Crispr streptomyces

Cinnamoyl moiety containing nonribosomal peptides represented by pepticinnamin E are a growing family of natural products isolated from different Streptomyces species and possess diverse bioactivities. The soil bacterium Streptomyces mirabilis P8-A2 harbors a cryptic pepticinnamin biosynthetic gene cluster, producing azodyrecins as major products. Inactivation of the azodyrecin biosynthetic gene cluster by CRISPR-BEST base editing led to the activation and production of pepticinnamin E ( 1 ) and its analogues, pepticinnamins N, O, and P ( 2 - 4 ), the structures of which were determined by detailed NMR spectroscopy, HRMS data, and Marfey's reactions...

We present the newly isolated Streptomyces sungeiensis SD3 strain as a promising microbial chassis for heterologous production of secondary metabolites. S. sungeiensis SD3 exhibits several advantageous traits as a microbial chassis, including genetic tractability, rapid growth, susceptibility to antibiotics, and metabolic capability supporting secondary metabolism. Genomic and transcriptomic sequencing unveiled the primary metabolic capabilities and secondary biosynthetic pathways of S. sungeiensis SD3, including a previously unknown pathway responsible for the biosynthesis of streptazone B1...

Cirratiomycin, a heptapeptide with antibacterial activity, was isolated and characterized in 1981; however, its biosynthetic pathway has not been elucidated. It contains several interesting nonproteinogenic amino acids, such as (2S,3S)-2,3-diaminobutyric acid ((2S,3S)-DABA) and α-(hydroxymethyl)serine, as building blocks. Here, we report the identification of a cirratiomycin biosynthetic gene cluster in Streptomyces cirratus. Bioinformatic analysis revealed that several Streptomyces viridifaciens and Kitasatospora aureofaciens strains also have this cluster...

The demand for discovering novel microbial secondary metabolites is growing to address the limitations in bioactivities such as antibacterial, antifungal, anticancer, anthelmintic, and immunosuppressive functions. Among microbes, the genus Streptomyces holds particular significance for secondary metabolite discovery. Each Streptomyces species typically encodes approximately 30 secondary metabolite biosynthetic gene clusters within its genome, which are mostly uncharacterized in terms of their products and bioactivities...

Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces . However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity...

Azoxy compounds are a distinctive group of bioactive secondary metabolites characterized by a unique RN═N+ (O- )R moiety. The azoxy moiety is present in various classes of metabolites that exhibit various biological activities. The enzymatic mechanisms underlying azoxy bond formation remain enigmatic. Azodyrecins are cytotoxic azoxy metabolites produced by Streptomyces mirabilis P8-A2. Here, we cloned and confirmed the putative azd biosynthetic gene cluster through CATCH cloning followed by expression and production of azodyrecins in two heterologous hosts, S...

The platensimycin (PTM), platencin (PTN), and platensilin (PTL) family of natural products continues to inspire the discovery of new chemistry, enzymology, and medicine. Engineered production of this emerging family of natural products however remains laborious due to the lack of practical systems to manipulate their biosynthesis in the native producing Streptomyces platensis species. Here we report solving this technology gap by implementing a CRISPR-Cas9 system in Streptomyces platensis CB00739 to develop an expedient method to manipulate the PTM, PTN, and PTL biosynthetic machinery in vivo...

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 Tn5 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...

Aborycin is a type I lasso peptide with a stable interlocked structure, offering a favorable framework for drug development. The aborycin biosynthetic gene cluster gul from marine sponge-associated Streptomyces sp. HNS054 was cloned and integrated into the chromosome of S. coelicolor hosts with different copies. The three-copy gul -integration strain S. coelicolor M1346::3 gul showed superior production compared to the one-copy or two-copy gul -integration strains, and the total titer reached approximately 10...

For a potential application of FK506 in the treatment of acute kidney failure only the FKBP12 binding capability of the compound is required while the immunosuppressive activity via calcineurin binding is considered as a likely risk to the patients. The methoxy-groups at C13 and C15 are thought to have significant influence on the immunosuppressive activity of the molecule. Consequently, FK506 analogs with different functionalities at C13 and C15 were generated by targeted CRISPR editing of the AT domains in module 7 and 8 of the biosynthetic assembly line in Streptomyces tsukubaensis...

BACKGROUND: Oviedomycin is one among several polyketides known for their potential as anticancer agents. The biosynthetic gene cluster (BGC) for oviedomycin is primarily found in Streptomyces antibioticus. However, because this BGC is usually inactive under normal laboratory conditions, it is necessary to employ systematic metabolic engineering methods, such as heterologous expression, refactoring of BGCs, and optimization of precursor biosynthesis, to allow efficient production of these compounds...

