Ribosylhopane,a Novel Bacterial Hopanoid,as Precursor of C35 Bacteriohopanepolyols in Streptomyces coelicolor A3(2)

Wild‐type Streptomyces coelicolor A3(2) produces aminobacteriohopanetriol as the only elongated C₃₅ hopanoid. The hopanoid phenotype of two mutants bearing a deletion of genes from a previously identified hopanoid biosynthesis gene cluster provides clues to the formation of C₃₅ bacteriohopanepolyols. orf14 encodes a putative nucleosidase; its deletion induces the accumulation of adenosylhopane as it cannot be converted into ribosylhopane. orf18 encodes a putative transaminase; its deletion results in the accumulation of adenosylhopane, ribosylhopane, and bacteriohopanetetrol. Ribosylhopane was postulated twenty years ago as a precursor for bacterial hopanoids but was never identified in a bacterium. Absence of the transaminase encoded by orf18 prevents the reductive amination of ribosylhopane into aminobacteriohopanetriol and induces its accumulation. Its reduction by an aldose‐reductase‐like enzyme produces bacteriohopanetetrol, which is normally not present in S. coelicolor.     

Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

Type II polyketide synthases iteratively generate a nascent polyketide thioester of the acyl carrier protein (ACP); this is structurally modified to produce an ACP‐free intermediate towards the final metabolite. However, the timing of ACP off‐loading is not well defined because of the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess TE activity in vitro with a model substrate, anthraquinone‐2‐carboxylic acid‐N‐acetylcysteamine. In order to investigate its function further, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was reconstituted in vitro in a stepwise fashion up to (S)‐DNPA, and the product of ActIV reaction was characterized as an ACP‐free bicyclic intermediate. These findings indicate that ActIV is a bifunctional CYC‐TE and provide clear evidence for the release timing of the intermediate from the ACP anchor.     

Oxygen transfer and uptake in Streptomyces coelicolor A3(2) culture in a batch bioreactor

This paper provides quantitative information on oxygen transfer as well as the kinetic and metabolic parameters related to oxygen uptake in Streptomyces coelicolor A3(2) cultured in a 20 dm³ computer controlled bioreactor using both defined and complex media. It is evident from the literature that production of antibiotics is strongly affected by the dissolved oxygen concentration. Many processes of antibiotic fermentations have been developed to the point at which the microbial oxygen demand exceeds the oxygen transfer capability of the existing fermentation facilities. As a consequence, the oxygen transfer rate has become the rate limiting factor in such processes. It is necessary to know the oxygen kinetic and metabolic parameters of an aerobic fermentation for a successful scale-up and operational control of the process. In the literature, information concerning the oxygen uptake kinetics of the Streptomyces cultures is scarce despite their industrial importance. This paper, therefore, provides useful quantitative information on oxygen transfer and uptake rates in S. coelicolor cultures. In the defined medium, the total oxygen uptake rates were in the range of 5–6 mmol O₂ dm⁻³ h⁻¹ throughout the active growth phase, the maximum specific oxygen uptake rate was 7·44 mmol O₂ g cell⁻¹ h⁻¹, the specific oxygen maintenance demand was 1·88 mmol O₂ g cell⁻¹ h⁻¹, and the k_La values were in the range of 40–100 h⁻¹. In the complex medium, however, the k_La values varied in the range of 18–70 h⁻¹. © 1998 Society of Chemical Industry     

Cross-Recognition of Promoters by the Nine SigB Homologues Present in Streptomyces coelicolor A3(2)

