Abstract:[Objective] To enhance the production of enramycin by Streptomyces fungicidicus. [Methods] In this study, the ribosomal S12 protein encoding gene rpsL which affects the secondary metabolism and biosynthesis of antibiotics in enramycin producing strain of S. fungicidicus F1 was changed through the method of site-directed mutation, during which the Lys43 was substituted for Asn and Arg, respectively. And then the growth characteristics, synthesis of antibiotic and flask fermentation performance of the modified strains were studied. [Results] The results showed that the growth characteristics and physiological and biochemical characteristics of the modified strains were significantly changed compared with the wild type strain. Sporulation cycle was shorten, spore was produced from wild type strain using MS medium under the condition of 28 °C for 5?7 days, but the modified strains can produced large amounts of spore only after 3 days in the same conditions. Enramycin yield was relatively increased. Under the flask formation condition, the enramycin production of the modified strains of L-M1(Asn43) and L-M2(Arg43) can reach a maximum of 1 334 U/ml and 1 456 U/mL, respectively. Compared with the wild-type strain, it was improved by 11.9% and 22.1% separately. [Conclusion] Through the genetic modification, the yield of enramycin was improved, which might provide a feasibility for the modification of other sites.

Abstract:[Objective] Universal stress protein (USP) belongs to an ancient protein family. In Streptomyces, its physiological function has not been reported yet. This work focused on the protein particularly. [Methods] Sequence alignment was performed. The association between cAMP and USP was detected using circular dichroism spectroscopy. The usp gene (SLI_7517) was also disrupted. Susceptibility of wild-type and the usp disruption mutant strains to the thiol oxidant, diamide, was analyzed. qPCR was conducted to detect the expression differences of glutathione peroxidase and thiol peroxidase genes between the wild-type and usp disruption mutant strains. [Results] USP from different Streptomyces species shares 90% similarity, with the highly conserved USP-like domain. The alteration of USP CD spectra was observed when USP was mixed with cAMP. Tolerance to diamide increased after usp was knocked out. The transcription level of glutathione peroxidase gene was up-regulated in the usp disruption mutant. [Conclusion] USP protein from Streptomyces lividans 1326 can bind to cAMP. It might be involved in the regulation process, that suppresses the transcription of glutathione peroxidase when the bacteria exposed to oxidative environment.

Abstract:[Objective] To construct a biocatalytic system for enzymatic synthesis of (S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine (DHTP), several stereoselective oxidoreductases were expressed from recombinant strains and explored on their properties of catalyzing asymmetric reduction toward N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP). [Methods] From available recombinant strains involving oxidoreductases, enzymes were purified by Ni-ion affinity chromatography and their catalytic activities and stereoselectivities were evaluated toward DKTP. Among them, CR2 was further characterized, which could catalyze highly stereospecific synthesis of (S)-DHTP. Then, the catalytic process of CR2 was studied for asymmetric reduction of DKTP under optimal conditions. [Results] Enzyme CR2 was obtained with high stereoselectivity and catalytic activity for (S)-DHTP production. Its kinetic parameters of Km and kcat/Km were determined as 0.135 mmol/L and 3.689 L/(mmol·s), respectively. For CR2, the optimal pH was pH 8.4 (0.1 mol/L triethanolamine buffer) and the optimal reaction temperature was 35 °C. It was more stable at the temperatures ranging from 10 °C to 45 °C and at the pH ranging from 7.5 to 8.5. Zn2+ improved the enzyme activity of CR2. When the reaction was carried out for about 6 h, the target product was achieved with the yield of 92.1% and the optical purity of 99.9%. [Conclusion] This work provides the research foundation for further improvement of the enzymatic conversion efficiency of asymmetric reduction of DKTP.

