Substrate specificity of two cytochrome P450 monooxygenases involved in lankamycin biosynthesis.

To elucidate the gross lankamycin biosynthetic pathway including two cytochrome P450 monooxygenases, LkmK and LkmF, we constructed two double mutants of P450 genes in combination with glycosyltransferase genes, lkmL and lkmI. An aglycon 8,15-dideoxylankanolide, a possible substrate for LkmK, was prepared from an lkmK-lkmL double mutant, while a monoglycoside 3-O-l-arcanosyl-8-deoxylankanolide, a substrate for LkmF, was from an lkmF-lkmI double mutant. Bioconversion of lankamycin derivatives was performed in the Escherichia coli recombinant for LkmK and the Streptomyces lividans recombinant for LkmF, respectively. LkmK catalyzes the C-15 hydroxylation on all 15-deoxy derivatives, including 8,15-dideoxylankanolide (a possible substrate), 8,15-dideoxylankamycin, and 15-deoxylankamycin, suggesting the relaxed substrate specificity of LkmK. On the other hand, LkmF hydroxylates the C-8 methine of 3-O-l-anosyl-8-deoxylankanolide. Other 8-deoxy lankamycin/lankanolide derivatives were not oxidized, suggesting the importance of a C-3 l-arcanosyl moiety for substrate recognition by LkmF in lankamycin biosynthesis. Thus, LkmF has a strict substrate specificity in lankamycin biosynthesis.

SrrB, a Pseudo-Receptor Protein, Acts as a Negative Regulator for Lankacidin and Lankamycin Production in Streptomyces rochei.

Streptomyces rochei 7434AN4, a producer of lankacidin (LC) and lankamycin (LM), carries many regulatory genes including a biosynthesis gene for signaling molecules SRBs (srrX), an SRB receptor gene (srrA), and a SARP (Streptomyces antibiotic regulatory protein) family activator gene (srrY). Our previous study revealed that the main regulatory cascade goes from srrX through srrA to srrY, leading to LC production, whereas srrY further regulates a second SARP gene srrZ to synthesize LM. In this study we extensively investigated the function of srrB, a pseudo-receptor gene, by analyzing antibiotic production and transcription. Metabolite analysis showed that the srrB mutation increased both LC and LM production over four-folds. Transcription, gel shift, and DNase I footprinting experiments revealed that srrB and srrY are expressed under the SRB/SrrA regulatory system, and at the later stage, SrrB represses srrY expression by binding to the promoter region of srrY. These findings confirmed that SrrB acts as a negative regulator of the activator gene srrY to control LC and LM production at the later stage of fermentation in S. rochei.

The genome sequence of Streptomyces rochei 7434AN4, which carries a linear chromosome and three characteristic linear plasmids.

Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics, lankacidin and lankamycin, and carries three linear plasmids, pSLA2-L (211 kb), -M (113 kb), and -S (18 kb), whose nucleotide sequences were previously reported. The complete nucleotide sequence of the S. rochei chromosome has now been determined using the long-read PacBio RS-II sequencing together with short-read Illumina Genome Analyzer IIx sequencing and Roche 454 pyrosequencing techniques. The assembled sequence revealed an 8,364,802-bp linear chromosome with a high G + C content of 71.7% and 7,568 protein-coding ORFs. Thus, the gross genome size of S. rochei 7434AN4 was confirmed to be 8,706,406 bp including the three linear plasmids. Consistent with our previous study, a tap-tpg gene pair, which is essential for the maintenance of a linear topology of Streptomyces genomes, was not found on the chromosome. Remarkably, the S. rochei chromosome contains seven ribosomal RNA (rrn) operons (16S-23S-5S), although Streptomyces species generally contain six rrn operons. Based on 2ndFind and antiSMASH platforms, the S. rochei chromosome harbors at least 35 secondary metabolite biosynthetic gene clusters, including those for the 28-membered polyene macrolide pentamycin and the azoxyalkene compound KA57-A.

Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4.