In contrast to Bacillus subtilis, Streptomyces coelicolor A3(2) contains nine homologues of stress response sigma factor SigB with a major role in differentiation and osmotic stress response. The aim of this study was to further characterize these SigB homologues. We previously established a two-plasmid system to identify promoters recognized by sigma factors and used it to identify promoters recognized by the three SigB homologues, SigF, SigG, and SigH from S. coelicolor A3(2). Here, we used this system to identify 14 promoters recognized by SigB. The promoters were verified in vivo in S. coelicolor A3(2) under osmotic stress conditions in sigB and sigH operon mutants, indicating some cross-recognition of these promoters by these two SigB homologues. This two-plasmid system was used to examine the recognition of all identified SigB-, SigF-, SigG-, and SigH-dependent promoters with all nine SigB homologues. The results confirmed this cross-recognition. Almost all 24 investigated promoters were recognized by two or more SigB homologues and data suggested some distinguishing groups of promoters recognized by these sigma factors. However, analysis of the promoters did not reveal any specific sequence characteristics for these recognition groups. All promoters showed high similarity in the -35 and -10 regions. Immunoblot analysis revealed the presence of SigB under osmotic stress conditions and SigH during morphological differentiation. Together with the phenotypic analysis of sigB and sigH operon mutants in S. coelicolor A3(2), the results suggest a dominant role for SigB in the osmotic stress response and a dual role for SigH in the osmotic stress response and morphological differentiation. These data suggest a complex regulation of the osmotic stress response in relation to morphological differentiation in S. coelicolor A3(2).     

Two pathways for pyrrole formation in coumermycin A(1) biosynthesis: the central pyrrole moiety is formed from L-threonine

Coumermycin A₁ is an aminocoumarin antibiotic produced by Streptomyces rishiriensis. It contains three pyrrole rings, that is, two terminal 5‐methyl‐pyrrole‐2‐carboxyl moieties and a central 3‐methylpyrrole‐2,4‐dicarboxylic acid moiety. The biosynthesis of the terminal pyrrole moieties has been elucidated previously. However, the biosynthetic precursors of the central pyrrole moiety have remained unknown, and none of the genes or enzymes involved in its formation has been identified. We now show that five genes, contained in a contiguous 4.7 kb region within the coumermycin biosynthetic gene cluster, are required for the biosynthesis of this central pyrrole moiety. Each of these genes was deleted individually, resulting in a strong reduction or an abolishment of coumermycin production. External feeding of the central pyrrole moiety restored coumermycin production. One of these genes shows similarity to L ‐threonine kinase genes. Feeding of [U‐¹³C,¹⁵N]L ‐threonine and ¹³C NMR analysis of the resulting compound unequivocally proved that threonine was incorporated intact into the central pyrrole (19 % enrichment) to provide the heterocyclic nitrogen as well as four of the seven carbons of this moiety. Therefore, this pyrrole is formed via a new, hitherto unknown biosynthetic pathway. A hypothesis for the reaction sequence leading to the central pyrrole moiety of coumermycin A₁ is presented.     

Aromatic Polyketide GTRI‐02 is a Previously Unidentified Product of the act Gene Cluster in Streptomyces coelicolor A3(2)

The biosynthesis of aromatic polyketides derived from type II polyketide synthases (PKSs) is complex, and it is not uncommon that highly similar gene clusters give rise to diverse structural architectures. The act biosynthetic gene cluster (BGC) of the model actinomycete Streptomyces coelicolor A3(2) is an archetypal type II PKS. Here we show that the act BGC also specifies the aromatic polyketide GTRI‐02 ( 1 ) and propose a mechanism for the biogenesis of its 3,4‐dihydronaphthalen‐1(2H)‐one backbone. Polyketide 1 was also produced by Streptomyces sp. MBT76 after activation of the act‐like qin gene cluster by overexpression of the pathway‐specific activator. Mining of this strain also identified dehydroxy‐GTRI‐02 ( 2 ), which most likely originated from dehydration of 1 during the isolation process. This work shows that even extensively studied model gene clusters such as act of S. coelicolor can still produce new chemistry, offering new perspectives for drug discovery.     

Reevaluation of the violacein biosynthetic pathway and its relationship to indolocarbazole biosynthesis

The biosynthetic pathways for violacein and for indolocarbazoles (rebeccamycin, staurosporine) include a decarboxylative fusion of two tryptophan units. However, in the case of violacein, one of the tryptophans experiences an unusual 1-->2 shift of the indole ring. The violacein biosynthetic gene cluster was previously reported to consist of four genes, vioABCD. Here we studied the violacein pathway through expression of vio genes in Escherichia coli and Streptomyces albus. A pair of genes (vioAB), responsible for the earliest steps in violacein biosynthesis, was functionally equivalent to the homologous pair in the indolocarbazole pathway (rebOD), directing the formation of chromopyrrolic acid. However, chromopyrrolic acid appeared to be a shunt product, not a violacein intermediate. In addition to vioABCD, a fifth gene (vioE) was essential for violacein biosynthesis, specifically for production of the characteristic 1-->2 shift of the indole ring. We also report new findings on the roles played by the VioC and VioD oxygenases, and on the origin of violacein derivatives of the chromoviridans type.     