Abstract:[Objective] We overexpressed β-xylosidase with heterologous expression by constructing a recombinant Pichia pastoris. [Methods] According to the codon usage frequency of highly expressed genes in P. pastoris, the Thermomyces lanuginosus β-xylosidase (Xyl43) gene was optimized and expressed in P. pastoris GS115, followed by characterization of Xyl43. Then, single factor experiments were used to optimize the fermentation conditions, in a 5-L fermenter. [Results] The optimized Xyl43 gene changed greatly with 78.17% of the sequence homology, and the GC content reduced from 52.8% to 44.6% with 222 bases substituted. The optimized gene was transplanted with an expression vector pPIC9K-OptXyl43 into P. pastoris GS115 to produce transformants. Then, a high xylosidase activity secreting recombinant P. pastoris GS115-Xyl43 was selected from the transformants on G-418 resistant plates, followed by shake flask cultivation. Basic enzyme properties of the recombinant xylosidase was analyzed as below: the protein molecular weight 51.5 kD, the optimal reaction temperature 55 °C, the optimum pH 7.0, and kinetic parameters Km=2.93 mmol/L and Vmax=157.9 μmol/(min·mg). β-xylosidase fermentation was optimized in shake flask as follows: methanol supply 1% (each 24 h), shaking speed 250 r/min, incubation time 144 h, incubation temperature 28 °C and initial pH 6.0. Under the optimal condition, the extracellular enzyme activity reached 42 U/mL with a protein content of 0.54 g/L. Further, in a 5-L fermenter, P. pastoris GS115-Xyl43 achieved 222.2 U/mL of xylosidase at 156 h (methanol induction for 96 h), with protein concentration at 2.36 g/L, which was 4.3 fold more than that in the shake-flask fermentation. [Conclusion] β-xylosidase can be expressed in P. pastoris GS115 with high level production and can be used as a candidate in various industrial applications.

Abstract:[Objective] The effects of the threonine absorption system deletion, containing TdcC, SstT and LIV-1, on the extracellular accumulation of threonine were investigated in Escherichia coli. [Methods] In this paper, the TdcC, SstT and LIV-1 systems single- and muti-deletion of E. coli W3110 were constructed by deleting the corresponding genes (tdcC, sstT, livJ). Subsequently, the plasmid harboring the operon gene cluster of threonine, pKKthrAC1034TBC, was transformed into both the wild type and recombinant strains. The ability of absorption and extracellular accumulation of threonine was then detected. [Results] Compared with W3110, the threonine absorption ability of T04 with tdcC and sstT deletion was decreased by 43.28%. Meanwhile, T04 with plasmid pKKthrAC1034TBC accumulated 1.09 g/L threonine in vitro, which was 2.7 folds of that of strain W3110 with pKKthrAC1034TBC. Further deletion of livJ gene in T04 generated T07, which resulted in 12.97% decrease of threonine absorption. However, the maximum extracellular threonine concentration of strain T07 with pKKthrAC1034TBC was only 0.63 g/L, which was decreased by 42.2% when compared with that of strain T04 with pKKthrAC1034TBC. [Conclusion] Deletion of both TdcC and SstT systems of E. coli significantly decreased threonine absorption and improved extracellular threonine accumulation. Though inactivation of LIV-1 system reduced threonine absorption, it would impair the accumulation of threonine in vitro.

