Aihua Mao

The CRISPR/Cas9 system is a cornerstone technology in genome editing. Delivery of pre-assembled Cas9 ribonucleoprotein (RNP) complexes exhibits distinct advantages, including reduced off-target effects and lower immunogenicity. Conventional methods for purifying Cas9 protein typically involve multi-step chromatography and the cleavage of fusion tag, which are time-consuming and result in diminished yields. In this study, we present a simplified, one-step purification strategy for functional Streptococcus pyogenes Cas9 (SpCas9) using the ubiquitin (Ub) fusion system in Escherichia coli. The N-terminal Ub fusion not only improves protein solubility but also facilitates high-yield production of the His-Ub-Cas9 fusion protein. Importantly, the Ub tag does not require proteolytic removal during purification, allowing direct one-step purification of the fusion protein via nickel-affinity chromatography. The purified His-Ub-Cas9 retains robust DNA cleavage activity in vivo, as validated in zebrafish embryos. This protocol greatly simplifies the production of functional Cas9 protein, facilitating its broad application in genome editing.

Four citrus pectins were extracted from different discarded citrus peels. PPG, CJ, and QC pectins presented degrees of esterification (DE) values that exceeded 80%; in contrast, WG pectin presented a notably lower DE, which was reduced to approximately 65%. All four pectins are composed of seven monosaccharides: galacturonic acid, arabinose, galactose, glucose, rhamnose, mannose, and xylose, with galacturonic acid emerging as the predominant constituent (more than 50%). The molecular weights (Mws) of the four pectins all exceeded 100 kDa. Two Aureobasidium strains (40 and 44), which were isolated from CWs, harboured genomes enriched with pectin-depolymerizing carbohydrate-active enzymes (CAZymes). Following temperature and pH optimization of the pectin-degrading enzyme systems in the two black yeast strains, both effectively depolymerized the four pectins into lower-Mw oligosaccharides. All four pectin oligosaccharide products exhibited significant radical scavenging capacities against DPPH, superoxide anion and hydroxyl radicals, demonstrating their effectiveness as natural free radical scavengers.

The genera Polaribacter and Algibacter, which are members of the Bacteroidota family Flavobacteriaceae, are well known for their arsenal of carbohydrate-active enzymes (CAZymes) and specialized polysaccharide utilization loci (PULs). In this study, two aerobic, gram-negative, and rod-shaped bacterial strains, designated strains DS7-9 ^T and PT7-4 ^T, were isolated from the surface of seaweed collected from the coastal zone of Fujian Province, China. The two strains exhibited broad tolerance ranges to salinity (0–4%), temperature (10–40 °C) and pH (5.0–10.0). The predominant fatty acids in strain DS7-9 ^T were iso-C_15:0 (53.7%) and iso-C_15:0 3-OH (10.9%), whereas those in strain PT7-4 ^T were iso-G-C_15:1 (33.2%) and iso-C_15:0 (27.1%). Additionally, the predominant menaquinone was MK-6. The 16S rRNA gene of strain DS7-9 ^T shares 97.5% similarity with that of Polaribacter haliotis KCTC 52418 ^T, whereas that of strain PT7-4 ^T is 97.0% sequence similarity with that of Algibacter mikhailovii KCTC 12710 ^T. Phylogenomic analysis revealed that the highest average nucleotide identity (ANI) value between strain DS7-9 ^T and its closest relative reached 84.7%, whereas the corresponding value for strain PT7-4 ^T was 84.2%. Genomic characterization revealed DNA G + C contents of 29.5% and 31.9% for strains DS7-9 ^T and PT7-4 ^T, respectively. Both strains possessed complete circular chromosomes, with genome sizes of 3.7 Mb (DS7-9 ^T) and 3.5 Mb (PT7-4 ^T). Bioinformatic analysis revealed that strain DS7-9 ^T harbors 5 PULs putatively associated with laminarin, ulvan, fucoidan, and alginate catabolism, whereas strain PT7-4 ^T possesses 7 PULs predicted to target ulvan, alginate, and starch metabolism. On the basis of their distinct physiological characteristics, two strains are suggested for classification into novel species: Polaribacter sargassicola sp. nov. (type strain DS7-9 ^T = MCCC 1K08670^T = KCTC 102076 ^T) and Algibacter ulvanivorans sp. nov. (type strain PT7-4 ^T = MCCC 1K08669^T = KCTC 102077 ^T).