Use of a halogenase of hormaomycin biosynthesis for formation of new clorobiocin analogues with 5-chloropyrrole moieties

The depsipeptide antibiotic hormaomycin, which is produced by Streptomyces griseoflavus W-384, contains a 5-chloropyrrole moiety. In the producer strain we identified the gene hrmQ that shows sequence similarity to FADH(2)-dependent halogenases. This gene was cloned and heterologously expressed in Streptomyces roseochromogenes var. oscitans DS12.976, which is the producer of the aminocoumarin antibiotic clorobiocin, which contains a 5-methylpyrrole moiety. For the present experiment, we used a mutant of this strain in which the respective pyrrole-5-methyltransferase had been inactivated. Expression of the halogenase hrmQ in this mutant strain led to the formation of two new clorobiocin derivatives that carried a 5-chloropyrrole moiety. These compounds were isolated on a preparative scale, their structures were elucidated by (1)H NMR spectroscopy and mass spectrometry, and their antibacterial activity was determined. The substrate of HrmQ is likely to be a pyrrole-2-carboxyl-S-[acyl carrier protein] thioester. If this assumption is true, this study presents the first experiment in combinatorial biosynthesis that uses a halogenase that acts on an acyl carrier protein-bound substrate.     

A type I/type III polyketide synthase hybrid biosynthetic pathway for the structurally unique ansa compound kendomycin

Kendomycin is a bioactive polyketide that is produced by various Streptomyces strains. It displays strong antibiotic activities against a wide range of bacteria and exhibits remarkable cytotoxic effects on the growth of several human cancer cell lines. In this study we cloned the corresponding biosynthetic locus from the producer Streptomyces violaceoruber (strain 3844-33C). Our analysis shows that a mixed type I/type III polyketide synthase pathway is responsible for the formation of the fully carbogenic macrocyclic scaffold of kendomycin, which is unprecedented among all of the ansa compounds that have been isolated so far. Heterologous expression of a gene set in Streptomyces coelicolor shows that 3,5-dihydroxybenzoic acid is an intermediate in the starter unit biosynthesis that is initiated by the type III polyketide synthase. The identification of the kendomycin biosynthetic gene cluster sets the stage to study a novel chain termination mechanism by a type I PKS that leads to carbocycle formation and provides the starting material for the heterologous expression of the entire pathway, and the production of novel derivatives by genetic engineering.     

Identification and Characterization of the Carbapenem MM 4550 and its Gene Cluster in Streptomyces argenteolus ATCC 11009

Nearly 50 naturally occurring carbapenem β‐lactam antibiotics, most produced by Streptomyces, have been identified. The structural diversity of these compounds is limited to variance of the C‐2 and C‐6 side chains as well as the stereochemistry at C‐5/C‐6. These structural motifs are of interest both for their antibiotic effects and their biosynthesis. Although the thienamycin gene cluster is the only active gene cluster publically available in this group, more comparative information is needed to understand the genetic basis of these structural differences. We report here the identification of MM 4550, a member of the olivanic acids, as the major carbapenem produced by Streptomyces argenteolus ATCC 11009. Its gene cluster was also identified by degenerate PCR and targeted gene inactivation. Sequence analysis revealed that the genes encoding the biosynthesis of the bicyclic core and the C‐6 and C‐2 side chains are well conserved in the MM 4550 and thienamycin gene clusters. Three new genes, cmmSu, cmm17 and cmmPah were found in the new cluster, and their putative functions in the sulfonation and epimerization of MM 4550 are proposed. Gene inactivation showed that, in addition to cmmI, two new genes, cmm22 and ‐23, encode a two‐component response system thought to regulate the production of MM 4550. Overexpression of cmmI, cmm22 and cmm23 promoted MM 4550 production in an engineered strain. Finally, the involvement and putative roles of all genes in the MM 4550 cluster are proposed based on the results of bioinformatics analysis, gene inactivation, and analysis of disruption mutants. Overall, the differences between the thienamycin and MM 4550 gene clusters are reflected in characteristic structural elements and provide new insights into the biosynthesis of the complex carbapenems.     

