Abstract:Lactic acid bacteria are an important class of food industry microorganisms. At present, the identification and excavation of functional genes of lactic acid bacteria mainly rely on traditional gene homologous recombination technology. Although this technology has high reliability, there are complicated operations and low efficiency, which severely restrict the genetic selection of lactic acid bacteria. CRISPR/Cas gene editing technology has greatly improved the editing efficiency of multi-species genomes, which provides opportunities for rapid functional gene identification and genetic improvement of lactic acid bacteria. However, the application of existing CRISPR/Cas gene editing technology in lactic acid bacteria still has many limitations. This article summarizes the application status of CRISPR/Cas gene editing technology in the genome of lactic acid bacteria and the urgent problems to be solved, and prospects the future development of the lactic acid bacteria genome editing technology, providing references for the identification and genetic improvement of lactic acid bacteria.

Abstract:Protein methylation modification is one of the ways of post-translational modification. In fungi, a variety of lysine methyltransferases perform methylation of lysines at specific sites on histones. The methylation of lysine on histones is related to fungal DNA replication, transcription, and heterochromatin formation. Methylation is involved in a variety of biological processes, such as fungal development, circadian rhythm regulation, secondary metabolic gene cluster expression, hydrolase synthesis, and pathogenic fungal virulence formation. We summarize the nomenclature, classification, domains, structures of the catalytic domain of KMTs, and the roles of methylation in different fungi. We also discuss the research limitations and the future research direction.

Abstract:Loop-mediated isothermal amplification (LAMP) is an emerging nucleic acid amplification technique under isothermal condition, which can combined with microfluidic chip to rapidly detect the pathogenic bacteria. It is of higher specificity, higher sensitivity and simpler operation. This article classified and introduced the LAMP-based microfluidic chip for the detection of pathogenic bacteria, based on the analysis of different end-point detection methods. And also, we analyzed the improvement and existing problems of this technology, which can provide great reference value for the further studies and industry development.

Abstract:Mycophenolic acid is one of the most widely used immunosuppressant in the world, and the market demand is huge. So far, the industrial production of mycophenolic acid is mainly through fungal fermentation, and the strains used for production are mostly mutagenized high-yielding Penicillium brevicompactum. This article systematically reviews the research and industrialization progress of mycophenolic acid. Five aspects including current research and application of mycophenolic acid, chemical synthesis and biosynthetic pathways, genetic regulation, fermentation production and market analysis are described. This review will provide valuable information for the development of novel derivatives of the drug, the improvement of yield, and the intelligent creation of advanced biotechnology in the future.

Abstract:Nitrogen is an essential element of life, and is one of the major nutrients limiting plant growth. To meet the increased food production demand in agriculture, chemical nitrogen requirements increase from year to year. Biological nitrogen fixation (BNF) plays an important role in agriculture. More than 60% of the fixed N on earth results from BNF. Associative nitrogen-fixing bacterium in major cereal crops is considered as one alternative in reducing the use of chemical nitrogen fertilizer. There has been a goal to increase biological nitrogen fixation and engineering such associations in non-legume crops to reduce the use of chemically derived fertilizer. The use of synthetic biology tools to boost biological nitrogen fixation has been considered as one of the best strategies to increase crop yield to face population growth and general agronomic crop demand. This review discusses the nitrogen fixation microorganisms, nitrogen-fixation island genes in associative nitrogen-fixing bacterium and nitrogen-fixing mechanisms. This review also elaborates on current research efforts involved in nitrogen fixation using synthetic biology tools. Moreover, this review also proposes new and emerging strategies to improve the nitrogenase activity and future perspectives in this research field.

Abstract:Hydrophobin is a class of small molecular weight proteins that have surface stability and can be self-assembled at the interface to form an amphiphilic protein membrane, thus changing the hydrophobicity of the interface. Previous research has shown that hydrophobin is non-toxic and immunogenic, can be used for material surface modification, food surface plasticity, drug target transport or biosensor signal accurate identification. In recent years, a small molecule secretion-type hydrophobin BslA (formerly YuaB) has been found in the biofilm of Bacillus subtilis. The research has shown that the Bacillus subtilis hydrophobin BslA has high expression yield, simple purification process, easy operation and large-scale production, so BslA has greater application advantage and development value. In this review, we summarize the information on the properties, functions and structure of BslA, and compare and analyze them with fungal hydrophobin, and systematically analyze their structural characteristics and application value.

Abstract:There are a large number of microorganisms in the intestine of fish, which play an key role in maintaining host health. The fish immune system can monitor and regulate the intestinal microbial composition and maintain the homeostasis of intestinal flora. At the same time, the intestinal commensal microorganisms regulate the fish immune system, control the excessive proliferation of pathogenic microorganisms, and ensure the health of the host. This paper reviewed the progress of interactions between intestinal microorganisms and the host immune system in fish, and focusing on the host immune system sensing intestinal microbes, host immune system shaping intestinal microbiota, probiotics regulating host immunity and intestinal microbiota, etc. It is suggested that the ideal probiotics should be derived from the gastrointestinal tract of fish and the non-host derived probiotics should be carefully selected in aquaculture application. This paper would provide theoretical supports for the future researches on the development and application of intestinal functional microbes in fish.

