A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-β-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-β-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-β-lysine molecules adenylated by ORF 19 are used to elongate an L-β-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-β-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.     

Functional analysis of eubacterial ent-copalyl diphosphate synthase and pimara-9(11),15-diene synthase with unique primary sequences

We have previously cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp. strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-CDP synthase and a GGDP synthase, respectively, by experiments using recombinant enzymes. In this study, ORF3, that did not show similarities with any other known proteins was expressed in Escherichia coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into PMD. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg2+ and Zn2+ for its activity. The optimum pH and temperature were 5.5 and 35 degrees C. The Km value was calculated to be 13.7 +/- 1.0 microM for GGDP and the kcat value was 3.3 x 10(-2)/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30 degrees C. The Km value for ent-CDP was estimated to be 2.6 +/- 0.2 microM and the kcat value was 1.4 x 10(-3)/sec.     

Isolation and sequence analysis of polyketide synthase genes from the daunomycin-producing Streptomyces sp. strain C5.

A contiguous region of about 30 kbp of DNA putatively encoding reactions in daunomycin biosynthesis was isolated from Streptomyces sp. strain C5 DNA. The DNA sequence of an 8.1-kbp EcoRI fragment, which hybridized with actI polyketide synthase (PKS) and actIII polyketide reductase (PKR) gene probes, was determined, revealing seven complete open reading frames (ORFs), two in one cluster and five in a divergently transcribed cluster. The former two genes are likely to encode PKR and a bifunctional cyclase/dehydrase. The five latter genes encode: (i) a homolog of TcmH, an oxygenase of the tetracenomycin biosynthesis pathway; (ii) a PKS Orf1 homolog; (iii) a PKS Orf2 homolog (chain length factor); (iv) a product having moderate sequence identity with Escherichia coli beta-ketoacyl acyl carrier protein synthase III but lacking the conserved active site; and (v) a protein highly similar to several acyltransferases. The DNA within the 8.1-kbp EcoRI fragment restored daunomycin production to two dauA non-daunomycin-producing mutants of Streptomyces sp. strain C5 and restored wild-type antibiotic production to Streptomyces coelicolor B40 (act VII; nonfunctional cyclase/dehydrase), and to S. coelicolor B41 (actIII) and Streptomyces galilaeus ATCC 31671, strains defective in PKR activity.     

Identification ofStreptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

A 50 kb region of DNA fromStreptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) fromStreptomyces griseus. Cultures ofEscherichia coli containing plasmids with ORF2, on a 2.1 kbBamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-d-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase fromStreptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism.     

Isolation and Characterization of Antibiotic LL-AC541

A new basic, water-soluble antibiotic designated LL-AC541, produced by a strain of Streptomyces hygroscopicus (NRRL 3111), was isolated as a hydrochloride salt; [alpha](D) (25), - 58 degrees , melting point, 200 to 215 C (decomposition). It is structurally related to streptothricin but does not contain beta-lysine. Isolation procedures, physicochemical properties, antimicrobial spectrum, and in vivo testing results are presented. Another very closely related antibiotic obtained from the same fermentation is briefly described.     

Biosynthesis of the lantibiotic Pep5. Isolation and characterization of a prepeptide containing dehydroamino acids