Streptomyces , a genus of Gram-positive bacteria, is known as nature's medicine maker, producing a plethora of natural products that have huge benefits for human health, agriculture, and biotechnology. To take full advantage of this treasure trove of bioactive molecules, better genetic tools are required for the genetic engineering and synthetic biology of Streptomyces . We therefore developed CUBIC, a novel CUmate-Based Inducible CRISPR interference (CRISPRi) system that allows highly efficient and inducible gene knockdown in Streptomyces ...

Streptomycetes have a strong ability to produce a vast array of bioactive natural products (NPs) widely used in agriculture and veterinary/human medicine. The recently developed CRISPR/Cas9-based genome editing tools have greatly facilitated strain improvement for target NP overproduction as well as novel NP discovery in Streptomyces . However, CRISPR/Cas9 shows high toxicity to the host, limiting its application in many Streptomyces strains with a low DNA transformation efficiency. In this study, we developed a low-toxicity CRISPR/Cas9D10A nickase (nCas9)-based genome editing tool in the model strain Streptomyces coelicolor M145...

Staurosporine is the most well-known member of the indolocarbazole alkaloid family; it can induce apoptosis of many types of cells as a strong protein kinase inhibitor, and is used as an important lead compound for the synthesis of the antitumor drugs. However, the low fermentation level of the native producer remains the bottleneck of staurosporine production. Herein, integration of multi-copy biosynthetic gene cluster (BGC) in well characterized heterologous host and optimization of the fermentation process were performed to enable high-level production of staurosporine...

CRISPR tools, especially Cas9n-sgRNA guided cytidine deaminase base editors such as CRISPR-BEST, have dramatically simplified genetic manipulation of streptomycetes. One major advantage of CRISPR base editing technology is the possibility to multiplex experiments in genomically instable species. Here, we demonstrate scaled up Csy4 based multiplexed genome editing using CRISPR-mcBEST in Streptomyces coelicolor . We evaluated the system by simultaneously targeting 9, 18, and finally all 28 predicted specialized metabolite biosynthetic gene clusters in a single experiment...

Eriodictyol is a hydroxylated flavonoid displaying multiple pharmaceutical activities, such as antitumoral, antiviral or neuroprotective. However, its industrial production is limited to extraction from plants due to its inherent limitations. Here, we present the generation of a Streptomyces albidoflavus bacterial factory edited at the genome level for an optimized de novo heterologous production of eriodictyol. For this purpose, an expansion of the Golden Standard toolkit (a Type IIS assembly method based on the Standard European Vector Architecture (SEVA)) has been created, encompassing a collection of synthetic biology modular vectors (adapted for their use in actinomycetes)...

Lincomycin is a broad-spectrum antibiotic and particularly effective against Gram-positive pathogens. Albeit familiar with the biosynthetic mechanism of lincomycin, we know less about its regulation, limiting the rational design for strain improvement. We therefore analyzed two-component systems (TCSs) in Streptomyces lincolnensis, and selected eight TCS gene(s) to construct their deletion mutants utilizing CRISPR/Cas9 system. Among them, lincomycin yield increased in two strains (Δ3900-3901 and Δ5290-5291) while decreased in other four strains (Δ3415-3416, Δ4153-4154, Δ4985, and Δ7949)...

Actinomycetes are a distinct group of filamentous bacteria. The Streptomyces genus within this group has been extensively studied over the years, with substancial contributions to society and science. This genus is known for its antimicrobial production, as well as antitumor, biopesticide, and immunomodulatory properties. Therefore, the extraordinary plasticity of the Streptomyces genus has inspired new research techniques. The newest way of exploring Streptomyces has comprised the discovery of new natural metabolites and the application of emerging tools such as CRISPR technology in drug discovery...

Globomycin is a cyclic lipodepsipeptide originally isolated from several Streptomyces species which displays strong and selective antibacterial activity against Gram-negative pathogens. Its mode of action is based on the competitive inhibition of the lipoprotein signal peptidase II (LspA), which is absent in eukaryotes and considered an attractive target for the development of new antibiotics. Despite its interesting biological properties, the gene cluster encoding its biosynthesis has not yet been identified...

The CRISPR/Cas9 system provides an efficient tool for engineering genomes. However, its application to Streptomyces genome engineering has been hampered by excessive toxicity associated with overexpression of Cas9 protein. As the level of Cas9 toxicity varies significantly between Streptomyces species, species-specific optimization of Cas9 expression is a strategy to mitigate its toxicity while maintaining sufficient double-strand break (DSB) activity for genome engineering. Using a pool of randomized constitutive promoters and a blue pigment indigoidine biosynthetic gene ( IndC ) as a reporter, we developed the CaExTun (<u>Ca</u>s9 <u>Ex</u>pression <u>Tun</u>ing) platform, which enables rapid screening of a large pool of promoter - Cas9 constructs to quickly recover the one with high DSB activity and no apparent toxicity...