Abstract:Rhamnolipid is an important family of biosurfactant synthesized by many natural or engineered bacteria. Due to its excellent chemo-physical properties and environmental friendliness, it has been extensively used in many application scenarios (e.g., microbial enhanced oil recovery (MEOR), bioremediation), in place of its chemically synthesized counterparts. In commercial production, rhamnolipid is mainly produced by Pseudomonas aeruginosa, a natural producer with pathogenicity. Its genetic context also limits further enhancement of production using fermentation optimization approach. As a solution, genetic engineering approach can improve the bio-safety, yield, and product performance of rhamnolipid production, which has attracted increasing attention. However, few review articles systematically discussed this topic. Herein, we summarized recent advances in rhamnolipid production using genetically engineered microorganisms, particularly focusing on engineering strategies applied (e.g., heterogeneous production, pathway engineering, gene expression engineering, protein engineering, chassis engineering). Other than high yields, tailor-made rhamnolipid production (producing rhamnolipid mixture of specific composition) was also discussed. Finally, we proposed several potential strategies which may benefit rhamnolipid production:(1) using CRISPR-based tools for genome engineering; (2) gene expression optimization using synthetic promoter library and T7 polymerases; (3) synthetic protein scaffold for enzyme colocalization; (4) learning from polyhydroxyalkanoates (PHA) bioengineering; (5) development of in vivo directed evolution platform and rhamnolipid biosensor; (6) analysis of genome resequencing data.

Abstract:[Objective] We aimed to discover and identify the uncharacterized active components of Agricultural Antibiotic 120 by comprehensive analysis of genome sequence and secondary metabolites of Streptomyces hygroscopicus var. beijingensis (Bacteria, which produced Agricultural Antibiotic 120).[Methods] The genome sequence of wild-type strain was analyzed by antiSMASH for the sake of identifying possible biosynthetic gene clusters of nystatin and toyocamycin. HPLC, LC-MS and Q-TOF were used to analyze the secondary metabolites of wild-type strains, using chemical standards as control. Biosynthetic gene cluster (BGC) deletion mutants were constructed, and the fermentation products were analyzed.[Results] Biosynthetic BGCs of nystatin and toyocamycin were identified, and these two compounds were proved to be another two bioactive ingredients of Agricultural Antibiotic 120, besides the known anisomycin and tetramycin.[Conclusion]] The multiple BGC deletion mutant deducted the effect of compounds with high abundance and could serve as a good material to mine more natural compounds originally produced by WT strain. Moreover, correspondence analysis of genome sequence and metabolite profile was an effective method to identify compounds encoded by the silent biosynthetic gene clusters hidden in the genome.

Abstract:[Objective] This experiment studied the microbial flora in healthy milk and mastitis milk from two dairy farms to explore the similarities and differences of milk flora in different dairy farms, and to assess whether their effects on milk metabolism are the same.[Methods] The 16S rRNA high-throughput sequencing technology was used to determine the 16S rRNA V4 region sequence of microorganisms in the milk of 6 healthy cows and 6 mastitis cows in two dairy farms. The structure and diversity of bacterial community were compared, and the differences in milk flora within and between the two farms were analyzed.[Results] A total of 4013234 Raw Reads were obtained from the four groups of milk samples, and 2887024 Clean Reads were obtained after filtering. No significant differences were found in Chao index, Ace index, Shannon index and Simpson index of Alpha diversity (P>0.05); Four groups of samples with Beta diversity are clustered respectively; In farm 1 and farm 2, the dominant bacteria causing cow mastitis were Klebsiella and Escherichia, respectively; the abundances of Escherichia and Staphylococcus in the healthy milk in farm 2 were significantly higher than those in farm 1; the abundance of Escherichia and Lactococcus in the mastitis milk in farm 2 were significantly higher than those in farm 1; the abundance of Psychrophilic bacteria in the healthy milk of the two dairy farms were 31.87% and 38.72% respectively; correlation analysis and functional prediction analysis showed that there was a significant difference in the relationship between the healthy milk and the dominant species of mastitis in the two dairy farms; significant differences in metabolic pathways were found in both Level 1 and Level 2 in farm 1, while no significant differences in metabolic pathways were found in farm 2.[Conclusion]] This experiment studied the similarities and differences of microflora between healthy milk and mastitis milk in two dairy farms, and provided a theoretical basis for the prevention of mastitis in the two dairy farms and the quality control of raw milk during cold chain transportation.

