Abstract:Secondary metabolites including β-lactam antibiotics, flusidic acid, cyclosporine and statins are produced by several groups of filamentous fungi. Secondary metabolism, growth and development process of filamentous fungi are regulated by global regulatory factor and specifically regulating factors, such as LaeA and velvet family. This review elaborates the structural features of the Velvet family proteins (VeA, VelB, VosA, VelC), analyzes the regulation of the sexual and asexual development and the secondary metabolism, and discusses possible molecular mechanisms. These results might provide a significant reference to reveal fungi regulatory networks and activate the silencing gene to produce novel compounds.

Abstract:Lignocellulose is a cheap, abundant and underutilized bioresource. Cellulases can break down cellulose into glucose that could further be fermented to bioethanol, thus has commercial potentials. The improvement of cellulase production helps the use of the abundantly available lignocellulose, benefits the further exploration of the potential of bioenergy, and assists the relief of the energy crisis. However, glycosylation has an important influence on catalytic activity, thermal stability and other properties. Therefore, the detailed knowledge on cellulase glycosylation is required. Reasonably maniputing cellulase glycosylation modifications can accelerate the hydrolysis of lignocellulose, and contribute to produce liquid biofuels.

Abstract:Bacterial lipoproteins are important components of cell membrane. They play important roles in bacterial physiology and virulence in gram-negative bacteria. The Lol (localization of lipoprotein) pathway discovered from E. coli is responsible for the transport of lipoprotein in gram-negative bacteria. Recent research found that surface-exposed lipoproteins are widespread in gram-negative bacteria. To better understand the current studies of lipoprotein secretion mechanisms in gram-negative bacteria, we reviewed functions and conservation of five Lol proteins, the difference of lipoprotein secretion signals in variant bacteria and possible transport mechanisms of surface-exposed lipoproteins.

Abstract:[Objective] Alanine dehydrogenase gene (ald) of Arthrobacter ureafaciens CZ31 was cloned and transformed into Escherichia coli Rosetta (DE3) to construct engineering bacteria CZR07 with soluble expression of ald, and the conditions of alanine dehydrogenase(AlaDH) production were optimized.[Methods] Genomic DNA of Arthrobacter ureafaciens CZ31 was extracted; A pair of specific primers was designed to obtain the ald gene, and then subcloned into expression plasmid, pET-28a-ald, and expressed in E. coli Rosetta. The recombinant protein, AlaDH, was purified by Ni²⁺ chromatography. Response surface methodology was used to optimize fermentation conditions.[Results] The length of ald gene was 1119 bp, encoding 372 amino acid residues, molecular weight of the target protein was 40 kDa according to SDS-PAGE analysis. Specific enzyme activity of the recombinant enzyme was 2.65 U/mg. The optimal induction conditions were:22℃, isopropyl-β-D-thiogalactoside 0.7 mmol/L, induction time 8 h. The specific activity of the enzyme was 15.23 U/mg under optimized conditions, about 5.75 times of the initial.[Conclusion] We optimized the induction conditions of the recombinant enzyme production through Box-Benhnken Design, and achieved the desired results, which provided a reference for the optimization of other genetic engineering bacteria.

Abstract:[Objective] To study the mechanism of HBx regulating the transcription of insulin growth factor binding protein 3 (IGFBP3).[Methods] RNA-sequencing method was used to screen differently expressing genes in HepG2 and HBV transgenic cell line HepG2-4D14. The mRNA of IGFBP3 was measured by reverse transcription and real-time PCR. To verify the activity of IGFBP3 promoter, cells were analyzed by luciferase assay. The binding of p53 and IGFBP3 promoter was measured by ChIP Assay.[Results] The level of IGFBP3 mRNA in HBV transgenic cell line HepG2-4D14 was significantly lower than that in HepG2 cells. The data of real-time PCR indicated that HBV HBx can down regulate the transcription of IGFBP3. By taking the approach of promoter luciferase assay on HCT116 and HCT116-p53^-/- cell lines, we found that HBx can inhibit the promoter activity of IGFBP3 in a p53-dependent manner. Our data also showed that HBx can significantly interfere with the binding of p53 to the promoter of IGFBP3. As IGFBP3 is a suppressor for cell growth, we postulate that HBx promotes the cell proliferation by reducing the level of IGFBP3.[Conclusion] HBx can inhibit the transcription of IGFBP3 in a p53-dependent manner.

