Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V-F CRISPR/Cas Nuclease AsCas12f1.

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. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.

Development of a CRISPR/Cas9D10A Nickase (nCas9)-Mediated Genome Editing Tool 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. We showed that in the presence of both targeting sgRNA and Cas proteins, utilization of nCas9 instead of Cas9 significantly reduced the toxicity to the host and greatly enhanced cell survival. Using this tool, we achieved deletion of single genes and gene clusters with efficiencies of 87-100 and 63-87%, and simultaneous deletion of two genes or gene clusters with efficiencies of 47 and 43%, respectively. The editing efficiency of nCas9 is comparable to that of the Cas9-mediated editing tool. Finally, the nCas9-based editing tool was successfully applied for genome editing in the industrial rapamycin-producing strain Streptomyces rapamycinicus, in which CRISPR/Cas9 cannot work well. We achieved the deletion of three tested genes with an efficiency of 27.2-30%. Collectively, the CRISPR/nCas9-based editing tool offers a convenient and efficient genetic modification system for the engineering of streptomycetes, particularly those with low DNA transformation efficiency.

Cellular differentiation into hyphae and spores in halophilic archaea.

Several groups of bacteria have complex life cycles involving cellular differentiation and multicellular structures. For example, actinobacteria of the genus Streptomyces form multicellular vegetative hyphae, aerial hyphae, and spores. However, similar life cycles have not yet been described for archaea. Here, we show that several haloarchaea of the family Halobacteriaceae display a life cycle resembling that of Streptomyces bacteria. Strain YIM 93972 (isolated from a salt marsh) undergoes cellular differentiation into mycelia and spores. Other closely related strains are also able to form mycelia, and comparative genomic analyses point to gene signatures (apparent gain or loss of certain genes) that are shared by members of this clade within the Halobacteriaceae. Genomic, transcriptomic and proteomic analyses of non-differentiating mutants suggest that a Cdc48-family ATPase might be involved in cellular differentiation in strain YIM 93972. Additionally, a gene encoding a putative oligopeptide transporter from YIM 93972 can restore the ability to form hyphae in a Streptomyces coelicolor mutant that carries a deletion in a homologous gene cluster (bldKA-bldKE), suggesting functional equivalence. We propose strain YIM 93972 as representative of a new species in a new genus within the family Halobacteriaceae, for which the name Actinoarchaeum halophilum gen. nov., sp. nov. is herewith proposed. Our demonstration of a complex life cycle in a group of haloarchaea adds a new dimension to our understanding of the biological diversity and environmental adaptation of archaea.

Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces.

GlnR, an OmpR-like orphan two-component system response regulator, is a master regulator of nitrogen metabolism in the genus Streptomyces In this work, evidence that GlnR is also directly involved in the regulation of antibiotic biosynthesis is provided. In the model strain Streptomyces coelicolor M145, an in-frame deletion of glnR resulted in markedly increased actinorhodin (ACT) production but reduced undecylprodigiosin (RED) biosynthesis when exposed to R2YE culture medium. Transcriptional analysis coupled with DNA binding studies revealed that GlnR represses ACT but activates RED production directly via the pathway-specific activator genes actII-ORF4 and redZ, respectively. The precise GlnR-binding sites upstream of these two target genes were defined. In addition, the direct involvement of GlnR in antibiotic biosynthesis was further identified in Streptomyces avermitilis, which produces the important anthelmintic agent avermectin. We found that S. avermitilis GlnR (GlnRsav) could stimulate avermectin but repress oligomycin production directly through the respective pathway-specific activator genes, aveR and olmRI/RII To the best of our knowledge, this report describes the first experimental evidence demonstrating that GlnR regulates antibiotic biosynthesis directly through pathway-specific regulators in Streptomyces Our results suggest that GlnR-mediated regulation of antibiotic biosynthesis is likely to be universal in streptomycetes. These findings also indicate that GlnR is not only a master nitrogen regulator but also an important controller of secondary metabolism, which may help to balance nitrogen metabolism and antibiotic biosynthesis in streptomycetes.

Three of four GlnR binding sites are essential for GlnR-mediated activation of transcription of the Amycolatopsis mediterranei nas operon.

In Amycolatopsis mediterranei U32, genes responsible for nitrate assimilation formed one operon, nasACKBDEF, whose transcription is induced by the addition of nitrate. Here, we characterized GlnR as a direct transcriptional activator for the nas operon. The GlnR-protected DNA sequences in the promoter region of the nas operon were characterized by DNase I footprinting assay, the previously deduced Streptomyces coelicolor double 22-bp GlnR binding consensus sequences comprising a1, b1, a2, and b2 sites were identified, and the sites were then mutated individually to test their roles in both the binding of GlnR in vitro and the GlnR-mediated transcriptional activation in vivo. The results clearly showed that only three GlnR binding sites (a1, b1, and b2 sites) were required by GlnR for its specific binding to the nas promoter region and efficient activation of the transcription of the nas operon in U32, while the a2 site seemed unnecessary.

[Comparison of four domestic quantity assay reagents for detecting the weak-reactive results of HBsAg].

To evalue the coincidence and correlation between the four domestic quantity assay reagents and with ARCHITECTi2000 immunoassay system. 185 weak-reactive serum samples and standard materials of different concentrations were tested by four domestic quantity assay reagents for HBsAg test and ARCHITECTi2000 immunoassay system. The coincidence, the precision and the correlations between different systems were analyzed. The coincidence rates of the results of 0.05-1.00 IU/ml samples between the four domestic quantity assay reagents and ARCHITECTi2000 immunoassay system were 25.93%, 35.19%, 51.85% and 18.52% respectively, and for those results of more than 1.00 to 10.00 IU/ml samples the coincidence rates were 71.76%, 87.79%, 95.42% and 69.47% respectively. The samples of 0.05 to 0.80 IU/ml weak-reactive serum samples detected by the i2000 system were all negative detected by the four domestic systems. The coincidence rates of more than 7.93 IU/ml serum samples detected by i2000 system were 100% detected by the four domestic systems. The correlations of the four domestic quantity assays were around 0.8629 to 0.9265. The analysis sensitivity of the four domestic quantity assay reagents were below the i2000 system. The results of under 0.80 IU/ml samples detected by i2000 system were disaccord with the results detected by the four domestic systems, whereas for the sapmples over 7.93 IU/ml the results were consistent.