Abstract:[Objective] The hemopoietic necrosis disease of Carassius auratus gibelio infected by Cyprinid herpesvirus 2 (CyHV-2) has caused huge economic losses to the farming industry of this fish. To reveal the mechanism of CyHV-2 infecting host cells might provide an important basis for the investigations of prevention and control technology for this disease.[Methods] In this study, we designed primers for the epitope rich region of CyHV-2 ORF25B, and amplified truncated ORF25B gene by polymerase chain reaction (PCR). Next, we cloned the amplified product into yeast two hybrid bait expression vector pGBKT7. Then the bait recombinant vector pGBKT7-tORF25B was constructed and we transformed it into yeast Y2H Gold. In addition, we detected the transcriptional self-activation and toxicity of the bait protein on the auxotroph medium. Using yeast two hybrid technique, we hybridized the bait strain pGBKT7-tORF25B/Y2H Gold with GiCB cDNA library.[Results] The truncated ORF25B gene was about 981 bp. It was suggested that the bait strain pGBKT7-tORF25B/Y2H Gold was obtained. And the bait vector was shown to have no toxicity to the yeast cells and no self-activation phenomenon to the report genes. There were four candidate host proteins interacting with tORF25B gene coded protein were preliminarily obtained.[Conclusion]] The results of this study have laid a foundation for further study on the protein function of CyHV-2 ORF25B and the mechanism of this virus invasion into host cells.

Abstract:[Objective] This study aims to investigate the production of alkaline xylanase from alkaliphilic bacterium Cellulomonas bogoriensis 69B4^T; clone the xylanase genes derived from this strain; and perform heterologous expression, purification, and characterization of the enzymatic properties.[Methods] We used the single-factor culture method to study the alkaline xylanase production of the strain and cloned five endo-xylanase genes through the homologous amplification. We expressed recombinant xylanases in Escherichia coli and purified them using the affinity chromatography. The enzymatic properties of purified enzymes were characterized using xylan as substrate.[Results] Five xylanases, i.e., Xyn370, Xyn393, Xyn425, Xyn466, and Xyn486, derived from C. bogoriensis69B4^T achieved heterologous expression in E. coli and obtained pure enzyme components. Their optimal temperature was 60℃, 50℃, 40℃, 40℃, and 60℃, and the remaining enzyme activity was more than 90% when kept at 50℃ for 2 h. The optimum pH for Xyn370, Xyn393, Xyn425, Xyn466, and Xyn486 were 7.0, 8.0, 8.0, 8.0, and 9.0, respectively. The five recombinant xylanases had good stability at a pH range of 5.0-9.0 and showed good tolerance to some metal ions and high salt concentrations, and the enzyme activity on beech xylan was the highest. All xylanases were endo-xylanases.[Conclusion]] The five recombinant xylanases expressed and purified in this paper have excellent salt and alkali resistance and are resistant to temperature, certain metal ions, and chemical reagents, providing novel enzymes for studying the alkali resistance mechanism and the industrial applications of the xylanase source.

Abstract:[Objective] Cytospora canker diseases are among the most important forest diseases, causing devastating economic losses and ecological damage. To understand the genome structure and genetic variation of Cytospora species with different host range, we performed a high-quality genome sequencing of Cytospora piceae and the corresponding comparative genomic analysis. Our study will provide a stepping stone to indicating the molecular mechanism of the interaction between Cytospora spp. and their hosts, and controlling the Cytospora canker disease.[Methods] The draft genome of Cytospora piceae was sequenced by PacBio sequencing technology, and was annotated. The genomic variation, host range determinants and the unique virulence-related gene families of the four Cytospora species were analyzed through comparative genomics tools.[Results] The assembled genome size of C. piceae was 39.25 Mb, with a GC content of 51.79%. The phylogenetic tree based on single-copy orthologue genes showed that C. piceae was closely related to Cytospora chrysosperma, and Valsa mali and Valsa pyri was in the same clade. GC content distribution analysis indicated repeat-induced point mutation activity in all Cytospora species, and C. piceae had the strongest repeat-induced point mutation activity. The carbohydrate active enzymes of all four Cytospora spp. was similar in number. Among plant cell wall degrading enzymes, the auxiliary activity family 3 and 7 related to lignin degradation expanded significantly, while the auxiliary activity family 5, the key enzymes for lignin degradation, was absent in Cytospora spp.. The number of genes in the glycoside hydrolase family 28 and 8 of key enzymes for pectin degradation in the C. piceae and C. chrysosperma genome was similar to that of V. mali and V. pyri. C. piceae and other three Cytospora species all had more major facilitator superfamily transporters and fewer ATP-binding cassette family transporters. In addition, C. piceae contained more Drug:H⁺ Antiporter-2, Pleiotropic Drug Resistance and Multidrug Resistance Exporter transporters, while V. mali contained less Drug:H⁺ Antiporter-1 transporters. Gene Ontology functional classification indicated that the genes of all Cytospora species concentrated on hydrolase activity, V. mali has the highest number of the genes of this class, and the biological processes were mainly related to carbohydrate metabolism, pectin catabolic and oxidation reduction processes. Among the secondary metabolism core genes, C. piceae had fewer polyketide synthases genes than V. mali and V. pyri. Among the four C. piceae specific secondary metabolites genes, three were nonribosomal peptide synthases genes.[Conclusion]] The carbohydrate active enzymes of four Cytospora species were similar in number, which showed strong pectin degradation ability. A complex pattern of presence or absence of nonribosomal peptide synthases genes in the secondary metabolites core genes, and the expanded multidrug transporters of four Cytospora species were observed, which indicated that they are likely to play an important role in host selection of Cytospora species.

