[Objective] The enzymatic depolymerization of polyethylene terephthalate (PET) involves the hydrolysis of PET catalyzed by PET hydrolases, leading to the production of terephthalic acid and ethylene glycol. In this study, we identified a new PET hydrolase through gene mining, with the goal of providing enzymes for efficient depolymerization of waste PET. [Methods] A previously uncharacterized functional enzyme, named as AePETase, was identified through gene mining. This enzyme demonstrated the ability to hydrolyze amorphous PET films. Subsequently, heterologous expression, purification, and enzymatic characterization were performed for AePETase. [Results] AePETase showed a T_m value of (53.0±0.7) ℃, and an optimal reaction temperature of 45 ℃. The optimal buffer was Na₂HPO₄-NaH₂PO₄ (100 mmol/L, pH 8.0), and the optimal enzyme concentration was 1 µmol/L. After 72 h, the hydrolysis of amorphous PET films by AePETase resulted in 13.4-fold more products than that by the reported IsPETase. [Conclusion] AePETase demonstrates significant hydrolytic activity on PET and represents a promising new enzyme for hydrolyzing PET.

Currently, the drugs available for treating fungal infections show limited number and off-target effects. Thus, there is an urgent need to develop new antifungal drugs. Fungal apoptosis-like cell death (ALCD) is a cell death phenomenon that occurs during the normal development stage of organisms. This article summarizes the characteristics, signaling pathways, and key factors involved in fungal ALCD and introduces the natural and synthetic drugs that can induce fungal apoptosis. The natural drugs include lipid peptides, farnesol, statins, and alkaloids from microorganisms, organic acids and essential oils from plants, and melittins from insects. Furthermore, this article establishes the basic molecular landscape of drug-induced fungal ALCD. This article provides a theoretical basis for formulating a new strategy for resisting pathogenic fungi and developing targeted antifungal drugs.

[Objective] To improve the overall quality of upper tobacco leaves. [Methods] We isolated a hemicellulase-producing bacterium from tobacco soil and identified it as Actinacidiphila bryophytorum. After optimizing the culture conditions of this strain, we prepared the crude enzyme liquid, which was sprayed on upper tobacco leaves. The physical properties and chemical composition of the treated tobacco leaves were then evaluated. [Results] The optimal fermentation conditions for the strain were an inoculation rate of 4%, pH 6.0, and incubation at 30 ℃ for 36 h. Enzyme preparations of different concentrations (50, 100, 150, 200 and 250 U/mL) were then sprayed on the surface of upper tobacco leaves, and the appearance quality, conventional chemical composition, lignocellulose content, and sensory scores of the tobacco leaves were evaluated. The results indicated that treatment of the enzyme preparation at 150 U/mL performed better than other treatments. Compared with the control group, the 150 U/mL treatment increased the overall score for the appearance quality (maturity, color, characteristics, and structure) of tobacco leaves by 5.15 points. Additionally, this treatment increased the total sugar content and reducing sugar content by 29.87% and 35.77%, respectively. Meanwhile, it decreased the content of nicotine, cellulose, hemicellulose, and lignin by 16.10%, 16.37%, 20.22%, and 17.13%, respectively. Furthermore, the aroma characteristics were enhanced, and off-flavors were reduced. [Conclusion] The enzyme preparation produced by the fermentation of A. bryophytorum S2 improves the appearance quality, increases the soluble total sugar and reducing sugar content, and reduces the lignocellulose content of tobacco leaves, thus improving the overall quality of tobacco leaves.

[Objective] To understand the molecular functions and potential applications of the significantly up-regulated gene cluster chr1_2605-chr1_2604 in response to tetrabromobisphenol A (TBBPA) stress, we investigated the roles of chr1_2605 and chr1_2604 in the specific recognition and efficient degradation of TBBPA. [Methods] Synthetic biology methods were employed to construct Sphingobiumxenophagum C1 (pBBR-2605-HiBiT) and Escherichiacoli BL21(DE3, pET30b-2604) as chassis cells for biosensing and degrading, respectively. The response characteristics of Chr1_2605 in the chassis cells to different pollutants were analyzed by the luciferase activity assay. Additionally, the degradation activity of TBBPA by Chr1_2604 in the chassis cells was determined by high-performance liquid chromatography. [Results] The xenobiotic-responsive element Chr1_2605 exhibited a highly specific response to TBBPA. The Chr1_2605-based chassis cell of S. xenophagum C1 (pBBR-2605-HiBiT) demonstrated high responsivity and sensitivity to TBBPA, with a limit of detection ranging from 0.010 to 0.050 μmol/L. The 2-oxoglutarate/Fe-dependent dioxygenase Chr1_2604 in the chassis cell of E. coli BL21(DE3, pET30b-2604) displayed the degradation rate of 44.415% for 2.0 mg/L TBBPA within 3 d [0.296 mg/(L·d)], which was significantly higher than those of most reported microbial strains under non-co-metabolic conditions. [Conclusion] The chr1_2605-chr1_2604 gene cluster can accurately recognize and degrade TBBPA. Specifically, the xenobiotic-responsive element Chr1_2605 specifically recognizes TBBPA, whereas the 2-oxoglutarate/Fe-dependent dioxygenase Chr1_2604 efficiently degrades TBBPA.

