Abstract:Terpenoids are mostly existing compounds in natural products like plants and microorganisms. The application prospect of terpenoids attracts much attention owing to more and more valuable terpenoids discovered. However, limited yield and high extraction cost of terpenoids restrict their wide applications. The rise of synthetic biology has provided new ideas for biosynthesis of valuable terpenoids using targeted and high efficient microbial cell factories. Although terpenoid synthases are widely used as target enzymes in metabolic regulation of terpenoids biosynthesis, many natural terpenoid synthases have some disadvantages, such as insufficient catalytic activity, poor substrate specificity, poor regio-or stereoselectivity, poor stability and so on, which unfavorably affect the yield of terpenoids. To solve above problems, directed evolution of terpenoid synthases has been applied, which will have profound impact on biosynthesis of terpenoids by microbial cell factories. This review summarizes recent advances and their applications in directed evolution of enzymes. Meanwhile, the strategies for directed evolution of terpenoid synthases are proposed.

Abstract:Flavivirus infection causes severe human disease, but no specific drugs are available to treat flavivirus infection. The N terminal region of non-structural protein 3 (NS3) of flavivirus and its cofactor NS2B constitute protease, and the polyprotein produced by virus is cleaved by protease into structural and non-structural proteins that are essential for the replication of the virus. NS2B-NS3pro represents an attractive drug target due to its critical role in the life cycle of flavivirus. Here we review the structural advances of NS2B-NS3pro of Zika Virus, Dengue Virus and West Nile Virus as well as the complex structures of some protease with inhibitors, these will provide necessary references for the future antiviral drug design.

Abstract:Salmonellosis is one of the most widespread food-borne diseases throughout the world, not only causes economic losses to the breeding industry, but also poses a threat to human health. Avian Salmonella enters the small intestine where infection can be established, inducing intestinal epithelial cells express a number of TLRs and inflammation. These lead to the expression of chemokines that attract immune effector cells to the sites of infection. Following translocation across the intestinal epithelial barrier, bacteria are phagocytosed by cells such as macrophages and DCs, macrophages are its preferred cell type. The adaptive immune response is stimulated by the innate immune system through antigen presentation to lymphocytes. Adaptive immunity can clear infection within 2-3 weeks following infection by a Th1 dominated response in the gut and deeper tissues. In contrast, avian systemic salmonellosis caused by the host-adapted serovar S. Pullorum is characterized by invasion from the intestine with multiplication in the spleen, liver and other organs. The stage of infection resulted in little inflammation in vivo or in vitro. S. Pullorum tends to induce an immune response that more closely resembled the Th2 response in mammals and would allow S. Pullorum to establish an intracellular carriage evading Th1-mediated clearance. This review compared the pathogenesis and immune response characteristics of Salmonella, especially the mechanisms by which S. Pullorum evades host immunity and produces the persistent carrier state. Finally, it will be helpful to provide new targets and new opinions for the prevention and control of avian salmonellosis.

Abstract:Specialized substrates are injected into a host cell through secretion system, to destroy various signal pathways, which is an effective way for bacterial colonization and infection. Vibrio parahaemolyticus being an important foodborne pathogen, its Type Ⅲ Secretion System (T3SS) and Type VI Secretion System (T6SS) are important virulence factors to the hosts. In this review, we summarize the specific roles of T3SS and T6SS effectors in pathogenicity and the related regulatory mechanisms. This paper provides a reference to further understand the diseases caused by Vibrio parahaemolyticus, to investigate its pathogenesis and find the pathogenic targets.

Abstract:[Objective] To study the potential of regulatory gene slnN upstream of salinomycin biosynthesis gene cluster. [Methods] We used genetic manipulation technique to knock out and overexpress the slnN gene in the original strain Streptomyces albus BK3-25. Then, using inhibition zone test and fermentation experiment, we detected the changes of the production of salinomycin biosynthesis in different derivative strains. At the same time, we used qRT-PCR technique to analyze the difference of the structural gene expression between the derived strains and the original strain. [Results] The production of salinomycin was increased by about 35% in the slnN gene-deleted strain (slnNDM), whereas the production of salinomycin was decreased by about 43% in the slnN gene-overexpressing strain (slnNOE). qRT-PCR analysis revealed that loss of slnN gene caused up-regulation of slnO and slnA1 genes. The slnN gene overexpression, on the one hand down-regulated the expression of slnO and slnA1 gene, on the other hand caused slnT1 and slnF gene up-regulation. [Conclusion] The slnN gene has a significant negative regulatory effect on the biosynthesis of salinomycin.