In vivo mutational analysis of the mupirocin gene cluster reveals labile points in the biosynthetic pathway: the "leaky hosepipe" mechanism

A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a beta-hydroxy-beta-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points.     

Generation of new landomycins with altered saccharide patterns through over-expression of the glycosyltransferase gene lanGT3 in the biosynthetic gene cluster of landomycin A in Streptomyces cyanogenus S-136

Two novel landomycin compounds, landomycins I and J, were generated with a new mutant strain of Streptomyces cyanogenus in which the glycosyltransferase that is encoded by lanGT3 was over-expressed. This mutant also produced the known landomycins A, B, and D. All these compounds consist of the same polyketide-derived aglycon but differ in their sugar moieties, which are chains of different lengths. The major new metabolite, landomycin J, was found to consist of landomycinone with a tetrasaccharide chain attached. Combined with previous results of the production of landomycin E (which contains three sugars) by the LanGT3- mutant strain (obtained by targeted gene deletion of lanGT3), it was verified that LanGT3 is a D-olivosyltransferase responsible for the transfer of the fourth sugar required for landomycin A biosynthesis. The experiments also showed that gene over-expression is a powerful method for unbalancing biosynthetic pathways in order to generate new metabolites. The cytotoxicity of the new landomycins--compared to known ones--was assessed by using three different tumor cell lines, and their structure-activity relationship (SAR) with respect to the length of the deoxysugar side chain was deduced from the results.     

Identification and Analysis of the Biosynthetic Gene Cluster for the Indolizidine Alkaloid Iminimycin in Streptomyces griseus

Indolizidine alkaloids, which have versatile bioactivities, are produced by various organisms. Although the biosynthesis of some indolizidine alkaloids has been studied, the enzymatic machinery for their biosynthesis in Streptomyces remains elusive. Here, we report the identification and analysis of the biosynthetic gene cluster for iminimycin, an indolizidine alkaloid with a 6-5-3 tricyclic system containing an iminium cation from Streptomyces griseus. The gene cluster has 22 genes, including four genes encoding polyketide synthases (PKSs), which consist of eight modules in total. In vitro analysis of the first module revealed that its acyltransferase domain selects malonyl-CoA, although predicted to select methylmalonyl-CoA. Inactivation of seven tailoring enzyme-encoding genes and structural elucidation of four compounds accumulated in mutants provided important insights into iminimycin biosynthesis, although some of these compounds appeared to be shunt products. This study expands our knowledge of the biosynthetic machinery of indolizidine alkaloids and the enzymatic chemistry of PKS.     

A biosynthetic pathway to isovaleryl-CoA in myxobacteria: the involvement of the mevalonate pathway

A biosynthetic shunt pathway branching from the mevalonate pathway and providing starter units for branched-chain fatty acid and secondary metabolite biosynthesis has been identified in strains of the myxobacterium Stigmatella aurantiaca. This pathway is upregulated when the branched-chain alpha-keto acid dehydrogenase gene (bkd) is inactivated, thus impairing the normal branched-chain amino acid degradation process. We previously proposed that, in this pathway, isovaleryl-CoA is derived from 3,3-dimethylacrylyl-CoA (DMA-CoA). Here we show that DMA-CoA is an isomerization product of 3-methylbut-3-enoyl-CoA (3MB-CoA). This compound is directly derived from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by a decarboxylation/ dehydration reaction resembling the conversion of mevalonate 5-diphosphate to isopentenyl diphosphate. Incubation of cell-free extracts of a bkd mutant with HMG-CoA gave product(s) with the molecular mass of 3MB-CoA or DMA-CoA. The shunt pathway most likely also operates reversibly and provides an alternative source for the monomers of isoprenoid biosynthesis in myxobacteria that utilize L-leucine as precursor.