[Objective] Listeria monocytogenes (Lm) is a Gram-positive foodborne pathogen causing human and animal listeriosis with high case fatality rates (20%-30%). Lm can grow at low temperatures, which brings great risks to food safety and human health. Exploring the mechanism of Lm growth at low temperatures will provide a theoretical basis for the formulation of measures to control Lm growth at low temperatures and the development of low-temperature growth inhibitors. Studies have shown that LisK/R, a two-component signal transduction system (TCS) in Lm, plays a role in regulating the growth of Lm at low temperatures, while the specific mechanism remains unclear. This study aims to reveal the mechanism of LisK/R in regulating the growth of Lm at low temperatures. [Methods] With the Lm strain LM201 isolated from foodstuff as the parental strain, we constructed three strains: ΔlisR (RR-deleted mutant of LisK/R), ΔlisRc (complementary strain of ΔlisR), and ΔlisKc (complementary strain of ΔlisK, which was constructed before this study in our laboratory). The growth curves of these strains were determined at 4 ℃. RNA-Seq was performed for ΔlisK and the parental strain growing at 4 ℃, and the obtained data were analyzed. We assayed the swarming area diameter on soft agar plate, flagellar biosynthesis by transmission electron microscopy, and transcriptional levels of flagellar genes by RT-qPCR for all the strains at 4 ℃. [Results] The growth of deletion mutants was slower than that of the parental strain (P<0.05), and the growth of complementary strains was consistent with that of the parental strain at 4 ℃. The RNA-Seq data showed that compared with that in the parental strain, the expression of flagellar genes in ΔlisK was up-regulated (P<0.05). At 4 ℃, the swarming area diameters of deletion mutants on soft agar plates were bigger than those of the parental strain and complementary strains (P<0.05); the deletion mutants had more flagella, while the parental and complementary strains had fewer flagella; the expression of flagellar genes in the deletion mutants was higher than that in the parental and complementary strains (P<0.05). [Conclusion] At 4 ℃, the LisK/R mutants with slow growth had more flagella, while the parental strain with rapid growth had fewer flagella. The results indicate that LisK/R inhibits the expression of flagellar genes in Lm to reduce flagellar production, and the energy is used to promote Lm growth at low temperatures. The specific molecular regulatory mechanism of LisK/R needs further study. This study provides new information for elucidating the mechanism of Lm growth at low temperatures.

[Objective] To investigate the antibiotic resistance and virulence of pet-derived Klebsiella pneumoniae in some areas of Guangdong Province. [Methods] Fecal swabs were collected from dogs and cats for strain isolation, and 16S rRNA and khe genes were amplified by PCR to identify the strains. The sensitivity of K. pneumoniae isolates to 17 antibiotics was determined by the agar diffusion method. PCR was employed to detect resistance genes to β-lactams (bla_SHV, bla_CTX, and bla_TEM), carbapenems (bla_KPC and bla_NDM), aminoglycosides (rmtB), quinolones (qnrS and oqxAB), sulfonamides (Sul1 and Sul2), amphenicols (floR), tetracyclines (tet(A)), and fosfomycin (fosA3) and some virulence genes (rmpA, maga, fimH, mrkD, uge, WabG, kfu, Aerobactin, and ureA). [Results] A total of 126 strains of K. pneumoniae were isolated from 428 fecal samples, with an isolation rate of 29.4%. The 126 isolates had high resistance rates to amoxicillin (75.40%), ampicillin (73.81%), and cotrimoxazole (61.90%). They were moderately sensitive to ceftazidime, amikacin, apramycin, and enrofloxacin, and they were sensitive to tigecycline, colistin, and meropenem. The detection rate of the resistance gene oqxAB was the highest, which was 86.51%, followed by those of the β-lactam resistance gene bla_SHV (73.81%) and the tetracycline resistance gene tet(A) (52.68%). Other resistance genes were detected to varying degrees (0.79%-46.03%) and the carbapenem resistance gene bla_KPC, colistin resistance gene mcr-1, and aminoglycoside resistance gene rmtB were not detected. Among the virulence genes, the urease gene urea, lipopolysaccharide-related gene uge, and fimbria-related gene fimH showed the detection rates of 100.00%, 95.54%, and 91.07%, respectively. Other virulence genes were detected to varying degrees (2.70%-8.90%), while the capsule-related gene magA and the fimbria-related gene mrkD were not detected. [Conclusion] In some areas of Guangdong Province, the antibiotic resistance of pet-derived K. pneumoniae is serious, but its pathogenicity is relatively weak. Monitoring of its drug resistance and pathogenicity should be strengthened. In addition, antibiotics should be strictly managed and rationally used in the clinical practice, so as to avoid the generation and dissemination of multidrug resistant strains of K. pneumoniae.

