Jianmei Che

In the post-antibiotic era, the Bacillus-based direct-fed beneficial microorganisms are emerging as a cornerstone for sustainable animal farming. This study aimed to screen and evaluate Bacillus strains with probiotic potential for use as feed additives. A total of 394 Bacillus strains were initially screened based on their extracellular enzyme production (cellulase, protease, and amylase) and antibacterial activities against Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Two strains, Bacillus velezensis FJAT-10508 and FJAT-13563, were selected and subsequently subjected to in vitro probiotic characterization, safety assessment, and whole-genome analysis. The results demonstrated that both strains exhibited α-hemolysis, acceptable antibiotic susceptibility profiles, absence of invasion and cytotoxicity effect on the Caco-2 cells, and no mobile virulence or antibiotic resistance genes, indicating their safety as probiotic candidates. High endospore-forming efficiencies (72.4–90.8%), strong auto-aggregation (74–85%) and co-aggregation abilities (52–82%) were observed. In addition, both strains showed considerable tolerance to simulated gastrointestinal conditions, with vegetative cell and endospore survival rates of 28.33–38.33% and 85–89.67% at pH 2.0, and 38.33–43.33% and 90.33–96.33% in 0.3% bile salts, respectively. Overall, B. velezensis FJAT-10508 and FJAT-13563 demonstrated robust in vitro probiotic properties, supporting their potential application as reliable Bacillus-based feed additives.

Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions, plays various roles in health and medical therapies. However, it is only found in a limited number of plant species in low concentrations, which hinders its development and utilization. Chalcone synthase (CHS) and stilbene synthase (STS) catalyze the same substrates to produce flavonoids and resveratrol, respectively. However, it remains unclear how CHS and STS compete in metabolite synthesis. In this study, two CHS2 mutant cell lines (MT1 and MT2) were generated using CRISPR/Cas9 genome editing. These CHS2 mutant cell lines exhibited abundant mutations in CHS2, leading to the premature termination of protein translation and subsequent CHS2 knockout. Amplicon sequencing confirmed comprehensive CHS2 knockout in MT1, whereas the wild-type sequence remained predominant in the MT2 cell line. Transcriptome and RT-qPCR results showed a significant downregulation of genes involved in flavonoid biosynthesis, including CHS2, CHS3, F3H, F3’H, DFR, FLS, LDOX, among others, resulting in decreased flavonoid accumulation, such as anthocyanins, proanthocyanidins, quercetin, and kaempferol. Conversely, STS genes involved in stilbenoid biosynthesis were upregulated competing with the flavonoid pathway. Consequently, there was a marked increase in stilbenoids, including resveratrol, piceatannol, piceid and pterostilbene, with a 4.1-fold increase in resveratrol and a 5.3-fold increase in piceid (a derivative of resveratrol) observed in CHS2 mutant cell lines. This research demonstrates that CHS2 mutation induces a shift from flavonoid biosynthesis towards stilbenoid biosynthesis, offering new insights into metabolite biosynthesis and regulation, as well as an alternative solution for natural resveratrol production, and a novel breeding approach for eliminating non-target agronomic traits using CRISPR-Cas9.

Brevibacillus brevis FJAT-0809-GLX has a broad spectrum of antimicrobial activity. Understanding the molecular basis of biocontrol ability of B. brevis will allow us to develop effective microbial agents for sustainable agriculture. In this study, we present the complete and annotated genome sequence of FJAT-0809-GLX. The complete genome size of B. brevis FJAT-0809-GLX was 6,137,019 bp, with 5688 predicted coding sequences (CDS). The average GC content of 47.38%, and there were 44 copies of the rRNAs operon (16S, 23S and 5S RNA), and 127 tRNA genes. A total of 11,162 genes were functionally annotated with the COG, GO, and KEGG databases, and 123 genes belonged to CAZymes. Genomic secondary metabolite analysis indicated 13 clusters encoding potential new antimicrobials. FJAT-0809-GLX was designated as B. brevis according to average nucleotide polymorphism (ANI) and phylogenetic analysis. The pangenome consisted of 7141 homologous genes, and 4469 homologous genes shared by B. brevis FJAT-0809-GLX, B. brevis NBRC100599, B. brevis DSM30, and B. brevis NCTC2611. The number of unique homologous genes of B. brevis FJAT-0809-GLX (419 genes) and B. brevis NBRC100599 (480 genes) were much more than those in B. brevis DSM30 (13 genes), and B. brevis NCTC2611 (6 genes). Nine gene clusters encoding for secondary metabolite biosynthesis were compared in the genome of B. brevis FJAT-0809-GLX with those of B. brevis NBRC100599, B. brevis DSM30 and B. brevis NCTC2611, and the gene clusters encoding for lantipeptide and transatpks-otherks only existed in genome of B. brevis FJAT-0809-GLX. The 11 BbPks genes were included in the B. brevis FJAT-0809-GLX genome, which contained the conserved PS-DH domain. The relative expression of BbPksL, BbPksM2, BbPksM3, BbPksN3, BbPksN4 and BbPksN5 reached a maximum at 120 h and then decreased at 144 h. Our results provided detailed genomic and Pks genes information for the FJAT-0809-GLX strain, and lid a foundation for studying its biocontrol mechanisms.

Gray mold disease, caused by Botrytis cinerea, has reduced grape’s output and market quality globally. In this study, the antifungal activity of a mixed microbial combination of Brevibacillus brevis FJAT-0809-GLX and Bacillus sp. strains was investigated. The results showed that the inhibition rate against B. cinerea was 85.10% when B. brevis FJAT-0809-GLX was mixed with a mixture of B. brevis FJAT-10623 and Bacillus velezensis FJAT-55034 at a proportion of 80%:20%, which was significantly higher than those of other combination proportions. The inhibitory rates of the mixed microbial combination diluted 0 times, 10 times, and 100 times were 89.14%, 88.10%, and 86.33%, respectively, with no significant differences between each other. The mixed microbial combination appeared to be temperature-insensitive and significantly stable from pH 3 to pH 7. Furthermore, it was discovered that its antifungal activity was significantly stable with UV radiation for 30 min, 60 min, and 90 min, with values of 84.82%, 83.89%, and 82.22%, respectively. An amount of 0.025 mol mL⁻¹ of KCl, CuSO₄, and MgCl₂ had no effects on the antifungal activity of the mixed microbial combination, while 0.025 mol mL⁻¹ of NaCl, ZnSO₄, FeSO₄, CaCl₂, and MnSO₄ reduced the inhibition rate. The mixed microbial combination demonstrated antifungal activities against a variety of fungi, with inhibition rates ranging from 68.78% to 85.10%. The grape fruits and grape leaves treated with the mixed microbial combination decayed at 27.27% and 48.34%, respectively. Additionally, the mixed microbial combination improved grape fruit resistance by increasing the activity of defense enzymes polyphenol oxidase (PPO) and catalase (CAT). Therefore, the results indicated that the mixed microbial combination had great biocontrol potential against gray mold in grape fruits.