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
Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear ...chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three
strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in
. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained.
The genomes of
species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids GLPs), rearrangements, and high GC content. To improve the quality of the
ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters.
Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong ...pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using
, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental
Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value.IMPORTANCEThere is a critical need to develop novel antibiotics to combat antimicrobial resistance.
species are very rich source of antibiotics, typically encoding 20-60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial
strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain
ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.
The removal of heavy metals by Streptomyces rimosus has been the subject of many investigations. This review paper focuses on the removal of heavy metals from aqueous solution through Streptomyces ...rimosus, produced from pharmaceutical industry as solid waste, as adsorbent, and discusses the effect of various process parameters like pH, temperature, metal concentration etc., on the metal removal efficiency of this bacterium. The paper also evaluates the different kinetic, equilibrium, and thermodynamic models used in Streptomyces rimosus sorption of heavy metals. Biomass characterization and sorption mechanisms as well as elution of metal ions are also discussed. The literature revealed that Streptomyces rimosus had a good affinity for binding lead and iron compared with other heavy metals. The adsorption of heavy metals is well described by Langmuir isotherm, which expresses the existence of monolayer adsorption. The kinetic data followed both pseudo first order and pseudo second order models. Thermodynamic studies showed spontaneous and exothermic nature of the sorption processes in most case. Dilute acids (HCl and H2SO4) are quite effective in desorption of heavy metals. Ion exchange played the chief role in the adsorption mechanism of metal, and carboxyl groups are mainly involved in this mechanism.
•Metal removal through biosorption is attractive.•Streptomyces rimosus biomass as potential adsorbent•In this paper, biosorption of heavy metals by Streptomyces rimosus is reviewed.•The need to switch batch to fixed columns studies for field applications•Ion exchange played the chief role in the adsorption mechanism of metal.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Precursor engineering is an effective strategy for the overproduction of secondary metabolites. The polyene macrolide rimocidin, which is produced by
Streptomyces rimosus
M527, exhibits a potent ...activity against a broad range of phytopathogenic fungi. It has been predicted that malonyl-CoA is used as extender units for rimocidin biosynthesis. Based on a systematic analysis of three sets of time-series transcriptome microarray data of
S
.
rimosus
M527 fermented in different conditions, the differentially expressed
acc
sr
gene that encodes acetyl-CoA carboxylase (ACC) was found. To understand how the formation of rimocidin is being influenced by the expression of the
acc
sr
gene and by the concentration of malonyl-CoA, the
acc
sr
gene was cloned and over-expressed in the wild-type strain
S
.
rimosus
M527 in this study. The recombinant strain
S
.
rimosus
M527-ACC harboring the over-expressed
acc
sr
gene exhibited better performances based on the enzymatic activity of ACC, intracellular malonyl-CoA concentrations, and rimocidin production compared to
S
.
rimosus
M527 throughout the fermentation process. The enzymatic activity of ACC and intracellular concentration of malonyl-CoA of
S
.
rimosus
M527-ACC were 1.0- and 1.5-fold higher than those of
S
.
rimosus
M527, respectively. Finally, the yield of rimocidin produced by
S
.
rimosus
M527-ACC reached 320.7 mg/L, which was 34.0% higher than that of
S
.
rimosus
M527. These results confirmed that malonyl-CoA is an important precursor for rimocidin biosynthesis and suggested that an adequate supply of malonyl-CoA caused by
acc
sr
gene over-expression led to the improvement in rimocidin production.
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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
An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic ...transformation system based on intergeneric conjugation was developed in
Streptomyces rimosus
M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10
-5
per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter
potrB
, and a constitutive promoter
permE"
commonly used for gene expression in streptomycetes were selected and their activity was analyzed using
gusA
as a reporter gene in
S. rimosus
M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in (3-glucuronidase (GUS) activity compared with the control promoter
permE
*. Promoter SPL-57 showed activity comparable to that of
permE
*. Promoter
potrB
, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control
permE
*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S.
rimosus
M527.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The transposon mutagenesis strategy has been employed to generate random insertion mutants and analyze the correlation between genes and secondary metabolites in the genus
Streptomyces
. In this ...study, our primary objective was to identify an unknown gene involved in rimocidin biosynthesis and elucidate its role in rimocidin production in
Streptomyces rimosus
M527. To achieve this, we established a random mutant library of
S. rimosus
M527 using a Tn
5
transposon-mediated random mutagenesis strategy. Among the 137 isolated mutants, M527-G10 and M527-W5 exhibited the most significant variations in antagonistic activity against the plant pathogenic fungus
Fusarium oxysporum
f. sp.
