Catalases play a key role in the defense against oxidative stress in bacteria by catalyzing the decomposition of H
O
. In addition, catalases are also involved in multiple cellular processes, such as ...cell development and differentiation, as well as metabolite production. However, little is known about the abundance, diversity, and distribution of catalases in bacteria. In this study, we systematically surveyed and classified the homologs of three catalase families from 2,634 bacterial genomes. It was found that both of the typical catalase and Mn-catalase families could be divided into distinct groups, while the catalase-peroxidase homologs formed a tight family. The typical catalases are rich in all the analyzed bacterial phyla except Chlorobi, in which the catalase-peroxidases are dominant. Catalase-peroxidases are rich in many phyla, but lacking in Deinococcus-Thermus, Spirochetes, and Firmicutes. Mn-catalases are found mainly in Firmicutes and Deinococcus-Thermus, but are rare in many other phyla. Given the fact that catalases were reported to be involved in secondary metabolite biosynthesis in several
strains, the distribution of catalases in the genus
was given more attention herein. On average, there are 2.99 typical catalases and 0.99 catalase-peroxidases in each
genome, while no Mn-catalases were identified. To understand detailed properties of catalases in
, we characterized all the five typical catalases from
ATCC 10970, the oxytetracycline-producing strain. The five catalases showed typical catalase activity, but possessed different catalytic properties. Our findings contribute to the more detailed classification of catalases and facilitate further studies about their physiological roles in secondary metabolite biosynthesis and other cellular processes, which might facilitate the yield improvement of valuable secondary metabolites in engineered bacteria.
Streptomyces
produces many valuable and important biomolecules with clinical and pharmaceutical applications. The development of simple and highly efficient gene editing tools for genetic ...modification of
Streptomyces
is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (Δ
pyrF
) resistant to the highly toxic uracil analog of 5-fluoroorotic acid (5-FOA) converted by PyrF, and a non-replicative vector pKC1132-pyrF carrying the complemented
pyrF
gene coding for orotidine-5′-phosphate decarboxylase. The
pyrF
gene acts as a positive selection and counterselection marker for recombinants during genetic modifications. Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing
pyrF
along with pKC1132-pyrF into the genome of the mutant Δ
pyrF
at the targeted locus. Double-crossover recombinants were generated, from which the
pyrF
gene, plasmid backbone, and targeted gene were excised through homologous recombination exchange. These recombinants were rapidly screened by the counterselection agent, 5-FOA. We demonstrated the feasibility and advantage of using this
pyrF
-based screening system through deleting the
otcR
gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in
Streptomyces rimosus
M4018. This system provides a new genetic tool for investigating the genetic characteristics of
Streptomyces
species.
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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
Oxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus. Despite its importance, little is known about the regulation of OTC biosynthesis, which hampered ...any effort to improve OTC production via engineering regulatory genes.
A gene encoding a Streptomyces antibiotic regulatory protein (SARP) was discovered immediately adjacent to the otrB gene of oxy cluster in S. rimosus and designated otcR. Deletion and complementation of otcR abolished or restored OTC production, respectively, indicating that otcR encodes an essential activator of OTC biosynthesis. Then, the predicted consensus SARP-binding sequences were extracted from the promoter regions of oxy cluster. Transcriptional analysis in a heterologous GFP reporter system demonstrated that OtcR directly activated the transcription of five oxy promoters in E. coli, further mutational analysis of a SARP-binding sequence of oxyI promoter proved that OtcR directly interacted with the consensus repeats. Therefore, otcR was chosen as an engineering target, OTC production was significantly increased by overexpression of otcR as tandem copies each under the control of strong SF14 promoter.