[Objective] To delineate the fungal diversity characteristics and disparities in the rhizosphere soil of naked barley from fields exhibiting different root rot incidences, thereby informing targeted and effective disease management strategies. [Methods] Rhizosphere soil samples of naked barley were collected from the healthy field and the fields with root rot incidences of 5%, 10%, 15%, and 20%, respectively. The 18S rRNA gene of fungi from the samples was amplified, and high-throughput sequencing was conducted on the Illumina-MiSeq platform. Following quality control, classification, and annotation, the data were analyzed for fungal diversity from various perspectives and taxonomic levels. [Results] The healthy sample and the sample with the root rot incidence of 5% had the highest fungal diversity, while the sample with the incidence of 10% exhibited the lowest diversity. Additionally, co-occurrence network analysis revealed more complex species interactions in the healthy sample and the sample with the incidence of 5%. The root rot incidence had a negative correlation with the relative abundance of Ascomycota but a positive correlation with the relative abundance of Basidiomycota and Glomeromycota. Dominant fungal classes were Agaricomycetes, Chytridiomycetes, and Sordariomycetes. Significant variations in the distribution of fungal groups with high average relative abundance were noted across samples. Specially, the healthy sample had the highest relative abundance of Eurotiales and the lowest relative abundance of Glomerales, which were converse in diseased samples. Arthrodermataceae was significantly enriched in the healthy sample, while Sordariaceae, Myxotrichaceae, and Olpidiaceae were preferentially associated with the samples exhibiting incidences of 10%, 15%, and 20%, respectively. At the genus and species levels, the composition of dominant fungal communities in the healthy sample and the sample with the incidence of 5% was similar, and it was similar in the samples with incidences of 10%, 15%, and 20%. FUNGuild predicted a decrease in the relative abundance of plant pathogens and an increase in the relative abundance of saprotrophs with the increase in disease incidence. [Conclusion] The progression of naked barley root rot is intricately linked to the disruption of the equilibrium within the rhizosphere fungal community. In the context of precision management of naked barley root rot, it is imperative to regulate and sustain the balance of the abundance of dominant fungal taxa in the rhizosphere.

The aggravating soil-borne diseases threaten the production of a variety of crops and undermine the sustainable development of agriculture. Streptomyces-based bio-agents have been widely explored as an environmentally safe and economically durable biocontrol resource. However, the biocontrol efficacy and influencing factors require further optimization. [Objective] Here we conducted a meta-analysis to evaluate the efficacy of Streptomyces-based bio-agents in controlling the two common soil-borne diseases and identify the key influencing factors. [Methods] The relevant articles were retrieved from Web of Science and CNKI with the keywords “Streptomyces” and “Fusarium wilt”, “Streptomyces” and “bacterial wilt”, “Streptomyces” and “Fusarium oxysporum”, and “Streptomyces” and “Ralstonia solanacearum (or Pseudomonas solanacearum)”. The articles containing comparable treatment groups (with Streptomyces application) and control groups (without Streptomyces application), disease incidence, sample sizes, and mean values were systematically selected. Finally, 76 articles (113 groups) on Streptomyces-based bio-agents for Fusarium wilt control and 19 articles (28 groups) on those for bacterial wilt control were obtained. [Results] After the application of Streptomyces-based bio-agents, the disease incidence of Fusarium wilt decreased from 75.58% to 24.49% (average control efficacy of 67.60%), and that of bacterial wilt decreased from 73.75% to 19.83% (average control efficacy of 73.11%). The soil density of Streptomyces, the final concentration ratio of Streptomyces to pathogen in soil, and climate types were vital for the biocontrol performance of Streptomyces-based bio-agents. Moreover, Streptomyces at the final concentration of 10⁷ CFU/g demonstrated the best biocontrol performance on both diseases. The biocontrol effect of Streptomyces on Fusarium wilt was better when the final concentration ratio of Streptomyces to pathogen was 1:1, whereas the biocontrol effect on bacterial wilt disease was better when the ratio was 10:1 or 100:1. Notably, Streptomyces demonstrated enhanced biocontrol effects on the both diseases in tropical monsoon climate regions. [Conclusion] The application dose of Streptomyces-based bio-agents in soil-borne disease management should be adjusted according to the species and concentration of the pathogen. In addition, it may be better to apply Streptomyces-based bio-agents in tropical monsoon climate regions.

