Chemical pesticides or insecticides with complex structures are highly abundant in the biosphere and have inevitable side effects on farmland, natural resources, and human health. Deltamethrin is the ...most popular and widely used pesticide that disrupts the cellular calcium channels. In the present study, isolated strains of bacteria were examined to determine the ones that were capable of degrading deltamethrin. Different species of bacteria were evaluated in terms of the capability to degrade deltamethrin. It is important to note that Streptomyces rimosus was able to degrade up to 200 mg/L deltamethrin concentration and could be grown in mineral salt medium agar containing deltamethrin to be used as a source of carbon and energy. The results demonstrated that there is a diversity of deltamethrin‐degrading bacteria in agricultural soil ecosystems. The application of these bacteria, especially S. rimosus, might be used as a bioremediation technique to decrease pesticide contamination of the ecosystem.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
In Streptomyces rimosus M527, the oxytetracycline (OTC) biosynthetic gene cluster is not expressed under laboratory conditions. In this study a reported‐guided mutant selection (RGMS) procedure was ...used to activate the cluster. The double‐reporter plasmid pAGT was constructed in which gusA encoding a β‐glucuronidase and tsr encoding a thiostrepton resistance methyltransferase were placed under the control of the native promoter of oxyA gene (PoxyA). Plasmid pAGT was introduced and integrated into the chromosome of S. rimosus M527 by conjugation, yielding initial strain M527‐pAGT. Subsequently, mutants of M527‐pAGT were generated by using ribosome engineering technology. The mutants harboring activated OTC gene cluster were selected based on visual observation of GUS activity and thiostrepton resistance. Finally, mutant M527‐pAGT‐R7 was selected producing OTC in a concentration of 235.2 mg/L. In this mutant transcriptional levels of oxysr genes especial oxyAsr gene were increased compared to wild‐type strain S. rimosus M527. The mutant M527‐pAGT‐R7 showed antagonistic activities against Gram‐negative and Gram‐positive strains. All data indicate that the OTC gene cluster was successfully activated using the RGMS method.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The polyene macrolide rimocidin, produced by
Streptomyces rimosus
M527, is highly effective against a broad range of fungal plant pathogens, but at low yields. Elicitation is an effective method of ...stimulating the yield of bioactive secondary metabolites. In this study, the biomass and filtrate of a culture broth of
Escherichia coli
JM109,
Bacillus subtilis
WB600,
Saccharomyces cerevisiae
, and
Fusarium oxysporum
f. sp.
cucumerinum
were employed as elicitors to promote rimocidin production in
S. rimosus
M527. Adding culture broth and biomass of
S. cerevisiae
(A3) and
F. oxysporum
f. sp.
cucumerinum
(B4) resulted in an increase of rimocidin production by 51.2% and 68.3% respectively compared with the production under normal conditions in 5-l fermentor. In addition, quantitative RT-PCR analysis revealed that the transcriptions of ten genes (
rimA
to
rimK
) located in the gene cluster involved in rimocidin biosynthesis in A3 or B4 elicitation experimental group were all higher than those of a control group. Using a β-glucuronidase (GUS) reporter system, GUS enzyme activity assay, and Western blot analysis, we discovered that elicitation of A3 or B4 increased protein synthesis in
S. rimosus
M527. These results demonstrate that the addition of elicitors is a useful approach to improve rimocidin production.
Key Points
• An effective strategy for enhancing rimocidin production in S. rimosus M527 is demonstrated.
• Overproduction of rimocidin is a result of higher expressed structural genes followed by an increase in protein synthesis.
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CEKLJ, DOBA, EMUNI, FZAB, GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Streptomyces
produce a broad spectrum of biologically active molecules such as oxytetracycline and rimocidin, which are widely used in human and animal treatments. microparticle-enhanced cultivation ...(MPEC) is one of the tools used for
Streptomyces
bioprocesses intensification by the control of mycelial morphology. In the present work, morphological changes of
Streptomyces rimosus
caused by the addition of 10 µm talc microparticles in MPEC were correlated with the biosynthetic activity of the microorganism. Comparing the runs with and without microparticles, major morphological changes were observed in MPEC, including the deformation of pellets, variation of their size, appearance of hyphae and clumps as well as the aggregation of mycelial objects. The presence of talc microparticles also influenced the levels of the studied secondary metabolites produced by
S. rimosus
. Comparing control and MPEC runs, the addition of talc microparticles increased the amounts of oxytetracycline (9-fold), 2-acetyl-2-decarboxamido-oxytetracycline (7-fold), milbemycin A
3
+4O (3-fold) and CE 108 (1.5-fold), while rimocidin (27-ethyl) and milbemycin β
11
+4O production was reduced. In summary, the addition of talc microparticles to
S. rimosus
cultivations led to the development of smaller morphological forms like hyphae and clumps as well as to the changes in the amounts of secondary metabolites.