Abstract:[Objective] To identify strain QTYC38, a fungus isolated from the gut of Pantala flavescens larvae, and study its phytotoxic and antimicrobial metabolites.[Methods] QTYC38 was identified by morphological observation and molecular biological analysis. Growth rate method and agar disc diffusion assays were used to test the antimicrobial activities. Petri-dish bioassay was used to test the phytotoxic activity. Bioactive components were isolated via chromatographic methods, and the structures were determined by mass spectrum and nuclear magnetic resonance analyses.[Results] QTYC38 was identified as Neosartorya aureola. Ethyl acetate extract of QTYC38 inhibited radical growth of Echinochloa crusgalli and Amaranthus retroflexus with the inhibition rates above 65% with the concentration of 100 μg/mL, The extract also inhibited Staphyloccocus aureus with the mean halo diameter of 22.7 mm with the concentration of 30 μg/filter. Four compounds were purified from the solid fermentation product:helvolic acid, aromatic lactones, questin and erogosterol. Helvolic acid inhibited the growth of Bacillus subtilis and S. aureus with the MIC values of 3.1 and 1.5 μg/mL, respectively. Questin and erogosterol inhibited Dothiorella gregaria and Fusarium graminearum at the concentration of 100 μg/mL, with the inhibition rates of 52.4% and 71.3%, respectively.[Conclusion] Strain QTYC38 could be potentially developed as a microbial herbicide and new antimicrobial agent.

Abstract:[Objective] The objective of this study was to screen lignin-degrading fungi and study their lignin-degrading enzyme using corn stover as substrate.[Methods] Lignin-degrading fungi were isolated from decayed corn stover from different latitude and longitude of Jilin Province by using guaiacol and aniline blue culture medium. Strains were identified by morphological screening followed by the phylogenetic analysis of ITS sequences deciphering their taxonomic status. Through the analysis of the activity of extracellular ligninase produced during the solid-state fermentation of corn stover, the most efficient stover-degrading fungi were selected.[Results] A highly efficient corn stover degrading fungus was isolated and named as W2 (Irpex lacteus W2). Manganese peroxidase produced after 4 to 8 days showed an ascending trend, and reached the peak value of 86.31 U/mL at 8 d, which was 88.20% higher than that of Phanerochaete chrysosporium (P<0.01). Laccase activity of this fungus was higher than that of Phanerochaete chrysosporium (45.86 U/mL) and reached 20.60 U/mL at 8 d, which was 40.76% higher than the control (P<0.05).[Conclusion] An efficient corn stover degrading fungus was isolated and identified as Irpex lacteus W2, with high activity of peroxidase and laccase during the degradation process.

Abstract:[Objective] To prepare mutants of Bacillus amyloliquefaciens JY06 stain with enhanced arginine consumption capability to reduce carcinogenic compound ethyl carbamate and its precursors in soy sauce.[Methods] Atmospheric and room temperature plasma (ARTP) and UV mutagenesis were used to mutate Bacillus amyloliquefaciens JY06, and a high-throughput screening approach was used to characterize mutants with high arginine utilization capability. The properties of mutants were studied following addition to soy sauce moromi during fermentation to initiate reduction of citrulline and ethyl carbamate.[Results] Twelve mutants with elevated arginine utilizing ability were obtained through ARTP and UV mutagenesis, of which C12 and E6 displayed the highest capacity for eliminating EC and citrulline. Compared with the JY06 parent strain, the addition of C12 or E6 to the moromi during soy sauce fermentation decreased citrulline content in soy sauce by 15.6% and 14.7%, and reduced ethyl carbamate by 19.3% and 13.1%, respectively.[Conclusion] Both ethyl carbamate and its precursor citrulline were significantly decreased in soy sauce by B. amyloliquefaciens JY06 mutants in the moromi during fermentation, demonstrating the potential of the mutants to control or eliminate toxic compounds in soy sauce and similar food products.

