Pseudorabies virus (PRV) is a member of the genus Varicellovirus in the Herpesviridae family. It primarily causes pseudorabies characterized by reproductive failure in sows, and neurological and respiratory symptoms in piglets, posing a significant threat to pig production. Vaccination is the most important measure to prevent PRV in pigs. However, due to variations of the virus and its latent infection characteristics, the effectiveness of traditional vaccines is compromised. Consequently, there is an urgent need for new drug preparations to assist vaccine immunization. It has been found that natural plant polysaccharides and small molecules such as flavonoids, phenols, and acids can inhibit PRV infection either by directly blocking the viral infection process or by regulating the immune response. In addition, host antiviral protein type Ⅰ interferon and its downstream interferon-stimulated genes have significant inhibitory effects on PRV infection. Host defense peptides, including antimicrobial peptides and defensins, also show good inhibitory effects on PRV infection. Interestingly, researchers have recently found that extracts and metabolites from bacteria and fungi also exhibit anti-PRV effects, and it is expected that these bacteria and fungi and their products could be applied for the prevention and treatment of viral diseases in the future. This study focused on the recent research progress of natural bioactive molecules against PRV infection, aiming to provide important references for the research and development of anti-PRV infection drugs.

In recent years, antimicrobial peptides (AMPs) are considered alternatives to antibiotics and have received increasing attention. AMPs have a broad antibacterial spectrum and extensive sources and are not prone to drug resistance. At present, AMPs are mainly produced with three methods: extraction from natural sources, chemical synthesis, and microbial expression via genetic engineering. The application of the former two methods is limited due to their complicated processes, high costs, and low yields. Microbial expression via genetic engineering is more economical, scientific, and effective than the above two methods. This article introduces and compares the various expression systems and summarizes the strategies for increasing the heterologous expression levels, with a view to providing theoretical support for large-scale production of AMPs with low costs.

[Objective] To mine the key enzyme genes associated with spinosad synthesis and the biosynthetic gene clusters (BGCs) in Saccharopolyspora spinosa at different developmental stages by transcriptomics, thus laying the groundwork for the construction of high-yield strains. [Methods] The transcriptomes of S. spinosa during the logarithmic phase (T2-48 h) and the stationary phase (T6-144 h) were compared. The results from qRT-PCR and transcriptome sequencing were mutually validated. Gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) enrichment were performed for the differentially expressed genes (DEGs). Central carbon metabolism analysis was performed. [Results] The transcriptome sequencing of S. spinosa revealed 2 542 DEGs, including 1 188 genes with significantly up-regulated expression and 1 354 genes with significantly down-regulated expression. GO annotation indicated that the DEGs were primarily involved in carboxylic acid metabolic process, oxoacid metabolic process, organic acid metabolic process, and amino acid metabolic process. KEGG enrichment analysis demonstrated DEGs were mainly involved in pathways such as glycine, serine, and threonine metabolism, oxidative phosphorylation, and arginine biosynthesis. Further analysis identified seven genes related to spinosad biosynthesis. Among them, accB, Pfk, G6PD, and dsdA showed significantly up-regulated expression, while GAPDH, aceE, DLAT involved in the consumption of spinosad precursors, as well as genes in the TCA cycle and arginine biosynthesis, exhibited significantly down-regulated expression. The results of qRT-PCR were consistent with the trends observed in transcriptome sequencing, which revealed 12 upregulated BGCs: BGC2 (43 846 bp), BGC4 (18 330 bp), BGC9 (20 501 bp), BGC18 (62 621 bp), BGC22 (19 626 bp), BGC25 (42 896 bp), BGC26 (40 086 bp), BGC28 (39 392 bp), BGC30 (20 282 bp), BGC31 (53 657 bp), BGC34 (20 787 bp), and BGC35 (40 232 bp). [Conclusion] This study elucidated DEGs in S. spinosa at different developmental stages through transcriptome analysis, and analyzed the biosynthetic pathways and BGCs of spinosad. These findings pave the way for optimizing the spinosad biosynthetic pathways and genetically modifying S. spinosa to enhance the spinosad production in subsequent studies.