Graphical abstract
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
High-yielding industrial
Streptomyces
producer is usually obtained by multiple rounds of random mutagenesis and screening. These strains have great potential to be developed as the versatile chassis ...for the discovery and titer improvement of desired heterologous products. Here, the industrial strain
Streptomyces rimosus
461, which is a high producer of oxytetracycline, has been engineered as a robust host for heterologous expression of chlortetracycline (CTC) biosynthetic gene cluster. First, the industrial chassis strain SR0 was constructed by deleting the whole oxytetracycline gene cluster of
S. rimosus
461. Then, the biosynthetic gene cluster
ctc
of
Streptomyces aureofaciens
ATCC 10762 was integrated into the chromosome of SR0. With an additional constitutively expressed cluster-situated activator gene
ctcB
, the CTC titer of the engineering strain SRC1 immediately reached 1.51 g/L in shaking flask. Then, the CTC titers were upgraded to 2.15 and 3.27 g/L, respectively, in the engineering strains SRC2 and SRC3 with the enhanced
ctcB
expression. Further, two cluster-situated resistance genes were co-overexpressed with
ctcB
. The resultant strain produced CTC up to 3.80 g/L in shaking flask fermentation, which represents 38 times increase in comparison with that of the original producer. Overall, SR0 presented in this study have great potential to be used for heterologous production of tetracyclines and other type II polyketides.
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CEKLJ, DOBA, EMUNI, FZAB, GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Actinomycetes have received considerable attention as biocontrol agents against fungal plant pathogens and as plant growth promoters. In this study, a total of 320 actinomycetes were isolated from ...various habitats in China. Among which, 77 strains have been identified as antagonistic activities against Fusarium oxysporum f. sp. cucumerinum which usually caused fusarium wilt of cucumber. Of these, isolate actinomycete M527 not only displayed broad‐spectrum antifungal activity but also showed the strongest antagonistic activity against the spore germination of F. oxysporum f. sp. cucumerinum. In pot experiments, the results indicated that isolate M527 could promote the shoot growth and prevent the development of the disease on cucumber caused by F. oxysporum f. sp. cucumerinum. The control efficacy against seedling fusarium wilt of cucumber after M527 fermentation broth root‐irrigation was up to 72.1% as compared to control. Based on 16S rDNA sequence analysis, the isolate M527 was identified as Streptomyces rimosus.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
As microbial genome sequencing becomes more widespread, the capacity of microorganisms to produce an immense number of metabolites has come into better view. Utilizing a metabolite/gene cluster ...correlation platform, the biosynthetic origins of a new family of natural products, the rimosamides, were discovered. The rimosamides were identified in Streptomyces rimosus and associated with their NRPS/PKS-type gene cluster based upon their high frequency of co-occurrence across 179 strains of actinobacteria. This also led to the discovery of the related detoxin gene cluster. The core of each of these families of natural products contains a depsipeptide bond at the point of bifurcation in their unusual branched structures, the origins of which are definitively assigned to nonlinear biosynthetic pathways via heterologous expression in Streptomyces lividans. The rimosamides were found to antagonize the antibiotic activity of blasticidin S against Bacillus cereus.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Clustered regularly interspaced short palindromic repeats, associated proteins (CRISPR/Cas), has been developed into a powerful, targeted genome-editing tool in a wide variety of species. Here, we ...report an extensive investigation of the type II CRISPR/Cas9 system for targeted gene editing in Streptomyces rimosus. S. rimosus is used in the production of the antibiotic oxytetracycline, and its genome differs greatly from other species of the genus Streptomyces in the conserved chromosome terminal and core regions, which is of major production and scientific research value. The genes zwf2 and devB were chosen as target genes, and were edited separately via single-site mutations, double-site mutations and gene fragment disruptions. The single-site mutation guided by sgRNA-1 or sgRNA-2, respectively, involved GG changing to CA, GC changing to AT, and GG changing to CC. The double-site mutations guided by sgRNA-1 and sgRNA-2 included deletions and/or point mutations. Consistently, all mutations occurred in the gRNA sequence regions. Deletion mutations were characterized by the absence of eight bases, including three bases upstream of the PAM (protospacer adjacent motif) sequence, the PAM sequence itself and two bases downstream of the PAM sequence. A mutant (zwf2-devB-) with a high yield of oxytetracycline was successfully obtained, whose oxytetracycline level was increased by 36.8 % compared to the original strain. These results confirm that CRISPR/Cas9 can successfully serve as a useful targeted genome editing system in S. rimosus.
Regulation of secondary metabolism involves complex interactions of both pathway-specific regulators and global regulators, which may trigger or repress the expression of genes involved in antibiotic ...biosynthesis. Similarly, many of these global regulatory proteins belong to two-component systems. In this study, a new two-component system (TCS) AfrQ1Q2 homologous to AfsQ1Q2 of Streptomyces coelicolor was acquired from the genome sequence of Streptomyces rimosus M4018 by using bioinformatics analysis. RT-PCR results showed co-transcription of afrQ1 (RR) and afrQ2 (HK) in S. rimosus. Consequently, the significant enhancement in oxytetracycline (OTC) yield in afrQ1-disrupted mutant was observed when cultivated in the defined minimal medium (MM) with glycine as the sole nitrogen source. In order to further investigate the regulation mechanism of AfrQ1Q2 in OTC production, the transcriptional levels of five biosynthesis and regulation related genes such as oxyB, otrB, otcG, otcR and otrC were tested by qRT-PCR, which indicated a significantly up-regulatory trend in the afrQ1-disrupted mutant. Meanwhile, a down-regulatory trend of each gene was tested in the complementary mutant as compared to wild type M4018. Moreover, these selected five genes were positively correlated with OTC production. Conclusively, these findings suggested that the TCS AfrQ1Q2 could be one of the global regulators, which negatively regulates OTC production via activating pathway specific regulators in S. rimosus M4018.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be ...cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation.
We achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease.
Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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