Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

Ginsenosides are a group of glycosylated triterpenes isolated from Panax species. Ginsenosides are promising candidates for the prevention and treatment of cancer as well as food additives. However, owing to a lack of efficient approaches for ginsenoside production from plants and chemical synthesis, ginsenosides may not yet have reached their full potential as medicinal resources. In recent years, an alternative approach for ginsenoside production has been developed using the model yeast Saccharomyces cerevisiae and non-conventional yeasts such as Yarrowia lipolytica and Pichia pastoris. In this review, various metabolic engineering strategies, including heterologous gene expression, balancing, and increasing metabolic flux, and enzyme engineering, have been described as recent advanced engineering techniques for improving ginsenoside production. Furthermore, the usefulness of a systems approach and fermentation strategy has been presented. Finally, the present challenges and future research direction for industrial cell factories have been discussed.

The taxonomic assignment ofBrevibacterium frigoritoleranstogether with the in-house environmental isolate EB93 was reassessed in this study using phylogenetic and phylogenomic approaches, and the detection of multiple molecular synapomorphies. Results from the reconstructed phylogenetic trees based on the 16S rRNA gene sequences, the concatenated protein sequences ofGyrA-GyrB-RpoB-RpoC, and the whole-genome sequences revealed the consistent exclusion ofB. frigoritoleransand the environmental isolate EB93 from the cluster formed by the type strains of the genusBrevibacterium. In addition,B. frigoritoleransand the environmental isolate EB93 were both observed to form a clade together with the type strains of the genusPeribacillus. The results from the analysis of the digital DNA–DNA hybridization, average nucleotide identity, average amino acid identity and the difference in the G+C content also corroborated with the phylogenetic inference, and thatB. frigoritoleransand the environmental isolate EB93 were of the same species. Furthermore, the presence of the molecular synapomorphies in the protein sequences noted in the description of the genusPeribacilluswere also observed inB. frigoritolerans, further strengthening its taxonomic affiliation in the genus. Based on the evidence from the multiple lines of analyses, we propose the reclassification ofBrevibacterium frigoritoleransas a member of the genusPeribacillusand assume the namePeribacillus frigoritoleranscomb. nov. (type strain DSM 8801^T=ATCC 25097^T=CCUG 43489^T=CIP 67.20^T=JCM 11681^T).

Fungal endophytes are ubiquitous in nature. They are known as potential sources of natural products, and possible agents for biocontrol attributing to their ability to produce a repertoire of bioactive compounds. In this study, we isolated fungal endophytes from three different tissues (needle, stem and root) of four Pinus species (Pinus densiflora, Pinus koraiensis, Pnus rigida, and Pinus thunbergii) across 18 sampling sites in Korea. A total number of 5872 culturable fungal endophytes were isolated using standard culturing techniques. Molecular identification based on the sequence analyses of the internal transcribed spacer (ITS) or 28S ribosomal DNA revealed a total of 234 different fungal species. The isolated fungal endophytes belonged to Ascomycota (91.06%), Basidiomycota (5.95%) and Mucoromycota (2.97%), with 144 operational taxonomic units (OTUs) and 88 different genera. In all sampling sites, the highest species richness (S) was observed in site 1T (51 OTUs) while the lowest was observed in site 4T (27 OTUs). In terms of diversity, as measured by Shannon diversity index (H’), the sampling site 2D (H′ = 3.216) showed the highest while the lowest H’ was observed in site 2K (H’ = 2.232). Species richness (S) in three different tissues revealed that root and needle tissues are highly colonized with fungal endophytes compared to stem tissue. No significant difference was observed in the diversity of endophytes in three different tissues. Among the four Pinus species, P. thunbergii exhibited the highest species richness and diversity of fungal endophytes. Our findings also revealed that the environmental factors have no significant impact in shaping the composition of the fungal endophytes. Furthermore, FUNGuild analysis revealed three major classifications of fungal endophytes based on trophic modes namely saprotrophs, symbiotrophs, and pathotrophs in four Pinus species, with high proportions of saprotrophs and pathothrops.

Pine wilt disease (PWD) is a destructive vector-borne forest disease caused by the nematodeBursaphelenchus xylophilus. To date, several options are available for the management of pine wilt disease; however constant development and search for natural products with potential nematicidal activity are imperative to diversify management options and to cope with the possible future emergence of resistance in parasitic nematodes. Here, a combined metabolomics and genomics approach was employed to investigate the chemical repertoire and biosynthetic potential of the bacterial endophytePeribacillus frigoritoleransBE93, previously characterized to exhibit nematicidal activity againstB. xylophilus.Feature-based molecular networking revealed the presence of diverse secondary metabolites. A cyclic imine heptapeptide, koranimine, was found to be among the most abundant secondary metabolites produced. Genome mining displayed the presence of several putative biosynthetic gene clusters (BGCs), including a dedicated non-ribosomal peptide synthase (NRPS) BGC for koranimine. Given the non-ribosomal peptide nature of koranimine, in silico molecular docking analysis was conducted to investigate its potential nematicidal activity against the target receptor ivermectin-sensitive invertebrate α glutamate-gated chloride channel (GluCl). Results revealed the binding of koranimine at the allosteric site of the channel—the ivermectin binding site. Moreover, the ligand-receptor interactions observed were mostly shared between koranimine and ivermectin when bound to the α GluCl receptor thus, suggesting a possibly shared mechanism of potential nematicidal activity. This study highlights the efficiency of combined metabolomics and genomics approach in the identification of candidate compounds.

Dps family proteins have the ferroxidase activity that contributes to oxidative stress resistance. In addition, a part of Dps family proteins including Escherichia coli Dps and Staphylococcus aureus MrgA (metallo regulon gene A) bind DNA and induce the structural change of the nucleoid. We previously showed that a mutated MrgA with reduced ferroxidase activity was unable to contribute to the hydrogen peroxide (H2O2) and UV resistance in S. aureus, suggesting that the nucleoid clumping by MrgA is not sufficient for the resistance. However, it remained elusive whether the nucleoid clumping is dispensable for the resistance. Here, we aimed to clarify this question by employing the E. coli Dps lacking DNA-binding activity, DpsΔ18. Staphylococcal nucleoid was clumped by E. coli Dps, but not by DpsΔ18. H2O2 stress assay indicated that Dps and DpsΔ18 restored the reduced susceptibility of S. aureus ΔmrgA. Thus, we concluded that the staphylococcal nucleoid clumping is dispensable for the Dps-mediated H2O2 resistance. In contrast, Dps was unable to complement S. aureus ΔmrgA in the UV resistance, suggesting the MrgA function that cannot be compensated for by E. coli Dps.