Pep5 is a tricyclic peptide antibiotic which contains the unusual amino acids dehydrobutyrine, lanthionine and 3-methyllanthionine. It is matured from a 60-amino-acid precursor peptide (pre-Pep5) deduced from the sequence of the structural gene pepA. To study the biosynthesis of Pep5 we tried to isolate the primary translation product. We identified a peptide in crude extracts of the Pep5-producing Staphylococcus epidermidis strain using antibodies raised against a synthetic 26-residue peptide representing the leader peptide region of pre-Pep5. The putative precursor was purified by reversed-phase HPLC. The isolated peptide did not react with antibodies directed against a C-terminal fragment of mature Pep5 containing two sulfide bridges. Neither lanthionine nor 3-methyllanthionine was detected in amino acid analysis of the isolated precursor. Its amino acid sequence was identical with the sequence predicted from pepA, but Edman degradation stopped at the first threonine residue of the prolantibiotic region indicating a posttranslational modification at this position. The molecular mass of the isolated peptide was 6575.4 +/- 1.7 Da, determined by ion-spray mass spectrometry. This is in agreement with a molecule being dehydrated at the four threonine and the two serine residues in the propeptide region; such a peptide has a calculated molecular mass of 6576.7 Da. The results strongly suggest that maturation of the lantibiotic Pep5 is initiated by selective dehydration of hydroxyamino acids in the propeptide region of the primary translation product and that thioether ring formation is not closely linked to dehydration.     

Development of a self-cloning system for Actinomadura verrucosospora and identification of polyketide synthase genes essential for production of the angucyclic antibiotic pradimicin.

A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 10(5) CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.     

Identification of Streptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

A 50 kb region of DNA fromStreptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) fromStreptomyces griseus. Cultures ofEscherichia coli containing plasmids with ORF2, on a 2.1 kbBamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-d-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase fromStreptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism. Dedicated to Professor Satoshi Ōmura, a pioneer in the field of antibiotics, on the occasion of his 60th birthday     

圈卷产色链霉菌尼可霉素生物合成基因——sanH和sanI的研究

通过反向遗传学方法克隆到圈卷产色链霉菌尼可霉素生物合成基因簇中约7.0kb的DNA片段。该片段除含有尼可霉素生物合成基因sanF外,对sanF上游约22kb的BglⅡDNA片段进行序列测定及分析表明,还含有两个完整的开放阅读框(ORF)。ORF1由1233个核苷酸组成,ORF2由195个核苷酸组成,它们分别编码由410个氨基酸残基和64个氨基酸残基组成的蛋白质,依次命名为sanH和sanI。蛋白序列数据库比较结果表明,SanH和SanI与浅灰链霉菌(\%Streptomyces griseolus)\%中共转录的细胞色素P450(cytochrome P450)和铁氧还蛋白(ferredoxin)有较高的同源性,一致性分别为46%和56%,相似性分别为62%和70%。基因功能研究表明,sanH基因的破坏虽不影响圈卷产色链霉菌产生的尼可霉素的生物活性,但该基因可能参与了尼可霉素羟基化反应的生物合成。     

Cloning of grisin resistance gene gsr and the study of its functioning in Streptomyces griseus Kr. strain

S. griseus Kr. is a commercial strain producing grisin, an antibiotic of the streptothricin group used as a feed additive. It was shown earlier that genetic instability of the strain was very high which was evident from a high frequency of nonreverting Grn- Grns mutants. With densitographic analysis of chromosomal DNA electrophoregrams and DNA-DNA hybridization it was revealed that the molecular basis of the genetic instability of the S. griseus strain was deletion of a DNA fragment about 20 kb in size containing a grisin resistance gene. The resistance gene designated as gsr was cloned to S. lividans TK 64 within the plasmid vector pIJ699. The restriction map of a cloned DNA fragment with a gsr gene was constructed and its similarity to that of a nat gene resistant to norseothricin, another streptothricin was observed. Introduction of a gsr gene within the multicopy plasmid pIJ699 into S. griseus 212, a highly productive strain synthesiing the antibiotic, led to an increase in its resistance and productivity. Proceeding from the preliminary data on possible linkage of a gsr gene and grisin biosynthesis genes, it appeared possible to use the cloned gene as a molecular probe in cloning the biosynthesis genes.     