Abstract:[Objective] In order to improve the D-1,2,4-butanetriol (BT) titer in recombinant Escherichia coli, two by-product pathways were blocked. [Methods] The xylAB, yagE and yjhH were knocked out by Red system and the cell growth, BT production and accumulation of byproduct of the resultant strains were detected. [Results] The biomass and BT titer of xylAB-deficient strain were repressed by 57% and 20%, with the BT yield per cell increased by 84%. In contrast, the biomass of yagE-deficient and yjhH-deficient strains was increased by 10% and 5%, respectively. The BT titers of these two strains showed increase of 36% and 14%, respectively. Co-knocking out these two genes led to the decrease of biomass by 21%, but the BT titer was improved by 184%, up to 2.44 g/L, and BT yield per cell was increased by 258%. Meanwhile, blocking these two by-product pathways simultaneously resulted in the decrease of biomass by 72% and raised BT titer per cell by 4 times, and the final BT titer was showed 43% improvement. The xylonate titers of recombinant strains were decreased, and the BT titer was further improved by pH-controlled fermentation, up to 3.11 g/L. [Conclusion] Although knocking out xylAB is beneficial to BT produce, the repressed xylose utilization ability through the PPP pathway and accumulated xylonate lead to the reduced biomass and BT titer. Knocking out the yagE or yjhH showed slightly positive effect on BT production. Further co-deficiency of these two genes shifts more 2-keto-3-deoxy-xylonate (KDX) into BT pathway, leading to significantly improved BT titer. Finally, blocking these two by-product pathways shows negative effect on cell growth but slightly improvement on BT production.

Abstract:[Objective] To evaluate the ability of Schizophyllum commune cfcc7252 in degrading Malachite Green (MG) dye. [Methods] Effect of oxygen demand, initial pH value, temperature, carbon source, nitrogen source, salinity and initial dye concentration on MG biodegradation by S. commune cfcc7252 was studied in flasks containing liquid cultures based on single factor experiment design. The toxicity of the biodegraded products of MG on plant seed germination and microbial growth was determined in petri dishes. [Results] Schizophyllum commune cfcc7252 could degrade MG under both aerobic and anaerobic conditions; 67.8% MG (initial concentration: 350 mg/L) was degraded under the culture condition of 10.0 g/L glucose, 5.0 g/L yeast extract, 0.01 mmol/L Zn2+ and pH 4.0 for 36 h. After continuous bleaching for 7 rounds, the degradation rate still reached over 95.4%. In addition, the degradation rate of S. commune cfcc7252 on MG was as high as 98% if the salinity of the culture was less than 10.20%. Toxicity test showed that the biodegraded products of MG were rarely toxic to plants of Vigna umbellate and Vigna unguiculata and microorganisms of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa. [Conclusion] Schizophyllum commune cfcc7252 has strong potential to treat MG contaminated wastewater.

Abstract:[Objective] In this study, the microbial community structure of Lidu ancient earthen cellar was determined to provide reference for further microbial diseases prevention and protection of the earthen ruins. [Methods] We collected 3 representative samples from Lidu ancient earthen cellular to construct clone libraries of the 16S rRNA gene and 18S rRNA-ITS intergenic region and subsequently analyzed the sequencing results in order to establish phylogenetic tree by software such as MEGA 3.0 and ClustalW 1.8. [Results] A total of 72 16S rRNA gene and 80 18S rRNA-ITS intergenic sequences were obtained. All 16S rRNA gene sequences were classified into 3 phylum and 11 genera and all 18S rRNA-ITS intergenic sequences were classified into 1 phyla and 7 genera. The major bacteria included Halomonas sp., Methylohalomonas sp. and Lactobacillus sp. The major fungi included Cyphellophora sp., Pichia sp., Exophiala sp. and Phialophora sp. [Conclusion] Among the microbes we identified in this earthen ruins, dominant bacteria are slat-tolerant, which mainly caused by the earthen cellar’s environmental condition. Major fungus is filamentous fungi and yeast. It is important to identify the microbes which could destroy the earthen ruins, because the result provides scientific basis for the study of reinforcement material against these microorganisms and the protection of the Lidu ancient earthen cellar.