[Objective] Human norovirus (HuNoV) is one of the most common pathogens causing acute gastroenteritis in humans worldwide. Currently, there are no approved vaccines to prevent this disease. This study aimed to prepare virus-like particles (VLPs) of HuNoV in the flashBAC baculovirus expression system, laying a foundation for the development of vaccines against HuNoV. [Methods] After codon optimization, the full-length gene sequence of the VP1 protein of HuNoV GⅡ.4 was synthesized and cloned into the baculovirus pBacPAK9 transfer vector to obtain the recombinant plasmid pBacPAK9-8his-VP1. After enzyme digestion and sequencing, the recombinant plasmid was co-transfected with the linear baculovirus plasmid (Bacmid) into SF9 cells to obtain a recombinant baculovirus carrying the VP1 gene. Hi-Five (HF) cells were infected by the recombinant baculovirus for protein expression, and the expression of VP1 was analyzed by SDS-PAGE and Western blotting. VP1 was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blotting. The purity of VP1 was examined by high performance liquid chromatography (HPLC). The VLPs were observed by transmission electron microscopy. [Results] A transfer plasmid pBacPAK9-8his-VP1 was constructed, and the VP1 protein, with a molecular weight of approximately 58 kDa, was mainly expressed in the cytoplasm of HF cells. The HPLC results showed that the purity of VP1 was over 99%. The VLPs with a regular shape, uniform sizes, and diameters of 30–40 nm were observed by transmission electron microscopy. [Conclusion] The VLPs of HuNoV GⅡ.4 were prepared with a baculovirus expression system, laying a foundation for the development of HuNoV vaccines.

[Objective] Monensin is a polyether antibiotic produced by Streptomyces cinnamonensis. To enhance the production of monensin by microbial fermentation, we employed metabolic engineering to strengthen the synthesis pathway of the key precursor methylmalonyl-CoA in S. cinnamonensis 2110. [Methods] Firstly, crotonyl-CoA reductase (CCR) was overexpressed to strengthen the acetoacetyl-CoA pathway. Subsequently, methylmalonyl-CoA mutase (MCM) was overexpressed to improve the succinyl-CoA pathway. Finally, an engineered strain with tandem overexpression of CCR and MCM was constructed and evaluated for the fermentation performance. [Results] The overexpression of CCR increased the strain biomass and monensin titer by 10.4% and 19.0%, respectively, after 10 days of shake-flask fermentation. The overexpression of MCM increased the monensin titer by 9.9%, whereas it did not increase the strain biomass after 10 days of shake-flask fermentation. The tandem overexpression of CCR and MCM increased the biomass and monensin titer by 9.4% and 26.8%, respectively, after 10 days of shake-flask fermentation. In a 5 L bioreactor, the engineered strain 2110-CCR-MCM reached the highest biomass of 54.6 g/L and monensin titer of 11.3 kU/mL, which increased by 12.7% and 36.2%, respectively, compared with those of the starting strain 2110. [Conclusion] CCR and MCM mediated the key metabolic pathway of monensin biosynthesis in S. cinnamonensis, and the overexpression of CCR and MCM was highly favorable for monensin synthesis. This study provides technical reference for the engineering of strains with high yields of other polyketides.

[Objective] To clarify the domestication characteristics and resistant mechanism of wild watermelon plants and provide a theoretical basis and technical support for building a new evaluation system for watermelon breeding and developing beneficial microorganisms, we compared the endophytic microbial community structure in roots between wild and cultivated watermelon varieties. [Methods] High-throughput sequencing was employed to reveal the community structures of endophytic microorganisms (bacteria and fungi) in the roots of wild and cultivated watermelon varieties. [Results] The phyla and genera of endophytic bacteria and fungi in the roots were significantly different between wild and cultivated watermelon varieties. The roots of wild watermelon varieties had significantly higher relative abundance of Nocardioides and Microbacterium than those of cultivated watermelon varieties. Actinoplanes, Mycobacterium, Lechevalieria, Amycolatopsis, Bradyrhizobium, and Rhodococcus were the special dominant endophytic bacterial genera in the roots of wild watermelon varieties. The relative abundance of unclassified_o__Chaetothyriale in the roots of wild watermelon varieties was significantly higher than that in cultivated watermelon varieties. unclassified__o_Chaetothyriales, Preussia, and unclassified_f__Microascaceae were the dominant endophytic fungi specific to wild watermelon varieties. [Conclusion] The beneficial bacteria, such as Nocardioides, Microbacterium, and Rhodococcus, which were capable of fixing nitrogen, solubilizing phosphorus, secreting siderophores, and producing bioactive substances and antibiotics, and the growth hormone-producing fungi, such as Preussia, were lost in the roots of cultivated watermelon varieties after the domestication of wild watermelon. Therefore, it can be concluded that parts of endophytic microorganisms in the roots disappear during the domestication process of watermelon varieties, which is a major reason for the weak resistance of cultivated watermelon varieties to stresses. In addition, the bacterial genera such as Nocardioides, Microbacterium, and Rhodococcus and the fungal genus Preussia can be taken as the candidate microbial resources for enhancing watermelon resistance.