Abstract:[Objective] The purpose of this study is to isolate and identify the MAs(III) demethylation strains. We cloned and expressed the arsI gene involved in MAs(III)-demethylation in Bacillus aryabhattai FJ-6 and characterized the ArsI protein.[Methods] The arsI gene fragment (432 bp) was amplified with PCR. Recombinant plasmid pET29a-arsI was constructed and transformed into Escherichia coli BL21(DE3) for heterologous expression, and the expressed protein was detected with SDS-PAGE. The enzyme activity of ArsI was determined by using HPLC-ICP-MS.[Results] The arsI gene was cloned and the recombinant plasmid pET29a-arsI was expressed. The molecular weight of the recombinant protein was 17.4 kDa.[Conclusion]] The activity of ArsI protein with MAs(III) demethylation was clarified.

Abstract:[Objective] This study aimed to investigate pathogenesis of diseased Acrossocheilus fasciatus cultured in Zhejiang province and analyze genetic characteristics of the pathogen.[Methods] We isolated and purified pathogens from diseased A. fasciatus and identified the pathogenic bacteria by analyzing the physiology biochemistry characteristics and 16S rRNA gene sequence. We also carried out artificial infection experiments to verify the pathogenicity of isolated strain to A. fasciatus. Then the genetic characteristics of the pathogen were analyzed by molecular serotyping, multilocus sequence typing (MLST) and PCR screening of surface protein antigen genes and virulence-related genes. Meanwhile, we tested its antimicrobial susceptibility by disc diffusion method.[Results] One predominant strain named ACRO-0708 was isolated from diseased A. fasciatus, which was gram-positive, chain-forming coccus and nonhemolytic, and was identified as Streptococcus agalactiae through the morphological characteristics, phenotypic characteristics and 16S rRNA sequence analysis. In Challenge trials, the strain ACRO-0708 exhibited high virulence in A. fasciatus with an intra-peritoneal LD₅₀ value of 6.47×10³ CFU/g. The strain ACRO-0708 was type of Ib-ST261, and the virulence-related genotype was sip⁺bibA⁺cfb⁺hylB⁺iagA⁺fbsA⁺fbsB⁺bac^-bca^-cylE^-scpB^-lmb^-. However, it did not carry any of the six surface protein antigen genes which were detected. The antimicrobial susceptibility results showed strain ACRO-0708 was susceptible to 8 agents including penicillin, ampicillin and other antibiotics. Meanwhile, it showed resistance to chloroamphenicol, trimethoprim-sulfamethoxazole and other 5 antibiotics.[Conclusion]] Our results demonstrated that S. agalactiae with molecular characteristics obviously different from the main epidemic strains of S. agalactiae in aquatic animals was the causative agent of the enzootic, and the disease could be prevented by using drugs such as penicillin and florfenicol in fisheries. To our knowledge, this is the first report of S. agalactiae as a pathogen of A. fasciatus.

Abstract:[Objective] Completely reconstruction of the biosynthetic pathway of whiE in Escherichia coli, isolation and purification of new compounds synthesized in the recombinant strain for elucidation of the biosynthetic pathway of whiE.[Methods] Construction of single-gene recombinant plasmids of whiE-ORFII, whiE-ORFVII, whiE-ORFI, and SDS-PAGE analysis of the fusion protein expression; isoschizomers Xba I/ Spe Ⅰ were adopted to assemble multiple genes in a single plasmid construct. The recombinant plasmid was then introduced into Escherichia coli BAP1 for heterologous expression, and the fermentation products was detected by high performance liquid chromatography (HPLC); the normal phase silica gel column and the reverse semi-preparative column were used sequentially to separate the fermentation products, the quadrupole high resolution mass spectrometry (Q-TOF MS) was used to identify the molecular weight of the fermentation products.[Results] whiE-ORFII, whiE-ORFVII and whiE-ORFI were solubly expressed; no new product was detected when these three genes were added to the strain BTw95 individually; two compounds ZYC-1 and ZYC-2 can be detected in the strains coupled with whiE-ORFII & whiE-ORFVII, whiE-ORFI & whiE-ORFVII as well as all the three genes. Q-TOF MS analysis in negative ion mode showed that the[M-H]^- of ZYC-1 is 419.0748 with a proposed molecular formula, C₂₃H₁₆O₈ and[M-H]^- of ZYC-2 is 465.0743 with a proposed molecular formula, C₂₄H₁₈O₁₀.[Conclusion]] This study achieved the heterologous reconstruction of the spore pigment whiE biosynthetic pathway in E. coli. Two dodecaketide type II polyketides were identified and isolated. The biosynthetic pathway of the spore pigment whiE was also proposed.