Abstract:[Objective] We modified genes responsible for xylose metabolism and riboflavin biosynthesis in Bacillus subtilis. [Methods] Genes responsible for riboflavin biosynthesis were overexpressed or co-overexpressed with genes of xylose metabolism in Bacillus subtilis. Recombinant strains were evaluated by measuring the riboflavin yield and biomass. Fermentation was done in shake flask and fermenter to produce riboflavin with sucrose as sole carbon source or a mixture of sucrose/xylose. Meanwhile, riboflavin yield, xylose consumption and biomass were analyzed. [Results] Overexpression of ribA gene increased riboflavin yield by 99% and reduced maximum biomass by 30% because of autolysis. With ribA and ribH genes co-overexpression, riboflavin yield increased by 280% without biomass decrease and autolysis. Through a 70 h batch fermentation with 6.5% xylose and 1.5% sucrose as carbon source in a 5 L fermenter, riboflavin yield of 3.6 g/L was obtained, 80% higher than that with 8% sucrose as carbon source. Riboflavin yield decreased significantly by overexpressing the genes involved in xylose metabolism. [Conclusion] Co-overexpression of ribA and ribH genes avoided the cytotoxicity effectively and further increased riboflavin production.

Abstract:[Objective] This study is aimed to constitutively express feruloyl esterase gene (AnfaeA) from Aspergillus niger in Pichia pastoris GS115. [Methods] The AnfaeA gene was amplified from A. niger by overlap extension PCR and cloned into the expression vector pGAP9K.The recombinant expression vector (pGAP9KAnfaeA) was linearized by Sal I, then transformed into P. pastoris GS115. Feruloyl esterase activity of the recombinant strain was determined by high-performance liquid chromatography (HPLC) and we optimized the fermentation conditions. [Results] We cloned AnfaeA gene (783 bp) and constitutively expressed AnfaeA gene in P. pastoris GS115. Feruloyl esterase activity reached its peak (5.72±0.10 U/mL) after 84 h fermentation, its specific activity was 59.75 U/mg, and the molecular weight of reAnfaeA was about 40 kD. The optimal fermentation conditions were as follows: glucose 40.0 g/L, tryptone 10.0 g/L, yeast extract 30.0 g/L, CaCO3 0.2 g/L, inoculum age 28 hours, inoculation level 3% (v/v), medium volume 50 mL in 250 mL flask. Under these conditions, feruloyl esterase activity in fermentation supernatant was 15.60±0.23 U/mL. [Conclusion] We constitutively expressed feruloyl esterase in P. pastoris GS115. It was helpful to study the constitutive expression system in P. pastoris and improve the fermentation production of feruloyl esterase.

Abstract:[Objective] To explore the effects of bio-control and growth-promoting of the siderophore involved with Ser gene in Act12, the siderophore synthase Ser gene knock-out mutant was generated. [Methods] For generating deletion mutant, suicide plasmid pKC1132 was chosen as a basic vector, kanamycin resistance gene as a marker, upstream and downstream of Ser gene as homology arms to construct the recombinant plasmid named pCT12. Then, it was transfered into Act12 by conjugal transfer. Putative deletion mutants were verified by PCR. Whereafter, biological analysis was conducted to assess the role of Ser gene in bio-control and growth promoting. [Results] The Ser deletion mutant ?ser was obtained by PCR and sequencing. Results of biological analysis showed that ?ser synthesizes less siderophores compared to wild strain Act12. Furthermore, the seed germination rate of Cucumis melo L and the antagonism against Macrophoma kawatsukai both significant declined. [Conclusion] These results suggest that Ser gene of Act12 has a certain impact on bio-control and growth promoting, which would establish the foundation for further mechanism study.

Abstract:[Objective] To identify a protease producing bacterium CAU342A isolated from soy sauce koji and optimize the fermentation conditions for protease production. [Methods] Strain CAU342A was identified based on its morphological characters, 16S rRNA gene sequence, physiological and biochemical characteristics. The fermentation conditions for protease production by strain CAU342A were optimized using the one-factor-at-a-time method and orthogonal test. [Results] strain CAU342A was identified as Pseudomonas aeruginosa, and the optimal fermentation conditions were obtained as follows: 3% vinasse, 1.5% yeast extract, 0.05% Tween-80, 0.5% NaCl, 0.7% K2HPO4, 0.3% KH2PO4, 0.04% MnSO4, the initial pH was 7.5 and incubation temperature was 30 °C for 72 h. Under the optimized fermentation conditions, the highest protease activity of 2 653.5 U/mL was achieved. The protease zymogram analysis showed that at least 4 proteases were secreted into the fermentation broth, and two major bands had molecular masses of 32 kD and 50 kD, respectively. [Conclusion] The high-level production of protease by Pseudomonas aeruginosa CAU342A enables its potential industrial applications.