Cloning and characterization of a regulatory gene of the SARP family and its flanking region from Streptomyces ambofaciens

In a search for activators of secondary metabolism we isolated a 12.6-kb DNA fragment from a genomic library of Streptomyces ambofaciens NRRL 2240 (the spiramycin producer ). Sequencing of 6 kb of the cloned fragment revealed a cluster of four ORFs (ORF1–4) whose deduced products showed similarities to those of other genes involved in polyketide biosynthesis, including a pathway-specific regulatory gene of the SARP family. The results of insertional inactivation of some of the cloned genes clearly indicate that the isolated cluster does not code for spiramycin production, suggesting that some other polyketide compound might well be produced by this strain. Received: 14 May 1999 / Accepted: 28 July 1999     

A novel N,N-8-amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) catalyzing the two terminal steps of roseoflavin biosynthesis in Streptomyces davawensis

Streptomyces davawensis synthesizes the antibiotic roseoflavin (RoF) (8-dimethylamino-8-demethyl-D-riboflavin). It was postulated that RoF is synthesized from riboflavin via 8-amino- (AF) and 8-methylamino-8-demethyl-D-riboflavin (MAF). In a cell-free extract of S. davawensis, an S-adenosyl methionine-dependent conversion of AF into MAF and RoF was observed. The corresponding N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity was enriched by column chromatography. The final most active fraction still contained at least five different proteins that were analyzed by enzymatic digestion and concomitant de novo sequencing by MS/MS. One of the sequences matched a hypothetical peptide fragment derived from an as yet uncharacterized open reading frame (sda77220) located in the middle of a (putative) gene cluster within the S. davawensis genome. Expression of ORF sda77220 in Escherichia coli revealed that the corresponding gene product had N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity. Inactivation of ORF sda77220 led to a S. davawensis strain that synthesized AF but not MAF or RoF. Accordingly, as the first identified gene of RoF biosynthesis, ORF sda77220 was named rosA. RosA (347 amino acids; 38 kDa) was purified from a recombinant E. coli strain (as a His(6)-tagged protein) and was biochemically characterized (apparent K(m) for AF = 57.7 ± 9.2 μm; apparent K(D) for AF = 10.0 μm; k(cat) = 0.37 ± 0.02 s(-1)). RosA is a unique enzyme and may be useful for a variety of applications.     

抗生素AGPM发酵过程中蛋白质表达差异的研究

由土壤中筛选到一能产生新型抗肿瘤抗生素AGPM的藤黄灰链霉菌株,应用蛋白质双向电泳方法,比较了藤黄灰链霉菌在发酵24h与72h蛋白质表达的差异,发现在发酵72h有17个蛋白质差异点出现,此外还发现一些蛋白质的含量明显高于24h的同种蛋白含量,这表明这些蛋白可能与抗生素AGPM的合成以及和菌体的生长等有关。     

与圈卷产色链霉菌分化有关的一个新基因—sawD的研究

距链霉菌发育分化控制启动子 Ｐ Ｔ Ｈ４ 直接控制的下游基因 ｐｒｏ Ｘ 间隔２４ 个碱基处存在一个部分开放阅读框 （ Ｏ Ｒ Ｆ） , 根据序列分析推测为丝氨酸蛋白酶的一部分。以此部分 Ｄ Ｎ Ａ 序列为探针, 在构建的圈卷产色链霉菌７１００ 的 Ｄ Ｎ Ａ 文库中克隆到一个与链霉菌发育和分化有关的新基因, 称之为ｓａ ｗ Ｄ。序列测定及分析结果表明, 在１３２０ｂｐ 的 Ｄ Ｎ Ａ 序列中有一个完整的开放阅读框 （ Ｏ Ｒ Ｆ） , 翻译起始位点为２１０ 位碱基处的 Ｇ Ｔ Ｇ, 终止密码子 Ｔ Ｇ Ａ 位于序列的９９９ 位碱基处。在距翻译起始位点 Ｇ Ｔ Ｇ 上游４ 个碱基间隔处有典型的核糖体结合位点区域 Ｇ Ａ Ｇ Ｇ Ｇ Ａ。在计算机蛋白文库中进行了同源性比较研究, 结果表明２６３个氨基酸的蛋白产物与 Ｃａｕｌｏｂａｃｔｅｒ ｃｒｅｓｃｅｎｔｕｓ 的依赖于 Ａ Ｔ Ｐ 的丝氨酸蛋白酶有４４７ ％ 的同源性, 其中存在功能活性区的丝氨酸保守位点 （ Ｇ Ｐ Ｓ Ａ Ｇ） 。基因功能研究表明, ｓａｗ Ｄ 在圈卷产色链霉菌发育分化中与气生菌丝分隔和色素的合成有关。该基因被阻断或破坏后, 使野生型圈卷产色链霉菌的分化停止在气生菌丝阶段, 不能形成具有灰色色素的孢子, 而出现白色     