Abstract:[Objective] This study is aimed to analyze the diversity of actinobacteria in soils of the Henan Yellow River wetland and screen actinobacteria strains which have antagonistic activity against plant pathogens. [Methods] High-throughput sequencing based on Illumina HiSeq technology was used to analyze the actinobacterial diversity and distribution in the Henan Yellow River wetland. We used eight selective media to isolate actinobacteria from 21 soil samples collected from Sanmenxia, Zhengzhou and Kaifeng Yellow River wetland. The cultivable actinobacteria were identified and phylogeny analyzed based on the 16S rRNA gene sequences. The active isolates were screened using seven plant pathogens as targets, and 5 functional genes were assayed, including polyketide synthases gene (PKS-I, PKS-Ⅱ), nonribosomal peptide synthetases gene (NRPS), 3-amino-5-hydroxybenzoic acid synthases gene (AHBA) and 3-hydroxy-3-methylglutaryl coenzyme A gene (HMG CoA). [Results] The high-throughput sequencing results indicated that the actinobacteria were abundant in Henan Yellow River wetland and there were significant differences among different wetland areas and different habitats in the same wetland. Nocardioides was the most frequently isolated genus, followed by the genera Streptomyces, CL500-29 marine group, Fodinicola, Mycobacterium and Micromonospora. In addition, there are still a lot of unknown groups. We isolated a total of 261 dereplicated strains, belonging to 7 orders, 9 families and 9 genera of the phylum Actinobacteria. They were including 97 putative species and eight unknown species. Among them, 86 actinobacteria strains have antagonistic activity against plant pathogens; 74 strains contain at least one gene which involved active metabolites synthesis. [Conclusion] Actinobacteria species diversity was abundant in Henan Yellow River wetland and new species have potential as biocontrol agents against plant diseases.

Abstract:[Objective] Curculio beetles are major parasitic insects of oak acorns, but the mechanism of their adaptation to high-tannin food (e.g., acorns) is not clear. In this study, we compared the structure and diversity of gut bacteria of the larvae of Curculio arakawai and C. dentipes infested in Mongolian oak (Quercus mongolica) and Liaodong oak (Q. liaotungensis), to elucidate the adaptive mechanisms of weevil larvae to high-tannin food. [Methods] We collected gut samples of weevil larvae for DNA extraction. V3-V4 areas were sequenced using the Illumina MiSeq technology 16S rRNA sequencing. We carried out sequence statistics of Operational Taxonomic Units (OTUs), analysis of species abundance, a and b diversity. [Results] There were 2054 OTUs and 2308 OTUs of the high quality sequences obtained from the guts of the 2 weevil species, in which 481 OTUs were shared. In the gut bacterial community of C. arakawai and C. dentipes, 27 phyla, 145 families and 274 genera were identified as the major taxonomic groups. At the phylum level, Proteobacteria, Bacteroidetes and Firmicutes were dominant in the guts of C. arakawai and C. dentipes. At the genus level, Pseudomonas (63.8%), Serratia (6%) and Acinetobacter (5.2%) were the main groups in the gut of C. arakawai, while Serratia (32%), Rahnella (24.2%), Aeromonas (6.8%) and Rickettsia (6.6%) were dormant in the gut of C. dentipes. Our a diversity analyses showed no significant differences between the gut bacteria of C. arakawai and C. dentipes; however, b diversity showed significant difference between the gut bacteria of C. arakawai and C. dentipes. [Conclusion] Differences in gut bacterial community may be related to differences in species of weevils and host tannin content. We also found several bacteria related to the degradation of tannins, e.g., Serratia marcescens and Lactococcus lactis, which may reflect the adaptive basis for two weevil species to use high tannin food.