[Objective] To explore the antifungal activity and inhibitory mechanism and identify the main antagonistic substances of the crude extract of Pseudomonas protegens FD6 in fermentation. [Methods] The plate confrontation test was employed to screen the optimal medium, extractant, and solvent and determine the antifungal activity stability of the crude extract. The main antifungal substances in the crude extract were identified by MALDI-TOF/TOF mass spectrometry, and the antifungal spectrum of the crude extract was explored. The inhibition mechanism of the crude extract against Botrytis cinerea was investigated by mycelial morphology observation, spore germination test, PI staining, and conductivity determination. [Results] The crude extract of FD6 in the potato juice medium without any carbon source displayed the strongest antifungal activity. The methanol-solved crude extract obtained with ethyl acetate as the extractant had the strongest inhibitory activity. The main antifungal substance of the crude extract was identified as a cyclic lipopeptide, orfamide A, based on mass spectrometry data. In addition, the crude extract displayed broad-spectrum inhibitory activities against Phomopsis amygdali, Fusarium graminearum, Colletotrichum gloeosporioides, and Monilinia fructicola. The antifungal activity of the crude extract was stable after treatment at 121 ℃ for 20 min, within the range of pH 4.0-10.0, and with protease K and trypsin. The mycelial morphology of B. cinerea became abnormal after treatment with the crude extract. The treatment with 2.0 mg/mL crude extract completely inhibited the conidial germination of B. cinerea, caused a 50% damage of the cell membrane, increased the conductivity, and led to leakage of intracellular nucleic acids and proteins. [Conclusion] The metabolites produced by P. protegens FD6 have broad-spectrum and stable antifungal activities, serving as lead compounds for exploration of new green fungicides.

[Objective] To explore the autophagy induced by the African swine fever virus (ASFV) protein E423R. [Methods] The RFP-LC3 fluorescent spots following the transfection of HeLa-Difluo^TM hLC3 cells with pCMV-Myc-E423R was quantified by the High-Content Analysis System, and the autophagic flux was assessed. The expression levels of key autophagy proteins, LC3-Ⅱ and SQSTM1/p62, in HeLa cells were analyzed via Western blotting. Co-localization of autophagosomes and lysosomes was examined by laser confocal microscopy. Additionally, Western blotting was employed to investigate the dose-dependent effect of E423R on LC3-Ⅱ expression and the regulatory effect of E423R on the AKT/mTOR/ULK1 signaling pathway. [Results] The High-Content Analysis System demonstrated a significant increase in RFP-LC3 fluorescence spots in the reporter cells expressing E423R, suggesting that E423R induced the activation of autophagy. Western blotting further confirmed that the expression of E423R significantly elevated the LC3-Ⅱ/β-actin ratio while decreasing the expression level of p62. Confocal microscopy results indicated that E423R enhanced the expression of GFP-LC3, promoted the co-localization of GFP-LC3 with Lyso-Tracker Red, and facilitated the fusion of autophagosomes and lysosomes. Additionally, the expression level of E423R exhibited a positive correlation with the expression level of LC3-Ⅱ in HeLa cells. Furthermore, E423R down-regulated the expression of p-AKT, p-mTOR, and p-ULK1 (Ser757) in the mTOR signaling pathway. [Conclusion] This study demonstrates that the ASFV E423R protein induces complete autophagy via the AKT/mTOR/ULK1 signaling pathway, exhibiting a dose-dependent effect on LC3-Ⅱ expression to a certain degree. These findings provide a foundation for further investigation into the infection and pathogenic mechanisms of ASFV.