cucumerinum
. Specifically, M527-G10 displayed a 72.93% reduction, while M527-W5 showed a 49.8% increase in rimocidin production compared to the wild-type (WT) strain
S. rimosus
M527. Subsequently, we employed a plasmid rescue strategy to identify the insertion loci of the transposon in the genomes of mutants M527-G10 and M527-W5, revealing a response regulator transcription factor (
rrt
) and a hypothetical protein (
hyp
), respectively. The roles of
rrt
and
hyp
in rimocidin biosynthesis were determined through gene deletion, overexpression in the WT strain, and complemented expression in the transposon mutants. Notably, the gene-deletion mutants M527-ΔRRT and M527-ΔHYP exhibited similar behavior in rimocidin production compared to the corresponding transposon mutants M527-G10 and M527-W5, suggesting that transposon insertions in genes
rrt
and
hyp
led to alterations in rimocidin production. Furthermore, both gene deletion and overexpression of
rrt
and
hyp
had no discernible effects on cell growth. These results reveal that genes
rrt
and
hyp
have positive and negative impacts on rimocidin production in
S. rimosus
M527, respectively.
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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
Increasing the self-resistance levels of Streptomyces is an effective strategy to improve the production of antibiotics. To increase the oxytetracycline (OTC) production in Streptomyces rimosus, we ...investigated the cooperative effect of three co-overexpressing OTC resistance genes: one gene encodes a ribosomal protection protein (otrA) and the other two express efflux proteins (otrB and otrC). Results indicated that combinational overexpression of otrA, otrB, and otrC (MKABC) exerted a synergetic effect. OTC production increased by 179% in the recombinant strain compared with that of the wild-type strain M4018. The resistance level to OTC was increased by approximately two-fold relative to the parental strain, thereby indicating that applying the cooperative effect of self-resistance genes is useful to improve OTC production. Furthermore, the previously identified cluster-situated activator OtcR was overexpressed in MKABC in constructing the recombinant strain MKRABC; such strain can produce OTC of approximately 7.49 g L
, which represents an increase of 19% in comparison with that of the OtcR-overexpressing strain alone. Our work showed that the cooperative overexpression of self-resistance genes is a promising strategy to enhance the antibiotics production in Streptomyces.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Streoptomyces rimosus M527 is a producer of the polyene macrolide rimocidin which shows activity against various plant pathogenic fungi. Notably, the regulatory mechanisms underlying rimocidin ...biosynthesis are yet to be elucidated.
In this study, using domain structure and amino acid alignment and phylogenetic tree construction, rimR2, which located in the rimocidin biosynthetic gene cluster, was first found and identified as a larger ATP-binding regulators of the LuxR family (LAL) subfamily regulator. The rimR2 deletion and complementation assays were conducted to explore its role. Mutant M527-ΔrimR2 lost its ability to produce rimocidin. Complementation of M527-ΔrimR2 restored rimocidin production. The five recombinant strains, M527-ER, M527-KR, M527-21R, M527-57R, and M527-NR, were constructed by overexpressing rimR2 gene using the promoters permE
, kasOp
, SPL21, SPL57, and its native promoter, respectively, to improve rimocidin production. M527-KR, M527-NR, and M527-ER exhibited 81.8%, 68.1%, and 54.5% more rimocidin production, respectively, than the wild-type (WT) strain, while recombinant strains M527-21R and M527-57R exhibited no obvious differences in rimocidin production compared with the WT strain. RT-PCR assays revealed that the transcriptional levels of the rim genes were consistent with the changes in rimocidin production in the recombinant strains. Using electrophoretic mobility shift assays, we confirmed that RimR2 can bind to the promoter regions of rimA and rimC.
A LAL regulator RimR2 was identified as a positive specific-pathway regulator of rimocidin biosynthesis in M527. RimR2 regulates the rimocidin biosynthesis by influencing the transcriptional levels of rim genes and binding to the promoter regions of rimA and rimC.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new ...microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well‐studied strains. Herein, we targeted Streptomyces rimosus, the widely‐used oxytetracycline producer, for the discovery of new natural products. Using MALDI‐MS‐guided high‐throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with backbone modifications and several non‐canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from “drained” strains.
Microbial producers of pharmaceuticals still code for dozens of yet‐to‐be identified natural products. Herein, three groups of cryptic metabolites were uncovered from Streptomyces rimosus, the industrial producer of oxytetracycline, using high‐throughput elicitor screening, notably momomycin, a novel cyclic peptide with antiproliferative properties.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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