Abstract:[Objective] St. Louis encephalitis virus (SLEV) is a single-stranded and positive-sense RNA virus, belonging to the Flaviviridae family. Non-structural protein 3 (NS3) encoded by flaviviruses plays an important role in virus replication and polyprotein precursor modification. NS2B is an essential element that acts as a co-factor to enhance the activity of NS3. Therefore, the NS2B-NS3 protease is considered as a crucial target for anti-flavivirus drug development. In the current study, we constructed recombinant plasmid of SLEV NS2B-NS3 protease, and expressed and purified the protein to screen inhibitors. [Methods] We obtained the NS2B-NS3 recombinant gene by PCR and subcloned the prokaryotic recombinant expression plasmid. The NS2B-NS3 protease was expressed in soluble form induced by isopropyl β-D-1-thiogalactopyranoside in Escherichia coli BL21(DE3). The fusion protein was purified with Ni⁺-NTA affinity column. Then the serine protease activity was determined by a fluorescence resonance energy transfer assay and the screening platform for inhibitors was constructed. [Results] The purity NS2B-NS3 protease was up to 95%. After screening for 700 old drugs, we found that procyanidine efficiently inhibited the enzymatic activity of the NS2B-NS3 protease. [Conclusion] Our current study provided a convenient and high-throughput method for screening of SLEV NS2B-NS3 protease inhibitors. Porcyanidine has been found to inhibit the activity of SLEV NS2B-NS3 protease for the first time, and might become a potential drug to treat SLEV infection.

Abstract:[Objective] To explore the relationship between soil fungal community structure and soil environmental factors of the degradation process of alpine grassland in Qilian Mountains.[Methods] We analysed the changes and diversity of fungal community structure in lightly, moderately and severely degraded grassland by Illumina Miseq PE250 high throughput sequencing technology, and redundancy analysis (RDA) of the relationship between soil fungal community structure and soil environmental factors was analysed.[Results] With the aggravated degree of deterioration, soil pH was increased, electrical conductivity was increased first and then decreased, soil water content, organic carbon, total nitrogen, total phosphorus and total potassium were gradually decreased. We obtained a total of 750575 effective sequences and 5788 OTUs by high throughput sequencing, the Chao1 and Shannon-Wiener index of fungal communities varied in different samples. The fungal communities' dominant phyla of all soil samples were Ascomycota, Basidiomycota, Zygomycota, Glomeromycota and Chytridiomycota. RDA analysis showed that soil available potassium, total nitrogen, available nitrogen and organic carbon were the important driving factors for the distribution of soil fungal community structure in different degraded alpine grassland of Qilian Mountains.[Conclusion] The soil fungal community structure was significantly different in different degraded alpine grassland, and soil environmental factors was a important factor influenced the distribution of soil fungal community structure in Qilian Mountains.

Abstract:[Objective] A thermostable β-xylosidase from the thermophile Caldicellulosiruptor owensensis OL (CoXyl B) was characterized. [Methods] Recombinant CoXyl B was heterogeneously expressed in Escherichia coli and then purified using Ni-NTA, anion exchange and Superdex 200 chromatography. Further, the hydrolysis activity of CoXyl B was studied using p-nitrophenyl-D-xyloside (pNPX) and xylooligosaccharide as substrates. [Results] The optimum temperature and pH of CoXyl B was 90℃ and pH 6.0, respectively. CoXyl B maintained stable at temperature between 40℃ and 70℃. After incubation at 70℃ for 1 h, CoXyl B retained more than 80% of its initial activity at both pH 5.0 and 6.0. Significantly, Ag⁺, SDS and PMSF exhibited negative effect on the activity of CoXyl B, whereas Mg²⁺, Li⁺ and EDTA significantly enhanced the activity of CoXyl B. The kinetic parameters of CoXyl B towards pNPX were k_cat of 5.0×10^-3 s^-1 and K_m of 1.9 mmol/L. Moreover, CoXyl B exhibited efficient hydrolysis activity towards xylobiose, xylotriose and xylotetraose. [Conclusion] Our work suggested the application potential of a new thermostable β-xylosidase (CoXyl B) in the area of xylan degradation at high temperature.