Reinvestigation of the metabolite profile in a disruptant of the quinoprotein dehydrogenase (orf23) gene revealed that the Orf23 protein catalyzes dehydrogenation of the C23-C25 lactate moiety to pyruvate during lankacidin biosynthesis in Streptomyces rochei 7434AN4. The dehydrogenase activity was expressed and detected in a soluble fraction of the Streptomyces lividans recombinant harboring orf23. The Orf23 protein preferentially converts lankacidinol to lankacidin C in the presence of pyrroloquinoline quinone (PQQ). Other lankacidinol derivatives, lankacidinol A and iso-lankacidinol, were also converted to the corresponding C-24 keto compounds, lankacidin A (=sedecamycin) and iso-lankacidin C. Addition of various divalent metal cations, especially Ca2+, enhanced the dehydrogenase activity, whereas EDTA completely inhibited. These findings confirmed that the quinoprotein dehydrogenase Orf23 functions at the final oxidation step of lankacidin biosynthesis.

Isolation and Biosynthesis of an Azoxyalkene Compound Produced by a Multiple Gene Disruptant of Streptomyces rochei.

Streptomyces rochei 7434AN4 predominantly produces lankacidin and lankamycin under normal culture conditions, thus suggesting that other biosynthetic gene clusters for secondary metabolites are silent. To identify the silent metabolites of 7434AN4, we constructed mutant KA57 with multiple disruptions of the transcriptional repressor srrB and the biosynthesis genes for both antibiotics. KA57 accumulated a compound (KA57A) with a strong UV absorption at 235 nm, not detected in the parent strain or other mutants. Various spectroscopic analyses revealed that KA57A is an azoxyalkene compound with the molecular formula C10 H20 N2 O3 and with the R configuration at C-2. Biosynthesis of KA57A was also studied by feeding with labeled acetates, amino acids, and 1-hexylamine. The hexenyl moiety (C1'-C6') was derived from fatty acid, whereas the 3-aminobutan-1,2-diol moiety (C1-C4) was derived from C-2 of acetate (C1) and serine (C2-C4). Incorporation of [1,1-(2) H2 ]-1-hexylamine indicated that C1'-C2' dehydrogenation occurs as the final step of biosynthesis.

Blockage of the early step of lankacidin biosynthesis caused a large production of pentamycin, citreodiol and epi-citreodiol in Streptomyces rochei.

In our effort to find the key intermediates of lankacidin biosynthesis in Streptomyces rochei, three UV-active compounds were isolated from mutant FS18, a gene disruptant of lkcA encoding a non-ribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrid enzyme. Their structures were elucidated on the basis of spectroscopic data of NMR and MS. Two compounds of a higher mobile spot on silica gel TLC (Rf=0.45 in CHCl3-MeOH=20:1) were determined to be an epimeric mixture of citreodiol and epi-citreodiol at the C-6 position in the ratio of 2:1. In contrast, the compound of a lower mobile spot (Rf=~0 in CHCl3-MeOH=20:1) was identical to a 28-membered polyene macrolide pentamycin. The yields of citreodiols and pentamycin in FS18 were 5- and 250-fold higher compared with the parent strain. Introduction of a second mutation of srrX, coding a biosynthetic gene of the signaling molecules SRBs, into mutant FS18 did not affect the production of three metabolites. Thus, their production was not regulated by the SRB signaling molecules in contrast to lankacidin or lankamycin.

The tap-tpg gene pair on the linear plasmid functions to maintain a linear topology of the chromosome in Streptomyces rochei.

Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2-L (211 kb), pSLA2-M (113 kb) and pSLA2-S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of pSLA2-L and pSLA2-M up to 3.1 kb from the ends and have homology to those of typical Streptomyces species. Mutant 2-39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of pSLA2-L from strain 51252, which carries only pSLA2-L, also resulted in terminal deletions in newly obtained mutants. The tap-tpg gene pair, which encodes a telomere-associated protein and a terminal protein for end patching, is located on pSLA2-L and pSLA2-M but has not hitherto been found on the chromosome. These results led us to the idea that the tap-tpg of pSLA2-L or pSLA2-M functions to maintain a linear chromosome in strain 7434AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap-tpg of pSLA2-M.

Chromosomal circularization of the model Streptomyces species, Streptomyces coelicolor A3(2).

