Reconstitution of the myxothiazol biosynthetic gene cluster by Red/ET recombination and heterologous expression in Myxococcus xanthus

Although many secondary metabolites exhibiting important pharmaceutical and agrochemical activities have been isolated from myxobacteria, most of these microorganisms remain difficult to handle genetically. To utilize their metabolic potential, heterologous expression methodologies are currently being developed. Here, the Red/ET recombination technology was used to perform all required gene cluster engineering steps in Escherichia coli prior to the transfer into the chromosome of the heterologous host. We describe the integration of the complete 57-kbp myxothiazol biosynthetic gene cluster reconstituted from two cosmids from a cosmid library of the myxobacterium Stigmatella aurantiaca DW4-3/1 into the chromosome of the thus far best-characterized myxobacterium, Myxococcus xanthus, in one step. The successful integration and expression of the myxothiazol biosynthetic genes in M. xanthus results in the production of myxothiazol in yields comparable to the natural producer strain.     

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters.Bacterial natural products are excellent lead compounds for drug discovery and have played major roles in the development of pharmaceutical agents in nearly all therapeutic areas (1, 7, 9). Unfortunately, the rate of discovery of new bacterial natural products has decreased, due in part to frequent rediscovery of known compounds (7). An enormous and currently inaccessible reservoir of new natural products is located in the biosynthetic pathways found in the genomes of uncultivated bacteria (18). Heterologous expression of these biosynthetic gene clusters represents a powerful tool for discovering new natural products (20, 21). Herein, we demonstrate that the deltaproteobacterium Myxococcus xanthus is an effective host for heterologous expression of aromatic polyketide biosynthetic pathways. This work expands the scope of polyketide biosynthetic pathways which can be heterologously expressed in M. xanthus and suggests that M. xanthus may be a suitable general host for heterologous expression.Molecular phylogenetic studies have shown that bacterial diversity is enormous, and the vast majority of the diversity is found in uncultivated bacterial species (18). Estimates suggest that 99% of bacteria from the environment are uncultivatable using standard techniques (2, 15, 16). Culture-independent analyses of metagenomic DNA libraries from soil and marine environments indicate that there is a wealth of natural product diversity in these uncultivated strains. For example, analysis of a soil metagenome for a highly conserved region of polyketide synthase genes showed that none of the sequences found were present in the known public databases (5). Polyketide synthases are key enzymes responsible for the production of the polyketide family of natural products in proteobacteria, actinobacteria, and “low-G+C Gram-positive bacteria” (4, 12, 19). Polyketide natural products have been developed into antibiotic, anticancer, and immunosuppressant clinical agents (1, 6, 8). Based on these observations, metagenomic DNA libraries are expected to possess a large number of new polyketide biosynthetic pathways, representing substantial new chemical diversity for drug discovery.Heterologous expression of biosynthetic pathways can play a major role in interrogating metagenomic DNA libraries for new polyketide biosynthetic pathways. Heterologous production of polyketides in hosts such as Streptomyces coelicolor and Streptomyces lividans is an important tool in the identification and characterization of these pathways (6, 8, 17). Results from these studies have shown that Streptomyces strains are good hosts for heterologous production of many polyketides, particularly those from actinomycetes. However, Streptomyces strains have proved to be poor hosts for expression of deltaproteobacterial polyketide biosynthetic pathways, such as those in myxobacteria (10, 17). As polyketide biosynthetic pathways in metagenomic DNA libraries contain both actinomycete- and deltaproteobacterium-derived pathways, a heterologous expression host competent to express pathways of both origins is needed.We examined the ability of the deltaproteobacterium M. xanthus to act as a general heterologous expression host. M. xanthus is a predatory bacterium that undergoes multicellular development in response to nutrient starvation. During development, M. xanthus is known to be an effective host for the heterologous expression of the deltaproteobacterium-derived epothilone D biosynthetic pathway and has been used for the production of epothilone D for clinical trials (17). M. xanthus has also been shown to be an excellent host for the heterologous expression of several other myxobacterial metabolites, including myxothiazol and myxochromide S (3, 11, 22). We demonstrate that M. xanthus can also heterologously express the Streptomyces rimosus oxytetracycline biosynthetic pathway, producing oxytetracycline. This is the first example of a polyketide from a nonmyxobacterial species heterologously expressed in a myxobacterium.To generate an M. xanthus strain capable of heterologously expressing oxytetracycline, the Streptomyces rimosus oxytetracycline biosynthetic pathway (Fig. ​(Fig.1)1) was