Physiological and chemical investigations into microbial degradation of synthetic Poly(cis-1,4-isoprene)

Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly(cis-1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for S. coelicolor 1A and P. citronellolis, whereas the molecular weight distribution for the control (S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as (6Z)-2,6-dimethyl-10-oxo-undec-6-enoic acid, (5Z)-6-methyl-undec-5-ene-2,9-dione, and (5Z,9Z)-6, 10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly(cis-1,4-isoprene) to methyl-branched diketones is proposed. It includes (i) oxidation of an aldehyde intermediate to a carboxylic acid, (ii) one cycle of beta-oxidation, (iii) oxidation of the conjugated double bond resulting in a beta-keto acid, and (iv) decarboxylation.     

Identification of Poly(cis-1,4-Isoprene) Degradation Intermediates during Growth of Moderately Thermophilic Actinomycetes on Rubber and Cloning of a Functional lcp Homologue from Nocardia farcinica Strain E1

The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50°C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.     

Characteristics of a Streptomyces coelicolor A3(2) Extracellular Protein Targeting Chitin and Chitosan

Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis. The chb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free Streptomyces lividans TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the S. lividans TK21 transformant during growth in the presence of glucose, N-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets α-chitin, β-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described.     

Oligo(cis-1,4-isoprene) aldehyde-oxidizing dehydrogenases of the rubber-degrading bacterium Gordonia polyisoprenivorans VH2

The actinomycete Gordonia polyisoprenivorans strain VH2 is well-known for its ability to efficiently degrade and catabolize natural rubber [poly(cis-1,4-isoprene)]. Recently, a pathway for the catabolism of rubber by strain VH2 was postulated based on genomic data and the analysis of mutants (Hiessl et al. in Appl Environ Microbiol 78:2874–2887, 2012). To further elucidate the degradation pathway of poly(cis-1,4-isoprene), 2-dimensional-polyacrylamide gel electrophoresis was performed. The analysis of the identified protein spots by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry confirmed the postulated intracellular pathway suggesting a degradation of rubber via β-oxidation. In addition, other valuable information on rubber catabolism of G. polyisoprenivorans strain VH2 (e.g. oxidative stress response) was provided. Identified proteins, which were more abundant in cells grown with rubber than in cells grown with propionate, implied a putative long-chain acyl-CoA-dehydrogenase, a 3-ketoacyl-CoA-thiolase, and an aldehyde dehydrogenase. The amino acid sequence of the latter showed a high similarity towards geranial dehydrogenases. The expression of the corresponding gene was upregulated > 10-fold under poly(cis-1,4-isoprene)-degrading conditions. The putative geranial dehydrogenase and a homolog were purified and used for enzyme assays. Deletion mutants for five aldehyde dehydrogenases were generated, and growth with poly(cis-1,4-isoprene) was investigated. While none of the mutants had an altered phenotype regarding growth with poly(cis-1,4-isoprene) as sole carbon and energy source, purified aldehyde dehydrogenases were able to catalyze the oxidation of oligoisoprene aldehydes indicating an involvement in rubber degradation.

     

Novel Type of Heme-Dependent Oxygenase Catalyzes Oxidative Cleavage of Rubber (Poly-cis-1,4-Isoprene)

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH₂— and —CH₂-COCH₃. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40°C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.     

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

Establishment of Tn5096-Based Transposon Mutagenesis in Gordonia polyisoprenivorans

The transposons Tn5, Tn10, Tn611, and Tn5096 were characterized regarding transposition in Gordonia polyisoprenivorans strain VH2. No insertional mutants were obtained employing Tn5 or Tn10. The thermosensitive plasmid pCG79 harboring Tn611 integrated into the chromosome of G. polyisoprenivorans; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn5096-mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn5096 as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly(cis-1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly(cis-1,4-isoprene). One rubber-negative mutant was disrupted in mcr, encoding an α-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(cis-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons.     