Abstract:[Objective] To explore the tolerance characteristic of selenium of Hypsizygus marmoreus mycelium strains. [Methods] To determine the average growth rate of mycelium at different concentrations of selenium by using the plate culture method and fitting the relationship between selenium and mycelial growth rate. Record the mycelium germination, the morphological characteristics of colony and do the restoring culture to verify. Using microscopic to observe the effect of selenium on the hyphae branch, size, lock-like joint morphology and surface structure. [Results] The results of experiment showed that when exogenous selenium concentration ≤50 mg/L, it promoted the growth of mycelium. The promoting effect vary in different strains, but compared to Control Check (CK), there is no significant difference (P>0.05). When exogenous selenium concentration ≥75 mg/L, the growth of H. marmoreus was inhibited. The maximum tolerance concentration of selenium is 150–200 mg/L. Restoring culture showed that this kind of inhibition could be restored; selenium concentration and the average growth rate accords with mycelium Cubic regression curve and the coefficient of determination (degree of fitting) is large. The results of microscopy observation indicated that when the concentration of exogenous selenium is low, the mycelia showed the following characteristic: even thickness, robust and full, more branches, smooth surface and distinct lock-like joint clear structure. At higher concentrations of exogenous selenium, thickness of the mycelium is irregular and it shrinked to long flat strip. The lock-like joint structure collapsed and wizened, the surface is uneven and became bamboo-like structure. The tip part of hyphae was gradually substituted by spherical conidia and some branches even were completely alienated into conidia or chlamydospores. [Conclusion] when the selenium concentration ≤50 mg/L, it has a promoting effect on the growth of mycelium; when selenium concentration ≥75 mg/L, it inhibited the growth. High concentration of selenium has toxic effects on the mycelia, but the toxic effects are reversible.

Abstract:[Objective] The aims of this study were to detect the source of p-cresol in strong flavor Chinese liquor; analyze the microbial community structure in pit mud, and separate strains producing p-cresol. [Methods] p-Cresol concentration in Daqu, pit mud and saccharogenic grains was detected by GC-MS. Microbial communities of different parts of pit muds were detected by highthroughput sequencing technology. Then strains that could produce p-cresol were separated. [Results] p-Cresol was only detected in pit mud, not in Daqu or saccharogenic grains. The concentration was 24.24 μg/g in bottom part of pit mud. The microbes mainly belonged to 8 classes in pit mud. Among them, Bacteroidia, Clostridia and Methanobacteria were all higher than 8%. The microbes mainly belonged to 11 genus, Clostridium, Aminobacterium, Methanobacterium, Methanobrevibacter, etc. After strains separation and volatile compounds detection, Clostridium aminovalericum, Clostridium ultunense and Clostridium purinilyticum could produce p-cresol, and were all higher than 0.20% in pit mud. [Conclusion] Clostridium was the primary microbial source of p-cresol.