Abstract:[Objective] Bioinformatics methods were used to analyze the whole blood transcriptome data of patients diagnosed with bacterial sepsis from public databases, and we explored the hub genes related to bacterial sepsis and their clinical significance.[Methods] The datasets GSE80496 and GSE72829 were obtained from Gene Expression Omnibus (GEO). GEO2R, gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) were applied to screen out the differentially expressed genes (DEGs) among the patients with bacterial sepsis, when compared to the healthy. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis were also performed on the key genes by R software, hubs genes were obtained by using String 11.0 and Cytoscape subsequently. In addition, the dataset GSE72809 was involved to verify the expression profiles of hub genes in whole blood samples among infants. And the relationships between gene expression and samples' baseline data (such as:gender, gestational age, birth weight, and antibiotic exposure) were also discussed in the study.[Results] 932 DEGs were screened out in GSE80496 dataset, while 319 DEGs were screened out in GSE72829 dataset. We identified 10 hub genes (MMP9, ITGAM, CSTD, GAPDH, PGLYRP1, FOLR3, OSCAR, TLR5, IL1RN and TIMP1). GSEA analysis shows key pathways (amino acid carbohydrate ribose metabolism, PPAR signaling pathway, glycan biosynthesis, autophagy regulatory pathway, complement/coagulation factor cascade reaction, nicotine and nicotinamide metabolism, unsaturated fatty acids biosynthesis and Alzheimer's disease pathway) and biological processes (steroid hormone secretion, adenylate cyclase activation, extracellular matrix degradation and metal ion transport) associated with bacterial sepsis.[Conclusion]] Through the analysis of GEO2R, GSEA combined with WGCNA, our study screened out 2 pivotal modules, 10 hub genes, several signaling pathways and biological processes closely related to bacterial sepsis, which may lay a theoretical basis for further research on the pathogenesis of bacterial sepsis.

Abstract:[Objective] Fast-preparation of effective microorganisms for perishable garbage compost.[Methods] Based on the Environmental Microbial Repository supported by the "Biological Resources Service Network Initiative", we selected excellent organic matters decomposition strains (protein, starch, cellulose, lignin, oil) according to microbial enzyme activity and perishable organic matters. The compost effective microorganisms (CM) were prepared according the antagonism between microbes. Subsequently, we studied the optimal fermentation condition of CM and the perishable garbage compost.[Results] The optimal fermentation condition for CM was inoculating 7% seed liquid in brown sugar medium and culturing for 48 h at 30℃. At this time, the activities of protease, amylase, cellulase, ligninase, and lipase in the fermentation solution were 181.76, 52.92, 1.57, 12.81, 9.94 U/mL, respectively. The pH, moisture content, and organic content of CM compost product meet the bioorganic fertilizer standard (NY 884-2012). The composting temperature of the CM group could reach 63.5℃ and maintain at the high temperature for 9-12 d, at the end of composting, the C/N, moisture content and organic matter degradation rate of CM compost product were 8.93, 28.7%, 30.4%. Compared with the control group, CM could accelerate the degradation of organic matters, and has a stronger nitrogen fixation capacity, shorten the decay time, improve the quality of compost products.[Conclusion]] It is a fast and effective method to prepare the effective microorganisms based on the Environmental Microbial Repository.

Abstract:[Objective] To establish a real-time quantitative method PCR (RT-qPCR) for the detection of Acetilactobacillus jinshanensis in the fermentation of Chinese traditional fermented vinegar and baijiu.[Methods] The specific gene in the Acetilactobacillus jinshanensis genome was screened for the design of PCR-sequence specific primer. The specificity and accuracy of specific primers were verified by PCR with isolated strains and vinegar Pei. The RT-qPCR method was established to analyze the content of Ac. jinshanensis in vinegar, jiupei and cupei from different regions.[Results] We designed a pair of primers with product size of 199 bp, GC content of 55% and T_m value of 59℃ with phenylalanine-tRNA ligase subunit α (encoding pheS gene) of Ac. jinshanensis. The RT-qPCR method has potent specificity, high sensitivity, good repeatability and versatility, and its detection range is 2.24-10.24 lg(copies/μL). The method can be applied for the detection of Ac. jinshanensis in the fermentation of cupei and jiupei from different regions.[Conclusion]] The RT-qPCR method can be used for the specific identification and accurate quantification of Ac. jinshanensis in the fermentation of vinegar and baijiu.