Effects of increased and deregulated expression of cell division genes on the morphology and on antibiotic production of streptomycetes

This paper describes the effects of increased expression of the cell division genes ftsZ, ftsQ, and ssgA on the development of both solid- and liquid-grown mycelium of Streptomyces coelicolor and Streptomyces lividans. Over-expression of ftsZ in S. coelicolor M145 inhibited aerial mycelium formation and blocked sporulation. Such deficient sporulation was also observed for the ftsZ mutant. Over-expression of ftsZ also inhibited morphological differentiation in S. lividans 1326, although aerial mycelium formation was less reduced. Furthermore, antibiotic production was increased in both strains, and in particular the otherwise dormant actinorhodin biosynthesis cluster of S. lividans was activated in liquid- and solid-grown cultures. No significant alterations were observed when the gene dosage of ftsQ was increased. Analysis by transmission electron microscopy of an S. coelicolor strain over-expressing ssgA showed that septum formation had strongly increased in comparison to wild-type S. coelicolor, showing that SsgA clearly influences Streptomyces cell division. The morphology of the hyphae was affected such that irregular septa were produced with a significantly wider diameter, thereby forming spore-like compartments. This suggests that ssgA can induce a process similar to submerged sporulation in Streptomyces strains that otherwise fail to do so. A working model is proposed for the regulation of septum formation and of submerged sporulation.     

Heme-Dependent Rubber Oxygenase RoxA of Xanthomonas sp. Cleaves the Carbon Backbone of Poly(cis-1,4-Isoprene) by a Dioxygenase Mechanism

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of ¹⁶O₂, ¹⁸O₂, H₂¹⁶O, and H₂¹⁸O. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of ¹⁸O-compounds. Incorporation of one ¹⁸O atom in ODTD was found if the cleavage reaction was performed in the presence of ¹⁸O₂ and H₂¹⁶O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H₂¹⁸O in the presence of ¹⁶O₂ indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of ¹⁸O₂, H₂¹⁶O, and alcohol dehydrogenase/NADH, incorporation of two atoms of ¹⁸O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.     

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.     

Latex Clearing Protein—an Oxygenase Cleaving Poly(cis-1,4-Isoprene) Rubber at the cis Double Bonds

Gordonia polyisoprenivorans strain VH2, a potent rubber-degrading actinomycete, harbors two latex clearing proteins (Lcps), which are known to be essential for the microbial degradation of rubber. However, biochemical information on the exact role of this protein in the degradation of polyisoprene was lacking. In this study, the gene encoding Lcp1_VH2 was heterologously expressed in strains of Escherichia coli, the corresponding protein was purified, and its role in rubber degradation was examined by measurement of oxygen consumption as well as by chromatographic and spectroscopic methods. It turned out that active Lcp1_VH2 is a monomer and is responsible for the oxidative cleavage of poly(cis-1,4-isoprene) in synthetic as well as in natural rubber by the addition of oxygen (O₂) to the cis double bonds. The resulting oligomers possess repetitive isoprene units with aldehyde (CHO-CH₂—) and ketone (—CH₂-CO-CH₃) functional groups at the termini. Two fractions with average isoprene contents of 18 and 10, respectively, were isolated, thus indicating an endocleavage mechanism. The activity of Lcp1_VH2 was determined by applying a polarographic assay. Alkenes, acyclic terpenes, or other rubber-like polymers, such as poly(cis-1,4-butadiene) or poly(trans-1,4-isoprene), are not oxidatively cleaved by Lcp1_VH2. The pH and temperature optima of the enzyme are at pH 7 and 30°C, respectively. Furthermore, it was demonstrated that active Lcp1_VH2 is a Cu(II)-containing oxygenase that exhibits a conserved domain of unknown function which cannot be detected in any other hitherto-characterized enzyme. The results presented here indicate that this domain might represent a new protein family of oxygenases.     

Streptomyces coelicolor A3(2) Lacks a Genomic Island Present in the Chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage HAU3 resistance (HAU3^r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis

We report the occurrence of an isomerase with a putative (βα)₈-barrel structure involved in both histidine and trypto-phan biosynthesis in Streptomyces coelicolor A3(2) and Mycobacterium tuberculosis HR37Rv. Deletion of a hisA homologue (SCO2050) putatively encoding N′-[(5′-phosphoribosyl)-formimino]-5 amino-imidazole-4-carboxamide ribonucleotide isomerase from the chromosome of S. coelicolor A3(2) generated a double auxotrophic mutant for histidine and tryptophan. The bifunctional gene SCO2050 and its orthologue Rv1603 from M. tuberculosis complemented both hisA and trpF mutants of Escherichia coli. Expression of the E. coli trpF gene in the S. coelicolor mutant only complemented the tryptophan auxo-trophy, and the hisA gene only complemented the histidine auxotrophy. The discovery of this enzyme, which has a broad-substrate specificity, has implications for the evolution of metabolic pathways and may prove to be important for understanding the evolution of the (βα)₈-barrels.     