Abstract:[Objective] To understand the number of ADP-ribosylation factor (ARFs) and their roles in Beauveria bassiana.[Methods] ARFs were identified by BLASTp searching against the database of B. bassiana proteins and analyzed using molecular phylogenetics. Role of an ARF in fungal growth, stress responses and virulence was characterized by analysis of the gene transcription pattern, and investigation of the genetically modified B. bassiana strains.[Results] At least six ARFs and ARF-like proteins (ARFLs) were identified in B. bassiana, which were distributed in different groups of yeast Saccharomyces cerevisiae and human Homo sapiens ARFs and ARFLs. One of ARFs, BBA_01574, designated BbarfA, was clustered in the group of human ARF3, ARF4 and ARF5. Transcription levels of BbarfA were obviously higher in the mature conidia or during the isotropic growth (swelling) phase of conidia than those during germ tube elongation phase. Antisense inhibition of BbarfA accelerated conidial germination and resulted in an increase in fungal virulence, whereas overexpression of BbarfA and the gene with site-mutation in GTP-binding sequences (BbarfA^Q71I) caused the opposite phenotypes. Although expression of the gene was induced by high salt, osmotic, oxidative and high temperature stresses, no obvious difference was noted in sensitivities to these adverse stresses in all the genetically modified transformants, which included strains suppressing (by antisense inhibition) or overexpressing BbarfA, overexpressing the genes with site-mutation in GTP-dissociating (BbarfA^D26G) or GTP-binding (BbarfA^Q71I) sequences, and the wild type strains.[Conclusion] ADP-ribosylation factor, BbarfA, was involved in conidial germination and virulence in B. bassiana.

Abstract:[Objective] To develop an FLP/FRT system for gene disruption in Candida amazonensis that can repeatedly use a single selectable marker, and to verify the effectiveness of this system by deleting the PDC gene encoding pyruvate decarboxylase.[Methods] Four promoters (SpXYLp, SpMAL6p, SpMAL1p and SpGAL1p) from Spathaspora passalidarum and ScGAL1p promoter from Saccharomyces cerevisiae were amplified and fused to the reporter gene of green fluorescent protein (gfpm) to study the regulation under corresponding inducible conditions. A strictly inducible promoter was selected to control the expression of the C. amazonensis-adapted FLP gene (caFLP), encoding the site-specific recombinase FLP. The promoter-caFLP fusion fragment was used to ligated with the hphm marker gene that conferred resistance to Hygromycin B, and the ligation product was flanked by direct repeats of the FLP recognition target (FRT). Then with the addition of the homologous arms, we constructed the PDC deletion cassette (PRFgHRP). The cassette was transformed into C. amazonensis CBS 12363 and transformants with hphm were derived. When the transformants were incubated into inducible medium, FLP-mediated recombination resulted in the deletion of DNA located between the repeats.[Results] SpMAL1p (induced by maltose) and SpGAL1p (induced by galactose) were identified to be strictly inducible promoters. SpGAL1p was used to regulate the expression of the FLP, and the PDC deletion cassette (PRFgHRP) was constructed and transformed into C. amazonensis successfully. After selection of Hyg-resistant transformant (designated as C. amazonensis PDC01) in which the deletion cassette was inserted into the PDC target gene, FLP expression was induced by growth of the transformant in galactose-containing medium, and Hyg-sensitive transformant in which hphm and caFLP flippers were excised from the genome was obtained, designated as C. amazonensis PDC02.[Conclusion] It is the first time to construct an FLP/FRT system for gene disruption in C. amazonensis, and we obtained a PDC mutant without resistant marker gene successfully through this system. These research results lay a good foundation for further metabolic engineering of C. amazonensis.