A SARP activator, OtcR, was identified in oxy cluster of S. rimosus; it was shown to directly activate five promoters from oxy cluster. Overexpression of otcR at an appropriate level dramatically increased OTC production by 6.49 times compared to the parental strain, thus demonstrating the great potential of manipulating OtcR to improve the yield of OTC production.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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
In Pseudomonas aeruginosa, quorum sensing (QS) regulates dozens of genes and proteins, many of which contribute to the virulence of this pathogen. QS inhibitory (QSI) compounds have been proposed as ...potential agents for treatment of bacterial infections. To search for Ps. aeruginosa QS inhibitors, we constructed an effective screening system, QSIS-lasI selector, based on the PlasI-sacB reporter, in which QS could be induced with 20 nM 3-oxo-N-(3S)-tetrahydro-2-oxo-3-furanyl-dodecanamide (3-oxo-C12-HSL). During screening of the crude extracts from 65 marine fungi, an isolate of Penicillium atramentosum was found to have QSI activity. Thin-layer chromatography assay of the fungal extracts for bioautographic identification of QSIS-lasI indicated that this fungus produced several QSI compounds, including QS inhibitors other than penicillic acid or patulin.
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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
Streptomycetes attract a lot of attention in metabolic engineering and synthetic biology because of their well-known ability to produce secondary metabolites. However, the available constitutive ...promoters are rather limited in this genus.
In this work, constitutive promoters were selected from a pool of promoters whose downstream genes maintained constant expression profiles in various conditions. A total of 941 qualified genes were selected based on systematic analysis of five sets of time-series transcriptome microarray data of Streptomyces coelicolor M145 cultivated under different conditions. Then, 166 putative constitutive promoters were selected by following a rational selection workflow containing disturbance analysis, function analysis, genetic loci analysis, and transcript abundance analysis. Further, eight promoters with different strengths were chosen and subjected to experimental validation by green fluorescent protein reporter and real-time reverse-transcription quantitative polymerase chain reaction in S. coelicolor, Streptomyces venezuelae and Streptomyces albus. The eight promoters drove the stable expression of downstream genes in different conditions, implying that the 166 promoters that we identified might be constitutive under the genus Streptomyces. Four promoters were used in a plug-and-play platform to control the expression of the cryptic cluster of jadomycin B in S. venezuelae ISP5230 and resulted in different levels of the production of jadomycin B that corresponded to promoter strength.
This work identified and evaluated a set of constitutive promoters with different strengths in streptomycetes, and it enriched the presently available promoter toolkit in this genus. These promoters should be valuable in current platforms of metabolic engineering and synthetic biology for the activation of cryptic biosynthetic clusters and the optimization of pathways for the biosynthesis of important natural products in Streptomyces species.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive ...repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (K D = 1.7 × 10–10 M) and oxytetracycline induced dissociation of the OtrR/DNA complex in a concentration-dependent manner. On the basis of these genetic parts, a synthetic inducible expression system Potr* was optimized. Induction of Potr* with 0.01–4 μM of oxytetracycline triggered a wide-range expression level of gfp reporter gene in different Streptomyces species. Benchmarking Potr* against the widely used constitutive promoters ermE* and kasOp* revealed greatly enhanced levels of expression when Potr* was fully induced. Finally, Potr* was used as a tool to activate and optimize the expression of the silent jadomycin biosynthetic gene cluster in Streptomyces venezuelae. Altogether, the synthetic Potr* presents a new versatile tool for fine-tuning gene expression in streptomycetes.
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IJS, KILJ, NUK, PNG, UL, UM
Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites. Here, it is employed to activate and manipulate the oxytetracycline (OTC) gene ...cluster and to alter OTC fermentation process. To achieve these goals, a fast-growing heterologous host
Streptomyces venezuelae
WVR2006 was rationally selected among several potential hosts. It shows rapid and dispersed growth and intrinsic high resistance to OTC. By manipulating the expression of two cluster-situated regulators (CSR) OtcR and OtrR and precursor supply, the OTC production level was significantly increased in this heterologous host from 75 to 431 mg/l only in 48 h, a level comparable to the native producer
Streptomyces rimosus
M4018 in 8 days. This work shows that
S. venezuelae
WVR2006 is a promising chassis for the production of secondary metabolites, and the engineered heterologous OTC producer has the potential to completely alter the fermentation process of OTC production.
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CEKLJ, DOBA, EMUNI, FZAB, GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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