The terminal inverted repeats of the linear plasmid SCP1 of Streptomyces coelicolor A3(2) possess a truncated copy of the transposon Tn 4811 of Streptomyces lividans 66

Streptomyces coelicolor A3(2), the best genetically studied streptomycete and Streptomyces lividans 66 are very closely related strains. This is further emphasized by our finding that a truncated copy of Tn4811 of S. lividans is present in the terminal inverted repeats of the S. coelicolor giant linear plasmid SCP1. The copy of Tn4811 in SCP1 lacks the first 1276 bp and shows only minor changes in the nucleotide sequence of the remaining 4.12 kb. Tn4811 exists in both ends of SCP1.     

The stereochemistry of hexahydroprenol, ubiquinone and ergosterol biosynthesis in the mycelium of Aspergillus fumigatus Fresenius

1. The mycelium of Aspergillus fumigatus has been shown to incorporate mevalonate into squalene, ubiquinone, ergosterol and hexahydroprenol. 2. The ³H/¹⁴C ratio in ubiquinone, biosynthesized from [2-¹⁴C-(4R)-4-³H₁]mevalonate, is the same as in the squalene; essentially no ³H was incorporated from [2-¹⁴C-(4S)-4-³H₁]mevalonate, indicating the biosynthesis of biogenetically trans-isoprene units. 3. The ³H/¹⁴C ratio for ergosterol (from `4R-mevalonate') was 3:5, showing that the proton at C-24 is not lost during alkylation of the side chain; it probably migrates to C-25. 4. As ³H from both mevalonates was incorporated into the hexahydroprenols the biosynthesis of both cis- and trans-isoprene units must occur. 5. The saturated ω- and ψ-isoprene units are shown to be biogenetically trans, as are two of the unsaturated residues. 6. The saturated α- and unsaturated β-isoprene residues are both biogenetically cis. 7. An inexplicable loss of approximately half of the olefinic protons from the cis-portion of hexahydroprenol occurs; possible reasons for this loss are discussed. 8. Increase in chain length of the hexahydroprenols is by a cis addition. 9. A biosynthesis of hexahydroprenols by addition of cis-isoprene units to all-trans-geranylgeranyl pyrophosphate, or a dihydro or tetrahydro derivative thereof, is suggested.     

The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity

The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, α- and β-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20–80). It showed pronounced activity towards p-nitrophenyl and α- and β-naphthyl esters of C₁₂–C₁₆. Higher activity was observed with α-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 °C) and thermal stability. CD analysis revealed predominance of α-helical structure (54% α-helix, 21% β-sheet) and a T_m value at 66 °C.     

Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp.

Antibiotics have either bactericidal or bacteriostatic activity. However, they also induce considerable gene expression in bacteria when used at subinhibitory concentrations (below the MIC). We found that lincomycin, which inhibits protein synthesis by binding to the ribosomes of Gram-positive bacteria, was effective for inducing the expression of genes involved in secondary metabolism in Streptomyces strains when added to medium at subinhibitory concentrations. In Streptomyces coelicolor A3(2), lincomycin at 1/10 of its MIC markedly increased the expression of the pathway-specific regulatory gene actII-ORF4 in the blue-pigmented antibiotic actinorhodin (ACT) biosynthetic gene cluster, which resulted in ACT overproduction. Intriguingly, S. lividans 1326 grown in the presence of lincomycin at a subinhibitory concentration (1/12 or 1/3 of its MIC) produced abundant antibacterial compounds that were not detected in cells grown in lincomycin-free medium. Bioassay and mass spectrometry analysis revealed that some antibacterial compounds were novel congeners of calcium-dependent antibiotics. Our results indicate that lincomycin at subinhibitory concentrations potentiates the production of secondary metabolites in Streptomyces strains and suggest that activating these strains by utilizing the dose-response effects of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. In addition to these findings, we also report that lincomycin used at concentrations for markedly increased ACT production resulted in alteration of the cytoplasmic protein (F_oF₁ ATP synthase α and β subunits, etc.) profile and increased intracellular ATP levels. A fundamental mechanism for these unique phenomena is also discussed.     

Unlocking Streptomyces spp. for Use as Sustainable Industrial Production Platforms by Morphological Engineering

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.