inserted via homologous recombination into the asgE locus of M. xanthus. The asgE locus of M. xanthus was amplified and inserted into the BglII site of pET28b (Novagen) to produce pMRH02. The oligonucleotides used for the amplification of the asgE locus were 5′-GACGAGATCTGTTGGAAGGTCGGCAACTGG-3′ and 5′-CTTAAGATCTTCCGTGAAGTACTGGCGCAC-3′. The asgE locus provides a chromosomal region for single-crossover homologous recombination into the M. xanthus chromosome. The 32-kb oxytetracycline pathway in S. rimosus was excised from pYT264 (24) and cloned into the EcoRI site of pMRH02 to produce pMRH08. M. xanthus DK1622 was electroporated under standard conditions (13) with pMRH08 to provide an M. xanthus ΔasgE Kan^r mutant. Positive selection for the chromosomal insertion was maintained throughout all experiments by use of kanamycin supplementation (40 μg/ml). This large genomic insertion significantly increased the doubling time for the strain (doubling time, ≈10 h).Open in a separate windowFIG. 1.Oxytetracycline biosynthetic pathway. (A) Enzymatic pathway responsible for formation of oxytetracycline. (B) Oxytetracycline biosynthesis gene cluster from S. rimosus.Oxytetracycline was heterologously produced in M. xanthus under standard rich medium culture conditions and detected in culture broth by liquid chromatography-mass spectrometry (LC-MS). A liquid culture of the mutant strain containing the oxytetracycline gene cluster was cultured for 10 days at 33°C in CTTYE (1.0% Casitone, 0.5% yeast extract, 10.0 mM Tris-HCl, 1.0 mM KH₂PO₄, and 8.0 mM MgSO₄; 100 ml). Acetone (10%, vol/vol) was added to the culture and vigorously mixed. The resulting mixture was extracted with 3 volumes of ethyl acetate to remove the organic soluble materials, including oxytetracycline. The organic extracts were concentrated in vacuo and resuspended in methanol (100 μl). LC-MS analyses were carried out using an Altima Hypersil C₁₈ column (3-μm particle size; 150 mm by 2.1 mm) with a linear gradient of water-acetonitrile (5 to 95%) with 0.05% formic acid over 90 min (0.20 ml/min), followed by positive-ion electrospray ionization (5,500 V) and analysis with a Shimadzu 2010A single quadrupole mass spectrometer. LC-MS analysis indicated that oxytetracycline was present in the fermentation broth (Fig. ​(Fig.2).2). The titer of oxytetracycline was determined to be approximately 10 mg per liter of fermentation broth. Quantification was performed in triplicate by LC-MS analysis using a standard curve generated from commercial oxytetracycline. Negative controls of M. xanthus DK1622 cultures processed under identical conditions did not contain detectable levels of oxytetracycline.Open in a separate windowFIG. 2.LC-MS ion extraction analysis of the molecular ion [M+H]⁺ of standard and culture extracts. (A) Oxytetracycline standard. (B) M. xanthus ΔasgE Kan^r mutant containing the oxytetracycline biosynthetic pathway. (C) Wild-type M. xanthus DK1622.These data indicate that M. xanthus can heterologously express the oxytetracycline polyketide synthase biosynthetic pathway in S. rimosus. Several factors affect the successful heterologous production of polyketide synthase pathways, including codon usage, mRNA stability, functionality of regulatory elements, and the presence of all necessary starter and extender units (14). As codon usages between M. xanthus and the genus Streptomyces are very similar and myxobacteria are known to produce polyketide products requiring a wide diversity of starter and extender units, neither codon usage nor starter and extender unit availability was considered likely to affect the ability of M. xanthus to heterologously express streptomycete biosynthetic pathways. As Streptomyces strains do not appear to be effective at heterologous expression of myxobacterial biosynthetic pathways, we were concerned that Myxococcus and Streptomyces strains may possess substantially different regulatory elements. Our data indicate that the regulatory elements present in streptomycete-derived biosynthetic pathways are sufficient to enable expression of the biosynthetic genes in M. xanthus. Further work exploring the regulatory elements present in myxobacterial polyketide biosynthetic gene clusters is needed to evaluate this hypothesis.This study demonstrates that M. xanthus can heterologously express streptomycete-derived polyketide biosynthetic pathways in addition to myxobacterial polyketide biosynthetic pathways. The observed titer of 10 mg/liter of culture broth is comparable to titers reported for the heterologous expression of myxobacterial polyketide biosynthetic pathways in myxobacteria (11) and streptomycete-derived polyketide biosynthetic pathways in Streptomyces (14, 23) and is sufficient for characterization of the polyketide product. Pseudomonas putida, which has a more favorable growth profile, has been shown to be a good host for heterologous expression of myxobacterial polyketide biosynthetic pathways, with product titers in the range of 0.6 to 40 mg/liter of culture broth (14, 21, 23). The observed breadth of polyketide pathways accessible and the titers of the polyketide products produced make M. xanthus an attractive potential candidate for a “universal” host for facilitating heterologous expression of polyketide biosynthetic pathways derived from environmental samples of metagenomic DNA.     

