organism, Streptomyces viridifaciens MG456-hF10. The gene fragment was then employed as a probe to clone the entire vlmH gene from an S. viridifaciens genomic library. Overexpression of the vlmH gene in Escherichia coli gave a soluble protein that was purified to homogeneity. The purified protein exhibited the catalytic activity expected for IBAH. The deduced amino acid sequence of IBAH exhibited the greatest similarity to the Sox/DszC protein from Rhodococcus sp. strain IGT38, a flavoprotein involved in the oxidation of dibenzothiophene to the corresponding sulfone. Significant similarities were also found between the amino acid sequence of IBAH and those of the acyl coenzyme A dehydrogenases.     

吸水链霉菌17997格尔德霉素部分生物合成基因簇的克隆和分析

吸水链霉菌17997(Streptomyceshy groscopicus17997)是我所从中国云南土壤中分离到的格尔德霉素(geldanamycin,GDM)产生菌,GDM具有良好的抗肿瘤和抗病毒活性,但其肝毒性和水溶性差的缺点限制了其在临床上的应用。为了实现对GDM结构的生物学改造,首先要获得GDM的生物合成基因。根据GDM后修饰基因——氨甲酰基转移酶基因(gdmN)的保守序列筛选S.hygroscopicus17997的柯斯质粒基因组文库,共获得6个阳性克隆,选择CT-4阳性柯斯质粒进行亚克隆和测序,又通过PCR延伸的方法获得了与CT4连锁的将近5kb的外源序列,共获得28.356kb的外源DNA序列,其中包含了13个可能阅读框架,通过同源比较证实该序列与S.hygroscopicusNRRL3602中的GDM生物合成基因有很高的同源性。为进一步研究GDM生物合成基因的功能,并通过组合生物学的方法改造GDM的结构奠定了基础。     

Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum.

The kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated. BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s-1 and 105 s-1 for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s-1 for BCO and 232 s-1 for SCO (in the presence of 1% Thesit and 10% 2-propanol). With reduced SCO the rate of Delta5-6-->Delta4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s-1). With oxidized SCO and BCO the rate of isomerization is much faster ( approximately 300 s-1), thus it is not rate-limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates approximately 250 s-1 and 1.3 s-1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme-product complexes.     

Pentalenolactone I and hygromycin A, immunosuppressants produced by Streptomyces filipinensis and Streptomyces hygroscopicus.

Immunosuppressants isolated from Streptomyces filipinensis and S. hygroscopicus were identified with pentalenolactone I and hygromycin A, respectively. The compounds as well as cyclosporin A showed immunosuppressant activity in the mixed lymphocyte reaction, and pentalenolactone I and cyclosporin A suppressed IL-2 production, however, hygromycin A did not. Hygromycin A may have immunosuppressant activity by a different mechanism from pentalenolactone I, cyclosporin A and tacrolimus.     

Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus

A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide-resistant plants.     

Streptomyces hygroscopicus has two glutamine synthetase genes.

Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII). The glnB gene was cloned from S. hygroscopicus DNA by complementation in an Escherichia coli glutamine auxotrophic mutant (glnA). glnB was subcloned in Streptomyces plasmids by insertion into pIJ486 (pMSG3) and pIJ702 (pMSG5). Both constructions conferred resistance to the tripeptide form of phosphinothricin (bialaphos) and were able to complement a glutamine auxotrophic marker in S. coelicolor. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of S. lividans(pMSG5) revealed a highly overexpressed 40-kilodalton protein. When GS was purified from this strain, it was indistinguishable in apparent molecular mass from the 40-kilodalton protein. The nucleic acid sequence of the cloned region contained an open reading frame which encoded a protein whose size, amino acid composition, and N-terminal sequence corresponded to those of the purified GS. glnB had a high G + C content and codon usage typical of streptomycete genes. A comparison of its predicted amino acid sequence with the protein data bases revealed that it encoded a GSII-type enzyme which had previously been found only in various eucaryotes (47 to 50% identity) and nodulating bacteria such as Bradyrhizobium spp. (42% identity). glnB had only 13 to 18% identity with eubacterial GSI enzymes. Southern blot hybridization experiments showed that sequences similar to glnB were present in all of the five other Streptomyces species tested, as well as Frankia species. These results do not support the previous suggestion that GSII-type enzymes found in members of the family Rhizobiaceae represent a unique example of interkingdom gene transfer associated with symbiosis in the nodule. Instead they imply that the presence of more than one gene encoding GS may be more common among soil microorganisms than previously appreciated.     