Abstract:[Objective] This study is aimed to screen and identify new lactic acid bacteria (LAB) with antagonistic activities against Campylobacter jejuni from chicken manure, to understand their intestinal probiotic properties, and to probe the effects of LAB’s cell-free extracts on the virulence factor, flagella formation of C. jejuni. [Methods] The inhibition activities of 40 strains isolated from chicken manure were analyzed based on an oxford cup method to screen the strains with excellent antagonistic inhibitory effects on the indicator bacteria C. jejuni. The 16S rRNA gene-based technique was used to identify the selected strains. The adherence abilities of LAB strains to human intestinal epithelial cell (HT-29) were evaluated. The tolerance of LAB strains to artificial gastrointestinal juices was tested. Scanning electron microscopy was used to observe the effects of LAB’s cell-free extract on the flagella formation of C. jejuni. [Results] Forty LAB strains were isolated from chicken manure, among which X13, X14 and G20 strain showed the strongest antagonistic activities against C. jejuni. The 16S rRNA gene sequence analysis showed that X13, X14, and G20 strain were respectively identified as Lactobacillus reuteri, Lactobacillus salivarius and Lactobacillus gallinarum. HT-29 cell adhesion tests showed that the adhesion index of X13, X14, and G20 were respectively 11.5, 20.3, and 14.3 cells per HT-29 cell, such results suggests that all these three strains showed strong adhesion abilities to HT-29 cell. Tolerance tests to artificial gastrointestinal juices showed that all these three strains showed good capabilities. SEM observation showed that the cell-free extracts of these three strains were able to inhibit the flagella formation of C. jejuni. [Conclusion] Three selected LAB strains from chicken manure have inhibit the growth and the flagella formation of C. jejuni in vitro effectively, which suggested that such three LAB strains could be used as potential animal protective probiotics for the control of C. jejuni infection in vivo.

Abstract:[Objective] Streptomyces sp. FR-008 was treated with nitrosoguanidine (NTG) to screen mutant with reduced genome in size and without any effect on its growth rate. The resultant mutants were measured on their potentiality to be further developed into chassis cells for heterologous production of secondary metabolites. [Methods] Treatment of the spore suspension of Streptomyces sp. FR-008 by NTG was immediately followed by screening of mutants that are sensitive to arsenite, indicative of loss of large linear plasmid. Pulsed-Field Gel Electrophoresis (PFGE) analysis of 103 strains that survived NTG treatment was used to detect the presence or loss of linear plasmids. Bioassay and HPLC analysis were used to compare the yield of candicidin component III for four mutants with that of the wild type strain. [Results] From 103 strains, 4 independent mutants named 10#, 42#, 59# and 115# with one linear plasmid cured were obtained, and the second-round NTG mutagenesis cured both two linear plasmids in XYS1. Fermentation analysis revealed that the yield of candicidin component III in 10# and 115# increased by 40% and 30%, respectively. [Conclusion] It is the first report that NTG is an effective reagent to cure the stable linear plasmids in Streptomyces, two strains from which the large linear plasmid were cured with enhanced yields of candicidin were obtained. NTG treatment can be used to cure the linear plasmids in some Streptomyces to efficiently reduce its genome, and therefore an effective approach in genetic breeding of antibiotic producer.

Abstract:[Objective] To improve avermectin yield, mutants of Streptomyces avermitilis 9-39 were screened and the fermentation medium was optimized. [Methods] Atmospheric and room temperature plasma (ARTP) technology was used to treat the spores of S. avermitilis 9-39. The method of streptomycin and kanamycin resistance combined with 96 deep-well plate screening was used for high-throughput screening of high-yielding mutants. Furthermore, based on single factor experiments, the fermentation medium for mutants was optimized by response surface methodology. [Results] A stable avermectin high-producing mutant K-1A6 was obtained and its avermectin production reached 4.22 g/L, 23.4% higher than that of the parent strain 9-39. Under the optimized medium fermentation, the yield of avermectin reached 5.36 g/L, 27.01% higher than that before optimization. [Conclusion] The yield of avermectin can be increased by ARTP mutagenesis, screening and fermentation medium optimization.