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition

Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes.     

Heterologous production of epothilones B and D in Streptomyces venezuelae

Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.     

New lessons for combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene cluster of Stigmatella aurantiaca DW4/3-1

The biosynthetic mta gene cluster responsible for myxothiazol formation from the fruiting body forming myxobacterium Stigmatella aurantiaca DW4/3-1 was sequenced and analyzed. Myxothiazol, an inhibitor of the electron transport via the bc(1)-complex of the respiratory chain, is biosynthesized by a unique combination of several polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS), which are activated by the 4'-phosphopantetheinyl transferase MtaA. Genomic replacement of a fragment of mtaB and insertion of a kanamycin resistance gene into mtaA both impaired myxothiazol synthesis. Genes mtaC and mtaD encode the enzymes for bis-thiazol(ine) formation and chain extension on one pure NRPS (MtaC) and on a unique combination of PKS and NRPS (MtaD). The genes mtaE and mtaF encode PKSs including peptide fragments with homology to methyltransferases. These methyltransferase modules are assumed to be necessary for the formation of the proposed methoxy- and beta-methoxy-acrylate intermediates of myxothiazol biosynthesis. The last gene of the cluster, mtaG, again resembles a NRPS and provides insight into the mechanism of the formation of the terminal amide of myxothiazol. The carbon backbone of an amino acid added to the myxothiazol-acid is assumed to be removed via an unprecedented module with homology to monooxygenases within MtaG.     

Microbial production host selection for converting second-generation feedstocks into bioproducts

Although many secondary metabolites with diverse biological activities have been isolated from myxobacteria, most strains of these biotechnologically important gliding prokaryotes remain difficult to handle genetically. In this study we describe the new fast growing myxobacterial thermophilic isolate GT-2 as a heterologous host for the expression of natural product biosynthetic pathways isolated from other myxobacteria. According to the results of sequence analysis of the 16S rDNA, this moderately thermophilic isolate is closely related to Corallococcus macrosporus and was therefore named C. macrosporus GT-2. Fast growth of moderately thermophilic strains results in shorter fermentation and generation times, aspects which are of significant interest for molecular biological work as well as production of secondary metabolites. Development of a genetic manipulation system allowed the introduction of the complete myxochromide biosynthetic gene cluster, located on a transposable fragment, into the chromosome of GT-2. Genetic engineering of the biosynthetic gene cluster by promoter exchange leads to much higher production of myxochromides in the heterologous host C. macrosporus GT-2 in comparison to the original producer Stigmatella aurantiaca and to the previously described heterologous host Pseudomonas putida (600 mg/L versus 8 mg/L and 40 mg/L, respectively).     