Abstract:[Objective] We studied the effects of dietary fucoidan on growth performance, nutrient digestibility, immune index in serum, and gut microbiota composition in weaned pigs. [Methods] A total of 36 weaned pigs (body weight 7.43±0.12 kg) were allocated to 3 dietary treatments, and piglets in each group received control diet, control diet containing antibiotic, control diet containing fucoidan for 28 days. Growth performance and nutrient digestibility were evaluated in piglets. Serum indices related to immunity were measured by spectrophotometric and ELISA assays. Gut microbiota composition was determined using 16S rRNA amplicon sequencing. [Results] A reduction in feed conversion ratio in piglets fed fucoidan supplementation on 0-14 days (P<0.05). The digestibility of neutral detergent fiber and acid detergent fiber were increased in fucoidan dietary treatment (P< 0.05). Compared with the control group, the production of interleukin 22 decreased in piglets treated with antibiotic and fucoidan intervention (P<0.05). Piglets provided with antibiotic and fucoidan treatments showed an increased tendency (P=0.07) in the population of Bacteroidetes members on day 14, whereas the abundance of Actinobacteria increased (P<0.05) on day 28. Specifically, there was an increase in the Bacteroides genus in diet containing fucoidan when compared to those in control and antibiotic groups. [Conclusion] Dietary fucoidan improved the fiber digestibility and the richness and diversity of Bacteroides genus, and decreased inflammatory cytokines of IL-22 production in weaned pigs. These changes should alleviate the post-weaning stress and contribute to the establishment of healthy and stable digestive system in weaned pigs.

Abstract:[Objective] A multi-epitope protein vaccine of foot-and-mouth disease virus (FMDV) serotype A was developed to provide a safe and effective candidate vaccine for the prevention and control of FMD in pigs. [Methods] We designed and synthesized multi-epitope immunogenic genes A10, IA10 and FA10 based on the results of previous experiments and the global epidemiological information of FMDV serotype A. The expressed proteins were emulsified with ISA 201 adjuvant to immunize pigs. Serum IgG antibody titers were detected by liquid phase blocking ELISA at 0, 14 and 28 days post-immunization. All pigs were challenged with virulent FMDV A/HNLY4/2014 after a single inoculation. [Results] Three recombinant proteins were successfully expressed in E. coli and specific bands of approximately 35, 57 and 64 kDa, which were consistent with the expected size of the recombinant proteins A10, IA10 and FA10, respectively. The recombinant proteins were also recognized specifically by the anti-FMDV (type A) antibodies. A10+201 induced lower level of total IgG antibodies than the inactivated vaccine, but higher than other groups. All pigs (100%) immunized with A10+201 and inactivated vaccine were completely protected against virulent FMDV challenge. Four out of five pigs (80%) immunized with IA10+201 and FA10+201 were protected and only one out of five pigs (20%) immunized with A10 and FA10 were protected. All pigs inoculated with PBS+201 were unprotected. [Conclusion] A10+201 conferred the greatest protection against FMDV serotype A challenge in pigs and can be further evaluated as a candidate vaccine.

Abstract:[Objective] In order to obtain highly efficient bacterial consortium that can rapidly decolorize azo dyes in high salinity environment. [Methods] A halophilic bacterial consortium was obtained by enrichment, and its community structure was determined by Illumina HiSeq2500 sequencing platform. The degradation characteristics were determined by spectrophotometry. The degradation mechanism was analyzed by GC-MS and Fourier Transform infrared spectroscopy. The toxicity of azo dye and degradation product were compared by micronuclear test. [Results] At 10% salinity, the bacterial consortium decolorizes methanil yellow G completely in 8 h. The bacterial consortium was composed of Zobellella, Rheinheimera, Exiguobacterium and Marinobacterium. At pH 6.0, with yeast powder as carbon sources, peptone or potassium nitrate as nitrogen sources and 1% NaCl were the optimal decolorization conditions. Decolorization metabolites of methanil yellow G were less toxic than original dye. The main degradation products of methanil yellow G were p-aminodiphenylamine and diphenylamine. In addition, the bacterial consortium also decolorized various azo dyes, such as acid magenta GR and direct lake blue 5B. [Conclusion] A halophilic bacterial consortium for fast degradation of azo dyes was obtained, with the application potential to treat high-salt printing and dyeing wastewater.