Abstract:[Objective] We aimed to perform and optimize the recombinant secretory expression of neutral endo-β-1,4-xylanase derived from Bacillus pumilus in Bacillus subtilis WB800 on the basis of important role of signal peptide and signal peptidase.[Methods] We amplified the full-length neutral endo-β-1,4-xylanase gene from B. pumilus genomic DNA and then ligated to the downstream of the P₄₃ promoter in the pWB980 vector. Thereafter, the recombinant vector was transformed into B. subtilis WB800 to construct the recombinant strain NZ-X. Signal peptides were screened among 23 signal peptides that were amplified from B. subtilis 168 genomic DNA. On this basis, we constructed two strains that overexpress two signal peptidases (SipS and SipT), respectively. And the effect of these two signal peptidases on the secretion of endo-β-1,4-xylanase was investigated.[Results] Neutral endo-β-1,4-xylanase was successfully secreted from the recombinant strain NZ-X and the enzyme activity in supernatant was 5.33 U/mL via shake flask fermentation. The results of signal peptide screening indicated that five signal peptides (YlaE, YfhK, EglS, YqxI, YpjP) were effective, and the enzyme activities were 7.15, 6.69, 6.36, 6.32, 6.18 U/mL, respectively. Among these signal peptides, the secretion of endo-β-1,4-xylanase was promoted mostly by SipS signal peptidase when fusion with YfhK signal peptide. The enzyme activity could be increased to 10.64 U/mL, which was 1.99 times than that of NZ-X.[Conclusion]] The secretion of heterologous proteins in B. subtilis could be improved effectively through signal peptide optimization and signal peptidase overexpression.

Abstract:[Objective] Giant panda is one of the first-class protected animals in China, which the population is facing continuous threats such as infectious diseases and habitat fragmentation. Bacterial infection and imbalance of bacterial community in the genitourinary system of giant panda could affect the reproductive health, in severe cases, it can also lead to abortion, which is one of the causes of reproductive disorders in giant panda. In this study, our aim was to examine the bacterial community composition of semen and prepuce secretion and isolate potential pathogenic bacteria of giant panda.[Methods] 13 of prepuce secretions and 12 of semen samples of giant panda were used to determine the bacteria species by 16S rRNA amplicon sequencing technique, bacterial separation and PCR methods.[Results] The results showed that the abundance of Firmicutes was the highest in the samples of prepuce and semen of giant panda at phylum level. Moreover, at the genus level, the bacterial community composition of prepuce was changed in different periods, Corynebacterium and Dolosicoccus were the most abundant microbiome in preputial samples at phase I with relative abundances of 15.45% and 12.40%, respectively. Streptococcus and Escherichia were the most abundant microbiome in preputial samples at phase II, with relative abundance of 37.94% and 9.68%, respectively. Bacteroides and Prevotella were the most abundant microbiome in seminal samples, with relative abundance of 14.40% and 12.88%, respectively. The results of bacterial diversity showed that the diversity in seminal samples was higher than that in preputial samples; there were no significant difference between preputial samples at stages I and stage II. Klebsiella pneumonia and other potential pathogenic bacteria were obtained by bacterial isolation and culture.[Conclusion]] In this study, we analyzed the bacterial community composition of the prepuce secretions in different periods and semen of male giant pandas, and the dominant microbiome were different. It was hypothesized that potential pathogenic bacteria were in the prepuce and semen of giant pandas, which may pose a threat to the reproductive system health of giant panda, and the pathogenicity needs further research.

Abstract:[Objective] The aim of this study is to obtain efficient nitrogen-fixing strains and fully utilize soil nitrogen-fixing microbial resources.[Methods] The Entisol with superior nitrogen fixation capability was selected to isolate nitrogen-fixing strains via enrichment and purification procedure. The identification of newly isolated strain at species level was performed by means of 16S rRNA gene phylogenetic analysis and whole genome correlation index comparison. Quantitative determination of nitrogen fixation activity of newly isolated strain was performed by acetylene reduction assay and ¹⁵N₂ tracer method. The plant growth promotion potentials of the nitrogen-fixing strain were preliminarily determined through culture characteristics and inoculation effects.[Results] A strain, designated P208, which could grow rapidly in nitrogen-free medium, was isolated from Entisol. Phylogenetic analyses based on 16S rRNA gene and 92 core genes revealed that strain P208 was most phylogenetically closely related to Azotobacter chroococcum IAM 12666^T (=ATCC 9043^T) (99.79% 16S rRNA gene similarities). The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values based on genome sequences of strain P208 and A. chroococcum ATCC 9043^T were higher than the thresholds for species circumscription (ANI>95%-96%, AAI>95%-96%, dDDH>70%), and the maximal unique matches index (MUMi) value was lower than the species circumscription threshold (<0.33). Therefore, The newly isolated strain P208 could be identified as A. chroococcum. The nitrogen fixation activity of A. chroococcum P208 was 2.61 times that of the type strain A. chroococcum ATCC 9043^T. In addition to nitrogen fixation, A. chroococcum P208 had plant growth promotion potentials in cultures, such as IAA production, phosphate solubilization and siderophore production. Inoculation with A. chroococcum P208 could promote root growth of rice and wheat seedlings under controlled conditions.[Conclusion]] A strain with superior nitrogen fixation and plant growth promotion potential was isolated and purified from the paddy soil with superior nitrogen fixation capability. It has relatively good development and application potential value, and could provide microbial resource for the study and utilization of biological nitrogen fixation.

