Abstract:Abstract:Lactic acid bacteria (LAB) are important organisms in the food industry． The study of microevolution of LAB is helpful in understanding of the biological function and mechanism of these microbes． With the development of molecular biology，a large number of technical means have emerged，such as multilocus sequence typing (MLST) and whole-genome re-sequencing，which enable the study of the phylogenetic and population evolution of LAB at genetic level． MLST has already been widely used on microevolution research of LAB to analyze the genetic diversity and population structure．Moreover，recently，as a result of the declining in sequencing cost，the advantage of whole genome sequencing technology is increasingly highlighted． This article elucidates the principle，methods and scientific significance of researching LAB microevolution，as well as introduces the application of whole genome sequencing in these aspects to provide new insights into further research．

Abstract:Abstract:