Abstract:[Objective] To explore the function of rmlB gene on antibiotic resistance, biofilm formation, and virulence in Listeria monocytogenes. [Methods] The rmlB gene was deleted through homologous recombination. Antibiotic resistancedifferences between the wild-type and the rmlB deletion mutant were compared. The biofilm formation ability of the mutant was measured using the microtiter plate assay. The transcription of major virulence genes in L. monocytogenes and hemolytic activity of the mutant were studied. [Results] Compared tothe wild-type strain, the deletion of rmlB increased susceptibility to envelope-acting antibiotics such as several cephalosporinsand bacitracin (P≤0.01) and decreased bacterial biofilm formation (P≤0.01).The transcription expression of the well-known virulencegene hly in L. monocytogenes and bacterial haemolytic activity were also significantly reduced in the rmlB deletion mutant (P≤0.01). [Conclusion] rmlB plays a crucial role in L. monocytogenes against the envelope-acting antibiotics, biofilm formation and virulence.

Abstract:[Objective] To investigate the interaction between plasminogen (Plg), lipoprotein(a) [Lp(a)] and dihydrolipoamide dehydrogenase (Lpd) on the surface of P. aeruginosa. [Methods] Recombinant Lpd and its mutants (rLpdK476A, rLpdK477A, rLpdΔKKR) were used to explore the interaction with enzyme-linked immunosorbent assay (ELISA) and affinity chromatography-binding assay followed by Western blot analysis. [Results] The recombinant rLpd and its mutants were expressed and purified from Escherichia coli. The results indicated that rLpd could specifically bind to Lp(a) but not to LDL. The binding capacity of rLpdΔKKR to Lp(a) is significantly lower than that of rLpd. The binding of rLpd to Lp(a) could be significantly inhibited by 1 mmol/L of EACA. 1 000 μg/L of Lp(a) could inhibit the interaction between rLpd and Plg. [Conclusion] Lpd could bind to Lp(a) and the 476th and 477th lysine residues of Lpd were main binding sites. Lp(a) could significantly inhibit the binding of Plg to Lpd.

Abstract:During long-term evolution of life in nature, bacteria and archaea have developed anadaptive defense system called clustered regularly interspaced short palindromic repeat sequences and CRISPR-associated genes (CRISPR-Cas) that protect organisms from invading viruses and plasmids. It has been recently used as a powerful gene-editing tool in a wide variety of important organisms. In this review, we summarize recent progresses on CRISPR–Cas9 system and its applications to gene editing, gene regulation, and in vitro DNA manipulations used as a programmable molecular clone tool.

Abstract:Epigenetics plays an important role in biological processes of microorganisms. DNA modification with restriction-modification system is a component of microbial immune system, and DNA modification without restriction system influences phenotypes by gene regulation. However, epigenetic information has not been collected for DNA sequence analysis routinely. Single molecule real time (SMRT) sequencing, based on analysis of DNA synthesis kinetics, enables the acquisition of DNA primary sequences and detection of modified nucleotides simultaneously. This technology provides a new platform for research on known DNA modifications and discovery of novel DNA modifications. This review summarized single molecule real time sequencing and its applications in microbial epigenetics.

Abstract:Because of its effectiveness in curing infections caused by multi-drug resistant Gram-negative bacteria, polymyxin is applied to clinic again. Its drug resistance is generally low among a variety of antibiotics. However, there are studies showing that the drug resistance of polymyxin increases. As the last resort for treating multi-drug resistant Gram-negative bacteria, the occurrence of polymyxin resistance is especially important. This article summarizes the present situation, the underlying mechanism and the counteracting measure for polymyxin resistance so as to provide guidance for scientific and rational use of polymyxin in clinic, for controlling and reducing the dosage of polymyxin and for the measures in the prevention of spread and pervasion of polymyxin resistant Gram-negative bacteria.