The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus

Plasmids with the aadA gene from plasmid R100, which confers resistance to the aminoglycosides spectinomycin and streptomycin in Escherchia coli, can be introduced into wild-type Myxococcus xanthus, strain DK1622, by electroporation. Recombinant M. xanthus strains with integrated plasmids carrying the aadA gene acquire resistance to high levels of these antibiotics. Selection for aadA in M. xanthus can be carried out independently of, or simultaneously with, selection for resistance to kanamycin. The kinds and frequencies of recombination events observed between integrative plasmids with aadA and the M. xanthus chromosome are similar to those observed after the transformation of yeast. Cleavage of integrative plasmid DNA at a site adjacent to a region of homology between the plasmid and the M. xanthus genome favors the targeted disruption of M. xanthus genes by allele replacement.     

Heterologous expression system in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> for fungal biosynthetic gene clusters of secondary metabolites

Fungal secondary metabolites have been considered promising resources in the search for novel bioactive compounds. Given the high potential of fungi as genetic resources, it is essential to find an efficient way to link biosynthetic genes to the product in a heterologous system, because many genes for the secondary metabolite in the original strain are silent under standard laboratory conditions. In a previous study, we constructed a heterologous expression system for a biosynthetic gene cluster using Aspergillus oryzae as the host. To make the host more versatile for the expression of secondary metabolism genes, the expression levels of a global regulator, laeA, were increased by placing the A. oryzae laeA gene under the control of the constitutive active pgk promoter. In the A. oryzae overexpressing laeA, two clusters of heterologous biosynthetic genes [the monacolin K (MK) gene cluster from Monascus pilosus and the terrequinone A (TQ) gene cluster from Aspergillus nidulans] were successfully overexpressed, resulting in the production of the corresponding metabolite, MK or TQ. The successful production of secondary metabolites belonging to different structural groups, namely MK as a polyketide and TQ as a hybrid of amino acid and isoprenoid, indicated that the laeA-enriched A. oryzae was a versatile host for the heterologous expression of the biosynthetic gene cluster.     

Protocols for yTREX/Tn5-based gene cluster expression in Pseudomonas putida

Bacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’: Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5-based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX-based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine-1-carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putidaKT2440 as well as the use of β-galactosidase-encoding lacZ as a reporter of production levels.     

Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase

Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase.     

Discovery of a new diol-containing polyketide by heterologous expression of a silent biosynthetic gene cluster from <Emphasis Type="Italic">Streptomyces lavendulae</Emphasis> FRI-5

The genome of streptomycetes has the ability to produce many novel and potentially useful bioactive compounds, but most of which are not produced under standard laboratory cultivation conditions and are referred to as silent/cryptic secondary metabolites. Streptomyces lavendulae FRI-5 produces several types of bioactive compounds. However, this strain may also have the potential to biosynthesize more useful secondary metabolites. Here, we activated a silent biosynthetic gene cluster of an uncharacterized compound from S. lavendulae FRI-5 using heterologous expression. The engineered strain carrying the silent gene cluster produced compound 5, which was undetectable in the culture broth of S. lavendulae FRI-5. Using various spectroscopic analyses, we elucidated the chemical structure of compound 5 (named lavendiol) as a new diol-containing polyketide. The proposed assembly line of lavendiol shows a unique biosynthetic mechanism for polyketide compounds. The results of this study suggest the possibility of discovering more silent useful compounds from streptomycetes by genome mining and heterologous expression.     

A simple and reliable method to conduct and monitor expression cassette integration into the Escherichia coli chromosome

We report a method for the integration of expression cassettes into the Escherichia coli chromosome using rare and dispensable sugar degradation gene loci as sites for integration. Clones carrying successfully recombined DNA fragments in the chromosome are easily screened using a solid differential medium containing the respective sugar compound. As an example for the heterologous expression of a complex natural product biosynthesis pathway, we show the stepwise chromosomal integration of the zeaxanthin biosynthesis pathway from Pantoea ananatis into E. coli.     