Abstract:[Objective] In order to study carbon metabolic repressors on cellulase activities and expression regulation in Trichoderma reesei, we constructed multi-targeting siRNA expression vectors to perform silencing carbon metabolic repressor cre1, cre2, cre3 and cre4 by simultaneous producing multi-targeting siRNAs. [Methods] According to previous studies and screening, the four best siRNAs targeting the cre1, cre2, cre3 and cre4 were selected and they were constructed a multi-targeting siRNA expression vector A. Additionally, according to 5 overlap common sequence in the cre1, cre2, cre3 and cre4, we designed and constructed the vector B. The two vectors were transformed the protoplasm of Trichoderma reesei QM9414 and selected on the hygromycin selection medium. Cellulase activities (CMC activity and filter paper activity) of transformants were detected and related gene expressions were also detected by RT-qPCR after incubation for 48 and 120 hours respectively. [Results] RT-qPCR results showed that the cre1, cre2, cre3 and cre4 expression levels in Trichoderma reesei were simultaneously silenced, and the cellulase activities were much higher than that of the staring strain. The CMC activity and filter paper activity of the multi-targeted inhibitor strains were 1.95-fold and 2.66-fold higher than those of the original strains. The cellulase-related genes expression levels were also increased significantly. The expression levels of cbh1, egl1 and xyr1 were 3.83-fold, 3.95-fold and 2.78-fold higher than those of the original strain. [Conclusion] Our results indicated that simultaneous silencing multi-targets of the carbon metabolic repressors in Trichoderma reesei is beneficial to release the glucose repressor effects and increase the expression and production of cellulase. These results provide evidence and techniques for study of regulation of carbon catabolite repressor genes on cellulase expression in Trichoderma reesei.

Abstract:[Objective] Marinomonas sp. FW-1 secretes high-activity arylsulfatase. This study was carried out to clone all the possible arylsulfatase in Marinomonas sp. FW-1 and investigate the motif information essential for arylsulfatase. [Methods] We used high-throughput sequencing to obtain the entire genome sequence of Marinomonas sp. FW-1. We carried out gene assembly, gene prediction, functional annotation and Clusters of Orthologous Groups clustering analysis. Thereafter, we cloned arylsulfatase genes in Escherichia coli and analyzed the activity of the recombinant proteins. [Results] The complete genome is of 3964876 bp in length, with an average Guanine and Cytosine content of 44.03%, encoding 3590 protein genes, containing 78 tRNA and 25 rRNA operons. Genome annotation indicated the presence of four possible arylsufatase genes. Among the four candidate genes, three genes contained C-X-P-X-R motif and their recombinant products were found to exhibit arylsulfatase activities. [Conclusion] This study revealed multiple arylsulfatase gene sequences in the complete genome Marinomonas sp. FW-1 strain, which predicted various potential applications of this strain. Moreover, C-X-P-X-R motif was confirmed to be essential for arylsulfatase activity.

Abstract:[Objective] To obtain effective secondary metabolites from endophyte J1 originated from Blumea balsamifera (L.) DC. [Methods] The endophytic fungus J1 was identified based on ITS gene phylogenies. Various chromatographic methods were used to isolate and purify compounds from the fermentation extract, and their structures were elucidated by NMR spectroscopy and MS analyses. [Results] J1 was determined as Diaporthe sp. by analyses of phylogenetic tree. Seven compounds were afforded from the rice culture of this fungus, and they were identified to be Dicerandrol A (1), Dicerandrol B (2), 4,6-dihydroxy-1H-isoindole1,3(2H)-dione (3), Cytochalasin H (4), Cytochalasin J (5), 4,6-dihydroxy-2,3-dihydro-1H-isoindol-1-one (6), Cerebroside C (7), individually. All of the seven metabolites were isolated from this fungus for the first time, and compound 1 displays potent inhibitory activity towards Bacillus subtilis KCTC 1021, with an MIC value of 0.125 μg/mL. [Conclusion] This study demonstrated that Diaporthe sp. contains many antimicrobial compounds and may serve as a potential microbial-origin antibacterial agent.

Abstract:[Objective] To develop a simple method to improve the enzymatic and antifungal activity of chitinase by fusion of chitin binding domain at both termini. [Methods] The chimeric and truncated chitinases were constructed using the unique glycoside hydrolase family 19 chitinase in Streptomyces alfalae ACCC40021 as template, and expressed in Escherichia coli. The 3,5-dinitrosalicylic acid (DNS) method was used to determine the enzyme activity with colloid chitin as the substrate. [Results] CatD_ChiB (catalytic domain), rChiB (N-terminal chitin-binding domain) and DChBD_ChiB (double chitin-binding domain) were successfully constructed and expressed in E. coli BL21(DE3). Compared to CatD_ChiB and rChiB, DChBD_ChiB improved the binding ability and activity towards colloidal chitin, α-chitin and chitin from Aspergillus niger. Furthermore, antifungal activity was enhanced against plant pathogenic fungus Trichoderma longibranchiatum. [Conclusion] A simple, feasible and efficient method was developed to improve the enzymatic and antifungal activity of chitinase by fusion of chitin binding domain on both C-and N-terminus of protein.