Abstract:[Objective] To obtain oligomers of (S)-carbonyl reductase Ⅱ with strong activity and stability, we used sortase A from Staphylococcus aureus as molecular "stapler" to conjugate (S)-carbonyl reductase Ⅱ. The (S)-carbonyl reductase Ⅱ oligomers efficiently catalyzed the biotransformation of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol.[Methods] We cloned sortase A gene from S. aureus genome and expressed it in Escherichia coli. The recombinant enzyme was purified through Ni-affinity and gel filtration chromatography. Meanwhile, we added GGGGSLPETGG to C terminus of (S)-carbonyl reductase Ⅱ using genetic techniques, and purified recombinant SCRⅡ-GGGGSLPETGG to homogeneity. We determined the optimal reaction conditions of sortase A-mediated ligation of (S)-carbonyl reductase Ⅱ to oligomers. The enzyme characteristics of the generated oligomers were studied. And the oligomers-catalyzed biotransformation efficiency of (S)-1-phenyl-1,2-ethanediol was further detected.[Results] Oligomers showed a specific activity of 38.5 U/mg, 6-fold increase compared to (S)-carbonyl reductase Ⅱ. The optimal temperature and pH of ligation reaction by oligomers were 35℃ and 6.0 respectively. The relative activity was maintained over 90% at 50℃ for 1 hour. Denaturation test showed that the denaturation temperature of oligomers was 60.1℃, 10℃ higher than that of (S)-carbonyl reductase Ⅱ. Biotransformation results indicated that oligomers completely transformed 5 g/L 2-hydroxyacetophenone within 3 hours to generate (S)-1-phenyl-1,2-ethanediol with an optical purity of 100%. With oligomers, we reduced transformation duration for 16 folds compared to that of recombinant E. coli-(S)-carbonyl reductase Ⅱ cells.[Conclusion] This work first described sortase A-mediated the ligation of oxidoreductase and significantly improved catalytic efficiency and thermal stability of enzyme, suggesting sortase had great potential application in chiral synthesis.

Abstract:[Objective] RNA-seq technology was used to sequence the larval guts of Apis cerana cerana under stress of Ascosphaera apis. Subsequently, trend was analyzed for differentially expressed genes (DEGs) to obtain significant gene expression patterns, followed by transcriptome analysis of A. apis stressing the larval gut.[Methods] Infected honeybee larval guts were sequenced at Illumina HiSeq 2500 platform and in-depth analyses were done using corresponding biological software. Finally, RT-qPCR was conducted to validate RNA-seq data.[Results] A total of 41133932 high-quality clean reads were obtained. Trend analysis result showed that 22865 DEGs were grouped into 8 gene expression patterns, among them 16769 DEGs were assigned to 4 significant expression patterns including 2 up-regulated trends and 2 down-regulated trends. GO enrichment analysis result showed that all DEGs within significant up-and down-regulated patterns were enriched in 40 and 37 GO terms, respectively, and the mostly enriched one is cellular process (2486 unigenes). KEGG enrichment analysis result displayed that the DEGs within significant up-and down-regulated trends were enriched in 119 and 112 pathways, respectively, and biosynthesis of amino acids (127 unigenes) and ribosome (98 unigenes) were mostly enriched. A. apis facilitated its proliferation through enhancing the biosynthesis and the host could fight A. apis by inhibiting the protein synthesis of the fungal pathogen during the stress process. Furthermore, expression levels of 11 DEGs enriched in the pathogen's MAPK signaling pathway decreased when the stressing time of A. apis was prolonged, suggesting that A. c. cerana larvae could constrain the pathogen's replication by disturbing this pathway.[Conclusion] This is the first report of transcriptome investigation of A. apis infecting A. c. cerana larvae. Our data provide gene expression profiles of A. apis stressing the larval gut of A. c. cerana, as well lay the foundation of unraveling molecular mechanisms regulating the pathogenesis of A. apis.