Abstract:Selenium (Se) is an essential trace element which shows fundamental and important roles in medical health and industrial manufacturing. Selenium is normally a redox sensitive element that occurs in different forms including selenide [Se(?2)], elemental selenium [Se(0)], selenite [Se(+4)], and selenate [Se(+6)], all of which present in nature and organisms as organic or inorganic forms. Microorganisms play an important role in selenium transporter, reduction, oxidation, assimilation, and methylation in the environment. This review summarizes the importance of microorganism on the biogeochemical cycle of selenium, include: 1. Selenium cycle in environment; 2. Mechanism of selenium metabolism in microorganisms.

Abstract:Lipopolysaccharide plays an important role in the outer membrane of most Gram-negative bacterium, because it consists of the innate immune system for these bacteria. Lpt system is the only pathway to transport lipopolysaccharide in most Gram-negative bacteria. Among them, the outer membrane protein LptD is involved in the last step of lipopolysaccharide transportation, so it is regarded as a “vital gate”. LptD is involved in a variety of biological processes, including organic solvent tolerance, hydrophobic antibiotic resistance and membrane permeability. Recent studies have shown that the most important function of LptD is its role in the transportation of lipopolysaccharide. To help further understand LptD and reveal the Lpt mechanism in other gram-negative bacteria, we described the structure and functions of LptD in some Gram-negative bacteria.

Abstract:Microbiology experiment is an important basic practical course tailored for university undergraduates with biology relevant disciplines and also a fundamental skill for further biology professionals. With the encouragement of teaching reform in universities, comprehensive design experiments were carried out aiming to cultivate students’ comprehensive and creative abilities. However, many problems were discovered during the implementation process such as low participation rate of students, poor operational skills, non-flexible experiment program, and too simple assessment method. As a result, the expected learning outcomes were not obtained. In this study, we summarized the features of Microbiology Experiment teaching model, analyzed the problems existed in the teaching process, and provided advice to improve this model from the aspects of encouraging students’ participation, arranging the reasonable experimental settings, introducing research-orientated programs accompanied with corresponding teachers, and improving the evaluation approaches.

Abstract:Environmental Engineering Microbiology experiment is an important professional foundation required course in environment engineering. This course has an important influence on the study of professional courses. In order to guarantee the teaching quality of the course, a new teaching innovation system is established. The system consists of two parts: (1) Separation, screening, optimization of conditions, application effect and characteristics analysis of flocculant; (2) Analysis on actinomycetes, mold, yeast and protozoan in pollution control engineering. The two part of the experiment is both research and comprehensive. The first part is divided into five modules. These five modules are systematic and continuous and given a certain challenge. Through this part of the experiment, students can grasp the basic experimental techniques and research methods of bacteria. The second part concerns the filamentous microorganisms and eukaryotic microbes. This part of the experiment can enable students to grasp the basic experimental technology and application methods of these microorganisms. The system is rich in content, closely combined with professional and has greatly stimulated the enthusiasm of students to learn. Through the system, the students can fully obtain the basic experimental skills of Environmental Engineering Microbiology and helps to improve students’ comprehensive ability. The teaching effect is very remarkable.

Abstract:[Objective] We established an HPLC (High Performance Liquid Chromatography) method to determine Ganoderic acid C2, Ganoderic acid B, Ganoderic acid A, Ganodermanontriol, Ganoderic acid DM, Ganoderic acid T, Ganoderiol B, Ganoderic acid S, Ganoderic acid G, Ganoderic acid F, Ganoderic acid D, Ganoderenic acid B in Ganodema lingzhi. [Methods] Aglient ZORBAX SB-Aq (4.6 mm×250 mm, 5 μm) was adopted as the chromatographic column, with acetonitrile-0.01% acetic acid solution using gradient elution at 30 oC with a flow rate of 1 mL/min, and the UV detection wavelength was set at 252 nm. Composition and content of different varieties of triterpenoids from different areas were determined by the above designed method. [Results] Triterpenoids in different varieties of Ganodema lingzhi in one area and samples from different areas were significantly varied in their compositions and contents; the method was sensitive, reliable and reproducible. [Conclusion] This method can be used to determine triterpenoids.