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.     

Analysis of clustered genes encoding both early and late steps in daunomycin biosynthesis by Streptomyces sp. strain C5.

We recently described the isolation and sequence analysis of the daunomycin polyketide synthase biosynthesis genes of Streptomyces sp. strain C5 (J. Ye, M. L. Dickens, R. Plater, Y. Li, J. Lawrence, and W. R. Strohl, J. Bacteriol. 176:6270-6280, 1994). Contiguous to the daunomycin polyketide synthase biosynthesis gene region in Streptomyces sp. strain C5 are four additional genes involved in daunomycin biosynthesis, two of the products of which show similarity to different types of methyltransferases. The dauC gene, encoding aklanonic acid methyltransferase (AAMT), complements dauC-blocked mutants of Streptomyces sp. strain C5, restores in vitro AAMT activities to the mutant strains, and confers in vitro AAMT activity on Streptomyces lividans. Partial purification through gel filtration, followed by photoaffinity labeling of enriched AAMT with S-adenosyl-L-[3H-methyl]methionine, indicates that AAMT is a homodimer with an M(r) of ca. 48,000 (subunit M(r) of ca. 24,000), which corresponds with the size of the deduced gene product. The dauD gene, encoding aklanonic acid methyl ester cyclase, is divergently arranged with respect to dauC. Immediately downstream and apparently translationally coupled with dauD is the dauK gene, encoding carminomycin 4-O-methyltransferase. The dauK gene confers in vitro carminomycin 4-O-methyltransferase activity on S. lividans and is nearly identical to a similar gene isolated from Streptomyces peucetius and characterized. Directly downstream of dauK lies a gene encoding a deduced protein that is similar to the methyl esterases.     

Development of a Self-Cloning System for Actinomadura verrucosospora and Identification of Polyketide Synthase Genes Essential for Production of the Angucyclic Antibiotic Pradimicin

A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 10⁵ CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.     

Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tü22.

A 6.5 kb region of DNA from Streptomyces violaceoruber, which contains polyketide synthase (PKS) genes for production of the benzoisochromane quinone moiety of the antibiotic, granaticin, was cloned and sequenced. Of six open reading frames (ORFs) identified, four (ORFs 1-4) would be transcribed in one direction and two (ORFs 5 and 6) divergently from ORFs 1-4. ORF1 and ORF2, which show evidence for translation coupling, encode (deduced) gene products which strongly resemble each other and the Escherichia coli fatty acid ketoacyl synthase (condensing enzyme), FabB. We conclude that ORF1 (which contains a characteristic cysteine residue) functions as a condensing enzyme, possibly as part of a heterodimeric protein including the product of ORF2. The predicted ORF3 gene product strikingly resembles acyl carrier proteins (ACPs) of fatty acid synthase (FAS), particularly in the region of the active site motif, while the predicted ORF5 and ORF6 gene products resemble known oxidoreductases, suggesting that they function as reductive steps required during assembly of the granaticin carbon skeleton. Comparison of the deduced ORF4 gene product with available protein databases failed to elucidate its potential function. The overall conclusion is that the granaticin-producing PKS would consist of at least six separate enzymes involved in carbon chain assembly, thus resembling a Type II, rather than a Type I, FAS.