Abstract:[Objective] We isolated, identified and characterized lactic acid bacteria (LAB) from the intestinal digesta, mucosa and feces of piglets, to explore the potential probiotic stains for the swine production.[Methods] A total of 155 lactic acid bacteria were isolated. Four isolates with high lactate production were identified by conventional biochemical method and bacterial 16S rRNA sequence, and their potential probiotic properties were assessed.[Results] Four LAB isolates (L45, L47, L63 and L79), Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus johnsonii and Enterococcus facium were identified based on lactate production at 8 h (> 100 mmol/L) and final pH at 90 h (< 3.9). The 4 isolates grew rapidly in vitro. They tolerated low pH and bile salt. Strains L47 and L79 tolerated pH 2.5, and L47 tolerated 0.5% bile salts. When the 4 isolates were co-cultured with E. coli K88 and Salmonella, the growth of E. coli K88 and Salmonella was inhibited. L47 demonstrated stronger inhibition effect than other 3 isolates.[Conclusion] L47 strain presented a higher lactate production, better growth, higher tolerance against pH 2.5 and 0.5% bile salt, and stronger antimicrobial effect on E. coli K88 and Salmonella, indicating that L47 isolate may have the potential probiotic properties for swine production.

Abstract:[Objective] To study the effect of Nosema bombycis on the apoptosis of BmN cells and the expression level of BmIAPs under the condition without external apoptosis-inducing factor.[Methods] We observed the change of BmN cells at different times by using microscope after inoculation with N. bombycis. The relative expression level of BmCyt c in the N. bombycis infected BmN cells was detected by using real-time PCR. We searched databases of silkworm genome and Pfam finding information of Bombyx mori inhibitor of apoptosis proteins (BmIAPs). Real-time PCR was used to demonstrate the relative expression level of BmIAPs in N. bombycis infected BmN cells.[Results] Compared with the normal BmN cells, BmN cells infected by N. bombycis had no obviously change at 5 d post-inoculation. The growth of BmN cells was influenced by N. bombycis at 7 d post-inoculation. The vacuolization and apoptosis were found in most BmN cells at 12 d. However, the phenomenon was not found in N. bombycis infected BmN cells. Moreover, the relative expression level of BmCyt c was suppressed in N. bombycis infected BmN cells at most of the time, except for 7 d. The expression of BmCyt c was lowest compared with control groups (P<0.01) at 10 d and 12 d post-inoculation. Through searching the databases, we obtained four BmIAPs, including BmIAP-1, BmIAP-2, BmSurvivin-1 and BmSurvivin-2. The results of real-time PCR indicated that the relative expression level of BmIAP-1 and BmSurvivin-1 had up-regulating tendency at 10 d and 12d post-inoculation, especially at 12 d post-inoculation. But, BmIAP-2 and BmSurvivin-2 had down-regulated expression at most times.[Conclusion] Under the condition without external apoptosis-inducing factor, growth and apoptosis of BmN cells can be influenced by N. bombycis. According to the results of real-time PCR, we speculated that BmIAP-1 and BmSurvivin-1 may play a certain role in regulating the apoptosis of BmN cells infected by N. bombycis.

Abstract:[Objective] To highlight the significance of nitrite reductase in wastewater denitrification and shortcut nitrification, we studied wastewater denitrification by Escherichia coli expressing nitrite reductase.[Methods] Nitrite reductase was overexpressed by transformed a recombinant plasmid with nir gene in E. coli. Expression of nitrite reductase and metabolism of nitrite were studied by detecting the products in the whole cell of recombinant E. coli. To evaluate the function of recombinant E. coli used for shortcut nitrification, we measured the denitrification efficiency of the mixture consisting of the recombinant E. coli and YF14 that was a nitrifying and denitrifying bacterium.[Results] The recombinant E. coli could express nitrite reductase, reduced about 1 mmol/L nitrite within 2 hours and produced almost equal amounts of nitric oxide (OD₆₀₀=2.0 bacterial suspension). When the recombinant E. coli and YF14 were mixed in equal proportions, the ammonia nitrogen removal efficiency increased by about (36.0±7.4)% within 12 hours, and the maximum accumulated amount of nitrite decreased by about 37%. Recombinant E. coli (OD₆₀₀=1.0) significantly improved the ammonia nitrogen removal efficiency of activated sludge by about (31.0±5.7)% within 12 hours, and no nitrite and nitrate were detected. Oxygen supply affected ammonia nitrogen removal with optimal DO of (6.4±0.7) mg/L.[Conclusion] Recombinant E. coli with nitrite reductase can contribute to the shortcut nitrification capacity of wastewater.