Abstract:Pasteurella, mainly Pasteurella multocida, can cause a variety of diseases (pasteurellosis) and also human infection and diseases.[Objective] The aim of this work was to characterize the adhesion activity of Pasteurella glycolytic enzymes to host cells (rabbit kidney cells) and two common host molecules, plasminogen (Plg) and fibronectin (Fn).[Methods] The glycolytic enzymes of P. multocida were expressed by prokaryotic expression system, then purified, and their polyclonal antibodies were prepared. The adhesion activity of P. asteurella glycolytic enzymes was studied by cell surface localization detection, adhesion and adhesion inhibition experiments.[Results] Seven glycolytic enzymes except enolase and pyruvate kinase were found on the surface of P. multocida cells. The seven glycolytic enzymes all could adhere to rabbit kidney cells, but only PGI polyclonal antibodies could inhibit the adhesion of P. multocida. Far Western blotting showed that all nine glycolytic enzymes could bind to the host Plg and Fn. The recruitment inhibition experiments showed that the antibodies of phosophoglucose isomerase, aldolase and phosphoglycerate mutase could inhibit the binding of P. multocida to both Plg and Fn, while the antibodies of phosphofructokinase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase could only inhibit the binding of P. multocida to either Plg or Fn.[Conclusion]] The phosophoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and phosphoglycerate mutase of P. multocida play an important role in the adhesion of P. multocida to host cells or molecules. The completion of this study will deepen the understanding of molecular pathogenesis of Pasteurellosis, and provide basic data for new diagnostic markers, new vaccines, and new drug targets.

Abstract:[Objective] Isolation plant growth promoting rhizobacteria (PGPR) from disease-resistant tomato rhizosphere soil to control bacterial wilt caused by Ralstonia solanacearum and improve tomato growth, and explored the mechanisms.[Methods] PGPR bacteria with antibacterial activity was screened from tomato rhizosphere soil by inhibition zone method using a moderately pathogenic Ralstonia solanacearum HN4 as target; The highly similar strains were identified by BOX-PCR fingerprints, and the enzyme-producing activity of different genotype strains was evaluated by the plate transparent circle method; The 16S rRNA phylogenetic analysis was applied to identify the antagonistic strains with strong antibacterial ability and many kinds of enzyme production; The potential antagonistic bacteria were assessed for plant disease prevention and growth promotion in greenhouse, and the plant disease prevention and growth promotion related genes expression level were tested by real-time PCR.[Results] 29 strains were isolated and purified from tomato rhizosphere soil, of which 15 strains had antagonistic function against Ralstonia solanacearum. Moreover, four strains involved in controlling tomato bacterial wilt and promoting growth, were verified by the analysis of BOX-PCR fingerprinting and enzyme activity. In the result of 16S rRNA phylogenetic analysis, strain B2 was identified as Bacillus amyloliquefaciens. Strain B5 and B20 were identified as Bacillus subtilis, respectively. Strain B23 was identified as Bacillus velezensis. In greenhouse experiment, strain B2, B5, B20 and B23 inhibited Ralstonia solanacearum effectively, with the efficiency of 35.59%, 8.47%, 32.20% and 96.61%, respectively. Furthermore, all of them significantly increased the plant fresh weight, the total chlorophyll content and the number of root tips. The strain B2, B5 and B23 efficiently promoted plant height and root length of tomato. The strain B2, B20 and B23 efficiently promoted stem thickness of tomato. And strain B23 efficiently promoted the number of root forks. In the result of real-time PCR, strain B2, B20 and B23 upregulated the expression of PR1α and POD1 gene. Strain B2, B5 and B23 upregulated the expression of ctd1 gene. And strain B2, B5, B20 and B23 downregulated the expression of ERF2 gene.[Conclusion]] In this study, four biocontrol strains were isolated and identified to control tomato bacterial wilt and promote tomato growth, and can provide a theoretical basis for the targeted screening of plant growth-promoting and disease preventing bacteria.

Abstract:[Objective] To explore the molecular dynamics changes of cell division cyclin protein Cdc5 in budding yeast during mitosis at different temperatures.[Methods] In this study, Saccharomyces cerevisiae was used as the experimental material objective to explore the molecular dynamic changes of Cdc5 protein in the mitosis process of budding yeast at different temperatures by using living cell imaging method; draw the growth curve by measuring OD₅₉₅ to see whether the macro division is consistent with the micro dynamic changes of Cdc5 protein; use flow cytometry to detect the cell cycle changes.[Results] During cytokinesis, Cdc5 protein entered into daughter cells from mother cells and aggregated at bud neck. The aggregation time of Cdc5 protein at the bud neck was long at 25℃, and the aggregation time of Cdc5 protein at the bud neck was short at 37℃. There was a significant difference between them. However, there was no significant difference in the expression of Cdc5 protein between the two temperatures. At the same time, the temperature also affected the dynamics of Cdc5 protein during degradation process, including the occurence of frequency and time of the peak of fluorescence intensity of Cdc5 in mother cells and daughter cells. The growth curve results showed that the single cell division cycle of budding yeast affected its macroscopic cell growth, and the faster the division rate of budding yeast, the smaller the ratio of length to width of the daughter cell. The cell cycle results showed that the dynamic changes of Cdc5 protein at 37℃ were consistent with the cell cycle changes of budding yeast. The cell cycle results showed that the cell cycle of budding yeast changed from G₀/G₁ phase to S phase at 37℃, which also accelerated the division of budding yeast.[Conclusion]] This study was to explore the molecular dynamics of Cdc5 protein in the mitosis of budding yeast at different temperatures and the corresponding macro growth of budding yeast for the first time. The results showed that the temperature had an effect on the dynamics of Cdc5 protein, and its molecular dynamics was positively correlated with the division speed of budding yeast, which provided a basis for further study of its function in mitosis.