Streptomyces linear chromosomes frequently cause deletions at both ends spontaneously or by various mutagenic treatments, leading to chromosomal circularization and arm replacement. However, chromosomal circularization has not been confirmed at a sequence level in the model species, Streptomyces coelicolor A3(2). In this work, we have cloned and sequenced a fusion junction of a circularized chromosome in an S. coelicolor A3(2) mutant and found a 6-bp overlap between the left and right deletion ends. This result shows that chromosomal circularization occurred by nonhomologous recombination of the deletion ends in this species, too. At the end of the study, we discuss on stability and evolution of Streptomyces chromosomes.

ADP-ribosylation of guanosine by SCO5461 protein secreted from Streptomyces coelicolor.

The Streptomyces coelicolor A3(2) genome encodes a possible secretion protein, SCO5461, that shares a 30% homology with the activity domains of two toxic ADP-ribosyltransferases, pierisins and mosquitocidal toxin. We found ADP-ribosylating activity for the SCO5461 protein product through its co-incubation with guanosine and NAD(+), which resulted in the formation of N(2)-(ADP-ribos-1-yl)-guanosine ((ar2)Guo), with a K(m) value of 110 µM. SCO5461 was further found to ADP-ribosylate deoxyguanosine, GMP, dGMP, GTP, dGTP, and cyclic GMP with k(cat) values of 150-370 s(-1). Oligo(dG), oligo(G), and yeast tRNA were also ADP-ribosylated by this protein, although with much lower k(cat) values of 0.2 s(-1) or less. SCO5461 showed maximum ADP-ribosylation activity towards guanosine at 30 °C, and maintained 20% of these maximum activity levels even at 0 °C. This is the first report of the ADP-ribosylation of guanosine and guanine mononucleotides among the family members of various ADP-ribosylating enzymes. We additionally observed secretion of the putative gene product, SCO5461, in liquid cultures of S. coelicolor. We thus designated the SCO5461 protein product as S. coelicolor ADP-ribosylating protein, ScARP. Our current results could offer new insights into not only the ADP-ribosylation of small molecules but also signal transduction events via enzymatic nucleoside modification by toxin-related enzymes.

The butenolide signaling molecules SRB1 and SRB2 induce lankacidin and lankamycin production in Streptomyces rochei.

New signaling molecules that induce lankacidin and lankamycin production in Streptomyces rochei were extracted from the culture filtrate and purified by Sephadex LH20 and silica gel chromatography with the help of bioassay. Chiral HPLC and ESI-MS analyses indicated the presence of two active components--SRB1 and SRB2--and their molecular formulas were established to be C15H24O5 and C16H26O5, respectively. By extensive NMR analysis, SRB1 and SRB2 were determined to be 2-(1'-hydroxy-6'-oxo-8'-methylnonyl)-3-methyl-4-hydroxybut-2-en-1,4-olide and 2-(1'-hydroxy-6'-oxo-8'-methyldecyl)-3-methyl-4-hydroxybut-2-en-1,4-olide, respectively. These structures were finally confirmed by chemical synthesis and the absolute configuration at C-1' was determined to be R in each case. The synthetic 1'R isomers induced production of lankacidin and lankamycin at around 40 nM concentrations. SRB1 and SRB2 are therefore distinct from the well-known 2,3-disubstituted γ-butyrolactone molecules such as A-factor, virginia butanolide, and SCB1 and and belong, like avenolide, recently isolated from Streptomyces avermitilis, to the γ-butenolide family.

Isolation of borrelidin as a phytotoxic compound from a potato pathogenic streptomyces strain.

Streptomyces species strain GK18, isolated in Iran, induced deep-pitted lesions on potato tubers, lesions different from the raised lesions induced by the usual scab-causing phytotoxin, thaxtomin. In addition, neither thaxtomin production nor hybridization to its biosynthetic probe was detected for strain GK18, suggesting the production of a different phytotoxin. The active component was extracted with ethyl acetate from culture filtrate of strain GK18, purified by gel filtration and silica gel chromatography, and identified as an 18-membered macrolide, borrelidin, by spectroscopic analysis. The purified borrelidin induced necrosis on potato tuber slices and inhibited the growth of shoots and roots of radish seedlings. This is the first report on the phytotoxicity of borrelidin as a possible causative compound of potato scab disease.

pSLA2-M of Streptomyces rochei is a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L.