[Objective] To systematically analyze the enzymatic properties of transglutaminase (TGase) from Streptomyces mobaraensis CGMCC 4.1851 (strain XM4) and subsequently develop a high-yielding strain by engineering for achieving efficient expression of TGase in Streptomyces with reduced fermentation duration and enhanced production efficiency. [Methods] The pH of the fermentation broth and TGase activity were measured to assess the fermentation characteristics of strain XM4. TGase from XM4 was purified by alcohol precipitation combined with ion-exchange chromatography. The reaction conditions (pH, temperature, metal ions) were optimized for the enzyme, and the enzymatic kinetics were tested. The catalytic efficiency was evaluated by casein cross-linking experiments. Subsequently, genetic engineering was employed to enhance the modified strain through heterologous expression and replacement of the ribosome-binding site (RBS), followed by measurement of TGase production. [Results] TGase from strain XM4 exhibited good activity and stability within the range of pH 4.0–11.0, with the highest activity at 50 ℃ and pH 10.0. The modification realized efficient expression of TGase in S. mobaraensis, inceasing the production by 103.3% compared with the original strain and reducing the fermentation time to 24 h. [Conclusion] TGase from strain XM4 demonstrates excellent acid-base tolerance and thermal stability, demonstrating broad application prospects in the food industry, especially dairy processing. Additionally, the engineered strain enables efficient production of TGase, providing new options for the industrial production and application of TGase.

Porcine epidemic diarrhea virus (PEDV) is an enterovirus that can cause severe diarrhea and dehydration. The widespread epidemic of PEDV has caused huge economic losses to the pig breeding industry, which, however, lacks effective means for prevention and treatment. Nsp8 is an important non-structural protein involved in the replication of PEDV, while the host proteins interacting with Nsp8 remains unclear. [Objective] To screen the host proteins interacting with PEDV Nsp8 and explore the effects of the host proteins on the replication of PEDV, so as to provide a theoretical basis for discovering new key functional receptors or therapeutic targets of PEDV. [Methods] The eukaryotic expression plasmid of PEDV Nsp8 was successfully constructed with the eukaryotic expression vector pcDNA3.1(+). The host proteins interacting with PEDV Nsp8 were screened by co-immunoprecipitation, mass spectrometry, and laser confocal microscopy. The effects of the host proteins on PEDV replication were explored by overexpression and knockdown in LLC-PK cells. [Results] Thirty-six potential host proteins interacting with Nsp8 were screened by mass spectrometry, and the interaction between heat shock protein member 8 (HSPA8) and Nsp8 was verified. The overexpression of HSPA8 in LLC-PK cells inhibited the overexpression of Nsp8 in a dose-dependent manner. Meanwhile, it significantly inhibited the replication of PEDV in a dose-dependent manner at the protein and transcriptional levels. Interfering with endogenous HSPA8 expression significantly promoted the replication of PEDV. The 50% tissue culture infectious dose (TCID₅₀) and indirect immunofluorescence further proved that HSPA8 inhibited PEDV replication. [Conclusion] This study screened out the host protein HSPA8 interacting with PEDV Nsp8 and proved that HSPA8 could significantly inhibit PEDV replication, which provided a new idea for the design of HSPA8-targeted drugs for the prevention or treatment of PEDV.