Mining of a streptothricin gene cluster from Streptomyces sp. TP-A0356 genome via heterologous expression

Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces sp. TP-A0356. The ST cluster from S. sp. TP-A0356 was verified by successful heterologous expression in Streptomyces coelicolor M145. Two new ST analogs were produced together with streptothricin F and streptothricin D in the heterologous host. The ST cluster was further confirmed by inactivation of gene stnO, which was proposed encoding an aminomutase supplying -lysines for the poly-β-Lys chain formation. A putative biosynthetic pathway for STs is proposed based on bioinformatics analyses of the ST genes and experimental evidence.     

Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

Due to the worldwide prevalence of multidrug-resistant pathogens and high incidence of diseases such as cancer, there is an urgent need for the discovery and development of new drugs. Nearly half of the FDA-approved drugs are derived from natural products that are produced by living organisms, mainly bacteria, fungi, and plants. Commercial development is often limited by the low yield of the desired compounds expressed by the native producers. In addition, recent advances in whole genome sequencing and bioinformatics have revealed an abundance of cryptic biosynthetic gene clusters within microbial genomes. Genetic manipulation of clusters in the native host is commonly used to awaken poorly expressed or silent gene clusters, however, the lack of feasible genetic manipulation systems in many strains often hinders our ability to engineer the native producers. The transfer of gene clusters into heterologous hosts for expression of partial or entire biosynthetic pathways is an approach that can be used to overcome this limitation. Heterologous expression also facilitates the chimeric fusion of different biosynthetic pathways, leading to the generation of “unnatural” natural products. The genus Streptomyces is especially known to be a prolific source of drugs/antibiotics, its members are often used as heterologous expression hosts. In this review, we summarize recent applications of Streptomyces species, S. coelicolor, S. lividans, S. albus, S. venezuelae and S. avermitilis, as heterologous expression systems.     

海洋来源节菱孢菌ZSDS1-F3中PKS-NRPS类天然产物挖掘

【目的】以基因组信息为指导,定向激活海洋来源真菌Arthrinium arundinisZSDS1-F3中沉默的聚酮合成酶-非核糖体肽合成酶(PKS-NRPS)类生物合成基因簇,鉴定次级代谢产物结构。【方法】通过启动子工程和异源表达的策略激活实验室培养条件下沉默或低表达的生物合成基因簇,实现目标化合物的分离,通过HR-ESI-MS和NMR数据分析鉴定产物结构,结合基因重组和生物信息学分析结果推导化合物的生物合成途径。【结果】依据基因组生物信息学分析,从海洋来源真菌A. arundinis ZSDS1-F3中选取一个编码PKS-NRPS类次级代谢产物的生物合成基因簇开展研究,在宿主Aspergillus nidulansA1145中实现了基因簇的异源表达,从中分离到2个新化合物,并推导了其生物合成途径。【结论】基因组信息指导下的天然产物挖掘,可以目标明确地分离产物,加快真菌中新颖天然产物的发现步伐。     

Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage C31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.     

The mode of action of stigmatellin,a new inhibitor of the cytochrome b-c1 segment of the respiratory chain

The new antibiotic stigmatellin, obtained from the myxobacterium Stigmatella aurantiaca, was found to block the electron flow in the respiratory chain of bovine heart submitochondrial particles at the site of the cytochrome b-c₁ segment. Its inhibitory potency was identical with that of antimycin and myxothiazol, and like these antibiotics, stigmatellin caused a shift in the spectrum of reduced cytochrome b. Difference spectroscopic studies with the three inhibitors in various combinations indicated that the binding site of stigmatellin was different from that of antimycin, but more or less identical with that of myxothiazol. Experiments with 14 synthesized derivatives of stigmatellin showed that good inhibitory activity can be expected only if the side chain was kept relatively lipophilic, and the keto and the hydroxy groups of the chromone system were left intact.