In recent years, there has been extensive documentation of pathogenic fungi infecting Morchella sextelata. However, investigations of microorganisms with antagonistic properties against these pathogens are limited. This study successfully isolated two isolates of the genus Streptomyces (F16 and F19) from the rhizosphere soil of M. sextelata fruiting bodies, both of which exhibit potent antagonistic activity against Pestalotiopsis trachicarpicola, the causative agent of M. sextelata wilt disease. Comprehensive characterization, including physiological–biochemical tests and 16S rDNA sequence analysis, led to the identification of these isolates as Streptomyces sp. F16 and Streptomyces sp. F19. Both isolates significantly inhibited P. trachicarpicola through multiple mechanisms. The volatile compounds produced by these isolates effectively suppressed the conidial germination of P. trachicarpicola in vitro. Furthermore, fermentation filtrates at various dilutions exhibited pronounced antifungal activity against conidial germination, with Streptomyces sp. F16 showing 66.93% inhibition at a 50× dilution and Streptomyces sp. F19 achieving 49.22% inhibition under identical conditions. Field experiments have demonstrated the practical applicability of these antagonists. The topical application of fermentation filtrates (diluted 50×) from both isolates significantly reduced the incidence and severity of disease in M. sextelata cultivation. Notably, the yield improvements were substantial: fields treated with Streptomyces sp. F16 produced 299.6 g/m², whereas those treated with Streptomyces sp. F19 yielded 277.65 g/m². These yields significantly surpassed those of both the untreated control group (231 g/m²) and the P. trachicarpicola-inoculated group (134.93 g/m²). These findings indicate that the two isolates not only effectively control P. trachicarpicola but also increase the yield of M. sextelata.

Steroid hormones exhibit potent endocrine disrupting activity and have been shown to disrupt the equilibrium of aquatic ecosystems and pose a threat to public health through their persistent and carcinogenic effects. Pontibacillus chungwhensis HN14, a moderately halophilic bacterium with the capacity to effectively degrade various polycyclic aromatic hydrocarbons and other organic pollutants, was previously isolated. Additionally, the strain HN14 showed strong environmental adaptability under various environmental stress conditions. In this study, the steroid degradation by strain HN14 was studied for the first time. We demonstrated that strain HN14 could degrade estradiol (E2) to maintain the growth of the strain and could convert E2 to estrone. Additionally, the efficient substrate degradation efficiency of P. chungwhensis HN14 under high salinity and high substrate concentration conditions was demonstrated. Furthermore, a 17β-hydroxysteroid dehydrogenase, 17β-HSD(HN14), was identified in strain HN14. Comparative analysis reveals that 17β-HSD(HN14) shares approximately 38% sequence identity with 17β-HSDx from Rhodococcus sp. P14. In addition, 100 µg of purified 17β-HSD(HN14) could effectively convert about 40% of 0.25 mM of E2 within 1 h period, with an enzyme activity of 17.5 U/mg, and catalyze the dehydrogenation of E2 and testosterone at the C-17 position. The characterization of purified enzyme properties reveals that 17β-HSD(HN14) exhibits exceptional structural robustness and enzymatic efficacy even under high salinity conditions of up to 20%. Overall, this study enhances our comprehension of steroid biodegradation in strain HN14 and contributes novel ideas and theoretical underpinnings for advancing bioremediation technologies targeting steroid pollution in high-saline environments.