快速鉴定格尔德霉素产生菌——吸水链霉菌17997中的洋橄榄叶素

对格尔德霉素产生菌吸水链霉菌17997的发酵液乙酸乙酯提取物进行了硅胶板TLC 初步分离和NaOH溶液喷涂显色,对显红色、具有抗革兰阳性菌活性的条带进行了HPLC分析,提示抗革兰阳性菌活性化合物可能为大环二内酯类抗生素洋橄榄叶素;以dTDP-葡萄糖-4,6-脱水酶 (Tgd) 基因保守区设计PCR引物,扩增了吸水链霉菌17997基因组DNA中的tgd并进行了序列分析,表明吸水链霉菌17997含有洋橄榄叶素生物合成基因簇中的tgd基因;对NaOH溶液喷涂显红色的化合物进行LC-(+)-ESI-MS分析,证实     

Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602

We illustrate the use of a PCR-based method by which the genomic DNA of a microorganism can be rapidly queried for the presence of type I modular polyketide synthase genes to clone and characterize, by sequence analysis and gene disruption, a major portion of the geldanamycin production gene cluster from Streptomyces hygroscopicus var. geldanus NRRL 3602.     

Amides from Streptomyces hygroscopicus and their antimicrobial activity [corrected

Three amides, N-salicyloyl-2-aminopropan-1,3-diol (1) and 1-acetyl-N-salicyloyl-2-aminopropan-3-ol (2) including a natural product, N-salicyloyl-2-aminopropan-1-ol (3) were isolated from an ethyl acetate extract of the culture filtrate of Streptomyces hygroscopicus [corrected] The structures of these compounds were unambiguously established by interpretation of their spectral data including, a series of 1D and 2D-NMR and MS analyses. Compounds 1-3 showed significant antibacterial activity against a wide range of Gram positive and Gram negative bacteria.     

Purification and characterization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus

A hygromycin B phosphotransferase activity from Streptomyces hygroscopicus has been highly purified by ammonium sulphate fractionation followed by affinity column chromatography through Sepharose-6B-hygromycin-B. The combined active fractions showed a single protein band (41 kDa) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. When gel electrophoresis was performed under non-denaturing conditions, the single protein band promoted in situ phosphorylation of hygromycin B, indicating that this protein corresponded to the purified hygromycin B phosphotransferase. The enzyme has been purified 236-fold and approximate Km values of 0.56 microM for hygromycin B and ATP, respectively, were deduced.     

吸水链霉菌发酵生产谷氨酰胺转胺酶的补料研究

在吸水链霉菌(Streptomyces hygroscopicus)分批发酵研究的基础上,通过在菌体生长阶段指数流加葡萄糖,进行高细胞密度培养,获得了较高的菌体量;待菌体生长进入产酶期后,通过补加氮源,为产酶提供充足的氮源,其中通过流加蛋白质氮源,可以减少蛋白酶对成熟MTG的分解,促进产酶。结果表明,8~16 h采用较高的的比生长速率(0.15 h-1),后期降低比生长速率(0.10 h-1),此时得到的菌体量较高,可达到36 g/L,比分批发酵下的菌体量提高了80%。同时在培养基中添加50g/L的豆饼粉,最终酶活可达到5.79U/ml,提高了83%。     

Cloning and sequencing of the coenzyme B(12)-binding domain of isobutyryl-CoA mutase from Streptomyces cinnamonensis, reconstitution of mutase activity, and characterization of the recombinant enzyme produced in Escherichia coli.

Isobutyryl-CoA mutase (ICM) catalyzes the reversible, coenzyme B(12)-dependent rearrangement of isobutyryl-CoA to n-butyryl-CoA, which is similar to, but distinct from, that catalyzed by methylmalonyl-CoA mutase. ICM has been detected so far in a variety of aerobic and anaerobic bacteria, where it appears to play a key role in valine and fatty acid catabolism. ICM from Streptomyces cinnamonensis is composed of a large subunit (IcmA) of 62.5 kDa and a small subunit (IcmB) of 14.3 kDa. icmB encodes a protein of 136 residues with high sequence similarity to the cobalamin-binding domains of methylmalonyl-CoA mutase, glutamate mutase, methyleneglutarate mutase, and cobalamin-dependent methionine synthase, including a conserved DXHXXG cobalamin-binding motif. Using IcmA and IcmB produced separately in Escherichia coli, we show that IcmB is necessary and sufficient with IcmA and coenzyme B(12) to afford the active ICM holoenzyme. The large subunit (IcmA) forms a tightly associated homodimer, whereas IcmB alone exists as a monomer. In the absence of coenzyme B(12), the association between IcmA and IcmB is weak. The ICM holoenzyme appears to comprise an alpha(2)beta(2)-heterotetramer with up to two molecules of bound coenzyme B(12). The equilibrium constant for the ICM reaction at 30 degrees C is 1.7 in favor of isobutyryl-CoA, and the pH optimum is near 7.4. The K(m) values for isobutyryl-CoA, n-butyryl-CoA, and coenzyme B(12) determined with an equimolar ratio of IcmA and IcmB are 57 +/- 13, 54 +/- 12, and 12 +/- 2 microM, respectively. A V(max) of 38 +/- 3 units/mg IcmA and a k(cat) of 39 +/- 3 s(-1) were determined under saturating molar ratios of IcmB to IcmA.