The 113,463-bp nucleotide sequence of the linear plasmid pSLA2-M of Streptomyces rochei 7434AN4 was determined. pSLA2-M had a 69.7% overall GC content, 352-bp terminal inverted repeats with 91% (321/352) identity at both ends, and 121 open reading frames. The rightmost 14.6-kb sequence was almost (14,550/14,555) identical to that of the coexisting 211-kb linear plasmid pSLA2-L. Adjacent to this homologous region an 11.8-kb CRISPR cluster was identified, which is known to function against phage infection in prokaryotes. This cluster region as well as another one containing two large membrane protein genes (orf78 and orf79) were flanked by direct repeats of 194 and 566 bp respectively. Hence the insertion of circular DNAs containing each cluster by homologous recombination was suggested. In addition, the orf71 encoded a Ku70/Ku80-like protein, known to function in the repair of double-strand DNA breaks in eukaryotes, but disruption of it did not affect the radiation sensitivity of the mutant. A pair of replication initiation genes (orf1-orf2) were identified at the extreme left end. Thus, pSLA2-M proved to be a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L that might have been generated by multiple recombination events.

Crystal structure of the synergistic antibiotic pair, lankamycin and lankacidin, in complex with the large ribosomal subunit.

The structures of the large ribosomal subunit of Deinococcus radiodurans (D50S) in complex with the antibiotic lankamycin (3.2 Å) and a double antibiotic complex of lankamycin and lankacidin C (3.45 Å) have been determined, in continuation of previous crystallographic studies on lankacidin-D50S complex. These two drugs have been previously reported to inhibit ribosomal function with mild synergistic effect. Lankamycin, a member of the macrolide family, binds in a similar manner to erythromycin. However, when in complex with lankacidin, lankamycin is located so that it can form interactions with lankacidin in the adjacent ribosomal binding site. When compared to the well-documented synergistic antibiotics, Streptogramins A and B, the pair of lankacidin and lankamycin bind in similar sites, the peptidyl transferase center and nascent peptide exit tunnel, respectively. Herein, we discuss the structural basis for antibiotic synergism and highlight the key factors involved in ribosomal inhibition.

Giant linear plasmids in Streptomyces: a treasure trove of antibiotic biosynthetic clusters.

Many giant linear plasmids have been isolated from Streptomyces by using pulsed-field gel electrophoresis and some of them were found to carry an antibiotic biosynthetic cluster(s); SCP1 carries biosynthetic genes for methylenomycin, pSLA2-L for lankacidin and lankamycin, and pKSL for lasalocid and echinomycin. Accumulated data suggest that giant linear plasmids have played critical roles in genome evolution and horizontal transfer of secondary metabolism. In this review, I summarize typical examples of giant linear plasmids whose involvement in antibiotic production has been studied in some detail, emphasizing their finding processes and interaction with the host chromosomes. A hypothesis on horizontal transfer of secondary metabolism involving giant linear plasmids is proposed at the end.

Regulation of lankamycin biosynthesis in Streptomyces rochei by two SARP genes, srrY and srrZ.

Transcription and complementation experiments were carried out to analyze the regulatory hierarchy of two Streptomyces antibiotic regulatory protein (SARP) genes, srrY and srrZ, in the gamma-butyrolactone (GB)-dependent regulatory cascade in Streptomyces rochei 7434AN4. The srrY gene was transcribed in the srrZ mutant, while the srrZ gene was not in the srrY mutant. The SrrY protein was specifically bound to the promoter region of srrZ, where a possible SARP binding sequence was identified 26 bp upstream of the -10 sequence. Deletion of the repeat sequences from this region abolished its SrrY binding activity. In addition, complementation of srrZ restored lankamycin production in the srrY mutant. All of these results confirm that the SARP gene srrY directly regulates expression of the second SARP gene srrZ in a positive manner. This study gave the first confirmation of direct regulation of two SARP genes in the GB-dependent regulatory cascade in Streptomyces.

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics.

Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.

Extensive mutational analysis of modular-iterative mixed polyketide biosynthesis of lankacidin in Streptomyces rochei.

