The highly fibrinolytic enzyme-producing bacterium was identified as Bacillus subtilis DC27 and isolated from Douchi, a traditional fermented soybean food. The DFE27 enzyme was purified from the fermentation broth of B. subtilis DC27 by using UNOsphere Q column chromatography, Sephadex G-75 gel filtration, and high-performance liquid chromatography. It was 29 kDa in molecular mass and showed the optimal reaction temperature and pH value of 45 °C and 7.0, respectively, with a stable fibrinolytic activity below 50 °C and within the pH range of 6.0 to 10.0. DFE27 was identified as a serine protease due to its complete inhibition by phenylmethysulfony fluoride. The first 24 amino acid residues of the N-terminal sequence of the enzyme were AQSVPYGVSQIKAPALHSQGFTGS. The enzyme displayed the highest specificity toward the substrate D-Val-Leu-Lys-pNA for plasmin and it could not only directly degrade but also hydrolyze fibrin by activating plasminogen into plasmin. Overall, the DFE27 enzyme was obviously different from other known fibrinolytic enzymes in the optimum substrate specificity or fibrinolytic action mode, suggesting that it is a novel fibrinolytic enzyme and may have potential applications in the treatment and prevention of thrombosis.

Clubroot caused by Plasmodiophora brassicae is one of the most destructive diseases in cruciferous crops. Streptomyces alfalfae XY25^T, a biological control agent, exhibited great ability to relieve clubroot disease, regulate rhizosphere bacterial and fungal communities in Chinese cabbage, and promote its growth in greenhouse. Therefore, field experiments were carried out to investigate the effects of S. alfalfae XY25^T on clubroot and rhizosphere microbial community in Chinese cabbage. Results showed that the control efficiency of clubroot by S. alfalfae XY25^T was 69.4%. Applying the agent can alleviate soil acidification; increase the contents of soil organic matter, available nitrogen, available phosphorus, and available potassium; and enhance activities of invertase, urease, catalase, and alkaline phosphatase. During Chinese cabbage growth, bacterial diversity decreased first and then increased, and fungal diversity decreased gradually after inoculation with S. alfalfae XY25^T. High-throughput sequencing analysis showed that the main bacterial phyla were Proteobacteria, Bacteroidetes, Acidobacteria, and Planctomycetes, and the major fungal phyla were Ascomycota and Basidiomycota in rhizosphere soil. The dominant bacterial genera were Flavobacterium, Candidatus, Pseudomonas, Stenotrophomonas, Sphingomonas, Flavisolibacter, and Gemmatimonbacteria with no significant difference in abundance, and the major fungal genera were Monographella, Aspergillus, Hypocreales, Chytridiaceae, Fusarium, Pleosporales, Agaricales, Mortierella, and Pleosporales. The significant differences were observed among Pleosporales, Basidiomycota, Colletotrichum, two strains attributed to Agaricales, and another two unidentified fungi by using S. alfalfae XY25^T. Moreover, quantitative real-time PCR results indicated that P. brassicae content was significantly decreased after the agent inoculation. In conclusion, S. alfalfae XY25^T can affect rhizosphere microbial communities; therefore, applying the agent is an effective approach to reduce the damage caused by clubroot.

Background: Bacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability. Results: To address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNA_rep transcription was tightly controlled by rigorous promoter P_acoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5 days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL. Conclusions: We developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.

As a global cornerstone of animal protein, farmed shrimp production in China has been revolutionized by the Greenhouse Shrimp Farming Model (GSFM). However