Abstract:[Objective] In order to explore the evolutionary relationship and origin of natural plasmid of Lactobacillus plantarum.[Methods] We analyzed phylogenetic relationships and origins of 75 natural plasmids of L. plantarum by replication initiation protein (Rep) phylogenetic tree, genomic collinearity, genomic GC content and host range.[Results] Rep phylogenetic tree and genomic collinearity analysis simultaneously showed that all natural plasmids of L. plantarum could be divided into 6 families with close evolutionary relationships, 2 complex plasmids with special evolution form and 1 independent evolution plasmid pLP2140. The complex plasmids pMRI5.2 and pLP12-1 were fused by 2 different plasmids of the family 1-2 and family 5-6. Therefore, the natural plasmids of L. plantarum could be originated from 7 ancestors. The genomic collinearity analysis showed that the 6 family plasmids could be further divided into 17 subfamily groups with closer evolutionary relationships, and the phylogenetic relationships among plasmids could be clearly and effectively revealed at the subfamily level. Finally, the analysis of genomic GC content and host range provided further evidence for the phylogenetic relationship and origin of the natural plasmid of L. plantarum.[Conclusion] Therefore, the above research can accurately and effectively reveal the phylogenetic relationship and origin of the natural plasmids of Lactobacillus plantarum, which is of great reference value for the acquaintance and study of the evolution and origin of the natural plasmids of Lactobacillus plantarum. We proposed a more effective research strategy and analytical method of the evolution and origin of natural plasmids by comparing and combining the characteristics of the analytical methods of Rep phylogenetic tree and genome collinearity, and this method may be applicable to all bacterial natural plasmids, so it may have universal methodological significance for the study of the evolution and origin of natural plasmids.

Abstract:[Objective] Lipopeptides produced by Bacillus licheniformis FJAT-4 can strongly inhibit the mycelia growth of Fusarium oxysporum. The aim of this study was to identify the structure of antifungal lipopeptide, analyze the effect of culture medium and temperature on the antifungal lipopeptide production, and explain antifungal effect of lipopeptide against F. oxysporum. This study is helpful to understand the mechanisms of Bacillus licheniformis FJAT-4 in controlling F. oxysporum.[Methods] Crude lipopeptides were extracted by acid precipitation and resolved in methanol. Their structures were identified using liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Their antifungal activities were determined through the assay of inhibition zone experiment. Their effects against F. oxysporum were observed using scanning electron microscopy.[Results] Lipopeptides produced by B. licheniformis FJAT-4 were identified as C₁₇ fengycin A, C₁₇ fengycin B, C₁₇ fengycin B₂, C₁₆ fengycin A variant, C₁₆ fengycin B variant, C₁₃-C₁₅ surfactin and C₁₃-C₁₅ surfactin variants. Among them, C₁₃-C₁₅ surfactin variants with m/z 1048.6/1062.6/1076.6 were new compounds. Results showed that the culture mediums have relatively little influence on the composition of lipopeptides produced by FJAT-4. However, the culture temperature presented obviously impact on the production of antifungal lipopeptides, which can only be produced by strain FJAT-4 growing at higher temperature (30-40℃) in comparison to temperature between 20-25℃. The higher culture temperature was in favour of increasing the proportion of surfactins in lipopeptide mixture. The lipopeptides exhibited strong antifungal activity against several plant pathogenic F. oxysporum strains in a dosage-dependent mode. Observation under scanning electron microscopy showed that the antagonistic lipopeptides secreted by B. licheniformis FJAT-4 significantly led to abnormal mycelia and spore growth of F. oxysporum.[Conclusion] The lipopeptides produced by FJAT-4 was composed of fengycins and surfactins. These lipopeptides display strong antifungal activity against several strains of plant pathogenic F. oxysporum, and destroyed the mycelial structure of the pathogenic F. oxysporum.