Abstract:[Objective] The purpose was to explore the effect of porcine cathepsin S (cathepsin S, CTSS) on SVV (Seneca Valley virus, SVV) replication.[Methods] IBRS-2 cells were infected with SVV, and the regulation of endogenous CTSS expression by SVV infection was investigated by RT-qPCR at transcriptional levels. The effect of SVV infection on CTSS enzyme activity was determined by ELISA. The regulatory effects of overexpressed CTSS on SVV replication and antiviral cytokines induced by SVV were detected by Western blotting (WB) and RT-qPCR. The specific siRNA for CTSS was synthesized and the interference effect of siRNA on CTSS and the effect of CTSS interference on SVV replication were detected by WB and RT-qPCR.[Results] The expression of endogenous CTSS and the activity of CTSS enzyme were markedly up-regulated in IBRS-2 cells infected with SVV. SVV replication in IBRS-2 cells was significantly inhibited and the expression of host antiviral cytokines host antiviral cytokines was up-regulated by CTSS overexpression. siRNA-2947 down-regulate the expression of endogenous CTSS to promote SVV replication.[Conclusion]] CTSS inhibits SVV replication by enhancing the up-regulated expression of host antiviral cytokines. This study provided the reference basis for further exploring the role and mechanisms of host CTSS in anti-SVV innate immune response.

Abstract:[Objective] The aim of this study was to investigate the underlying mechanism for the regulation of protein disulfide isomerase (PDI) in protein folding and expression of exogenous proteins in yeast by co-expression of yeast PDI and its substrate protein chicken cystatin (cC). RNA-seq was carried out to screen for genes that are differentially expressed in Pichia pastoris (P. pastoris) and identify the key genes that could affect the expression of cC. This might provide theoretical support for the mechanistic analysis of high-level expression of exogenous protein and the construction of high-yield heterologous protein-expression strains.[Methods] PDI coding gene was transferred into GS115 and GS115 cC strains for the overexpression of PDI. RNA-seq was carried out to study the transcription of gene differences of two recombinant P. pastoris strains. Combined with the result of KEGG annotation, the data was analyzed to identify the differentially expressed genes between control and PDI/cC co-overexpressed cells, the result were verified by qRT-PCR and the functions in protein expression regulation were also clarified.[Results] The expression of cC was significantly increased in the PDI-overexpressing strain. A total of 373 differentially expressed genes were screened by RNA-seq analysis. Among them, 122 differentially expressed genes were assigned to the KEGG biological pathways, including 12 genes annotated into protein transport and catabolism pathway, 21 genes related to the protein folding sorting and degradation pathway, and 24 genes involved in the protein translation pathway.[Conclusion]] Overexpression of PDI in P. pastoris significantly increased the expression of the heterologous amyloidogenic protein cC. By analyzing the expression profiles of cC-overexpressed normal P. pastoris cells or PDI-overexpressed strain that co-overexpressed cC, some of the genes with significant transcriptional changes were preliminarily identified, laying a foundation for the transformation of yeast strains with high expression of amyloid protein.

Abstract:[Objective] To research the possibilities of biodegradation of brominated flame retardants by Irpex lacteus F17, we studied the characteristics and influence factors of aerobic degradation of tetrabromobisphenol A (TBBPA), and the degradation pathway was speculated based on the analysis of intermediates.[Methods] We determined the concentration of TBBPA during biodegradation by high-performance liquid chromatography (HPLC), and detected the intermediate products by gas chromatography-mass spectrometry (GC-MS).[Results] TBBPA could be biodegraded by I. lacteus F17 via co-metabolism, and glucose was confirmed to be the most appropriate co-metabolic matrix. Under the optimal degradation conditions which were 8 g/L glucose, 5% inoculum volume, pH 5.0, the degradation rate and the debromination rate were 85.5% and 14.6%, respectively, for 20 mg/L TBBPA. It was found that the degradation rate of TBBPA was related to the activity of manganese peroxidase (MnP). On the basis of GC-MS analysis, we detected seven intermediate products.[Conclusion]] I. lacteus F17 could degrade TBBPA effectively, and debromination, β-scission, hydroxylation, deprotonation, and oxidation were involved in the degradation mechanism of TBBPA by Irpex lacteus F17.