Extensive mutations of lankacidin synthase genes were carried out to analyze the modular-iterative mixed polyketide biosynthesis of lankacidin. Three ketoreductase domains (lkcC-KR, lkcF-KR1, and lkcF-KR2) were inactivated by in-frame deletion and site-directed mutagenesis of their active sites. The mutants ceased or diminished lankacidin production, indicating that the three KR domains are functional in lankacidin biosynthesis. However, all of the KR mutants failed to accumulate the expected unreduced metabolites. Mutational analysis of two tandemly aligned acyl carrier protein domains (lkcC-ACP1 and lkcC-ACP2) revealed that either ACP is sufficient for lankacidin production. Disruption and complementation experiments on three unique genes/domain (lkcD for acyltransferase, lkcB for dehydratase, and lkcC-MT for a C-methyltransferase domain) suggested that their gene products function iteratively during lankacidin biosynthesis.

Identification of a gene cluster of polyether antibiotic lasalocid from Streptomyces lasaliensis.

Elucidation of enzymatic polyether formation is a long-standing controversial issue in organic chemistry. To address this intriguing issue, identifying the actual substrate for epoxidation and sequential cyclization is essential. We selected the representative polyether ionophore, lasalocid, which has been proposed to undergo no modification at the late stage of biosynthesis. Cloning and a sequence analysis revealed seven polyketide synthase (PKS) genes, epoxidase and epoxide hydrolase genes for sequential ether formation, and several putative genes for supplying ethylmalonyl-CoA. Based on bioinformatic data, we propose the lasalocid biosynthetic pathway which involves characteristic aromatic ring formation and sequential cyclic ether formation. The finding of a thioesterase domain at the C-terminal of the seventh PKS indicates that intriguing oxidative cascade cyclization would occur after cleavage of the polyketide intermediate from PKS. Based on this observation, we have recently reported the enzymatic transformation of a bisepoxide intermediate to lasalocid with the recombinant epoxide hydrolase, Lsd19.

Gamma-butyrolactone-dependent expression of the Streptomyces antibiotic regulatory protein gene srrY plays a central role in the regulatory cascade leading to lankacidin and lankamycin production in Streptomyces rochei.

Our previous studies revealed that the srrX and srrA genes carried on the large linear plasmid pSLA2-L constitute a gamma-butyrolactone-receptor system in Streptomyces rochei. Extensive transcriptional analysis has now showed that the Streptomyces antibiotic regulatory protein gene srrY, which is also carried on pSLA2-L, is a target of the receptor/repressor SrrA and plays a central role in lankacidin and lankamycin production. The srrY gene was expressed in a growth-dependent manner, slightly preceding antibiotic production. The expression of srrY was undetectable in the srrX mutant but was restored in the srrX srrA double mutant. In addition, SrrA was bound specifically to the promoter region of srrY, and this binding was prevented by the addition of the S. rochei gamma-butyrolactone fraction, while the W119A mutant receptor SrrA was kept bound even in the presence of S. rochei gamma-butyrolactone. Furthermore, the introduction of an intact srrY gene under the control of a foreign promoter into the srrX or srrA(W119A) mutant restored antibiotic production. All of these results confirmed the signaling pathway from srrX through srrA to srrY, leading to lankacidin and lankamycin production.

Analysis of modular-iterative mixed biosynthesis of lankacidin by heterologous expression and gene fusion.

Lankacidin is a unique 17-membered macrocyclic antibiotic different from usual even-membered macrolides. Based on the gene organization of the lankacidin biosynthetic cluster coded on the linear plasmid pSLA2-L in Streptomyces rochei, we previously proposed a hypothesis of modular-iterative mixed polyketide biosynthesis for lankacidin. Two experimental evidences in this paper further strengthened this hypothesis. Heterologous expression of the lankacidin cluster (lkcA-lkcO) in Streptomyces lividans resulted in lankacidinol A production, indicating that the gene cluster is sufficient for the synthesis of the lankacidin skeleton. In addition, a gene fusant of lkcF and lkcG produced lankacidin at a similar level to the parent strain, suggesting that an iterative function of the LkcF protein is unlikely. These results are consistent with the hypothesis that LkcC is used four times and LkcA, LkcF and LkcG are used modularly to accomplish eight condensation reactions leading to the lankacidin skeleton.