[Objective] To provide candidate strains and effective strategies for the control of mulberry fruit sclerotiniose, we screened out the endophytic bacteria with biocontrol potential for mulberry fruit sclerotiniose from a resistant mulberry cultivar. [Methods] The endophytic bacteria antagonistic to mulberry fruit sclerotiniose were isolated from mulberry plants by the tissue culture and confrontation culture methods. The antagonistic strain was identified based on morphological features, physiological and biochemical characteristics, and the phylogenetic relationship based on 16S rRNA gene sequences. The antimicrobial spectrum and control efficiency to detached mulberry fruits were determined to evaluate the application potential of the antagonistic strain. Furthermore, we observed the inhibitory effect of the fermentation supernatant of the strain on the mycelial growth of the pathogen, measured the variations in glycogen and reactive oxygen species accumulation of the pathogen treated with the antagonistic strain, and determined the expression of pathogen-related genes after treatment with the antagonistic strain to decipher the antagonistic mechanism of this strain. [Results] An endophytic bacterial strain C1R32 with strong and stable antagonistic activity on Sclerotinia sclerotiorum PZ-2 (the pathogen of mulberry fruit sclerotiniose) was isolated from a healthy mulberry branch. C1R32 showed similar morphological features and physiological and biochemical characteristics with Bacillus. The phylogenetic analysis based on 16S rRNA gene sequences revealed that C1R32 was located in the same clade with B. subtilis. Therefore, strain C1R32 was identified as B. subtilis. B. subtilis C1R32 had antagonistic activities against a variety of phytopathogens including S. sclerotiorum. The suspension and fermentation supernatant of B. subtilis C1R32 showed the control effects of 52.94% and 46.43%, respectively, on sclerotiniose of detached mulberry fruits. The cell-free fermentation supernatant of B. subtilis C1R32 caused the hypha swelling and distorting, cell wall breaking, and cytoplasm leakage of S. sclerotiorum PZ-2. Moreover, B. subtilis C1R32 inhibited S. sclerotiorum PZ-2 by reducing glycogen accumulation, promoting reactive oxygen species burst, and influencing the expression of genes associated with antioxidant activity. [Conclusion] We isolated an endophytic B. subtilis strain capable of controlling mulberry fruit sclerotiniose from a resistant mulberry cultivar and preliminarily explored its antagonistic mechanism, providing potential strain resources for the biocontrol of mulberry fruit sclerotiniose.

Beneficial bacteria in the gut affect human health, and it is generally believed that the assemblage of healthy gut flora is achieved through vertical transmission of by breastfeeding in early infancy. There is limited evidence for the difference in the composition of beneficial bacteria across different mother-infant cohorts and the presence of population-specific microbial taxa. [Objective] To investigate Lactobacillus spp. and the vertical transmission and genetic differences of the dominant species Lacticaseibacillus paracasei among mother-infant cohorts of different ethnic groups, providing a theoretical basis for developing personalized probiotic regimens. [Methods] Lactobacillus strains were isolated from 39 mother-infant pairs of three ethnic groups without mixed marriage in China and identified by repetitive extragenic palindromic PCR (rep-PCR) and groEL sequences. The genetic differences of 83 strains of L. paracasei, a representative species, were analyzed by multilocus sequence typing (MLST). [Results] The species and abundance of Lactobacillus varied among the mother-infant pairs of different ethnic groups. A total of 945 Lactobacillus strains were isolated, belonging to 15 species of 4 genera. L. rhamnosus (20.07%), L. paracasei (16.54%), and L. casei (11.90%) were dominant species in the Han ethnic group, while L. casei (13.55%), L. paracasei (12.69%), and Ligilactobacillus salivarius (11.47%) were dominant bacteria in Uighur ethnic group in Hotan. The dominant species in the Li ethnic group in Hainan were Limosilactobacillus oris (24.55%), L. paracasei (15.85%), and Lactobacillus gasseri (10.87%). The 83 strains of L. paracasei were classified into 11 phylogenetic groups by rep-PCR and 31 sequence types (STs) by MLST, demonstrating ethnic specificity. L. paracasei isolates from the same mother-infant pair had the same STs, and isolates from the mother-infant pairs of the same ethnic group had higher genetic similarity. [Conclusion] Lactobacillus species varied in the mother-infant pairs of different ethnic groups, and L. paracasei strains from the same origin displayed higher genetic similarity, which supported vertical transmission at strain level and ethnic specificity.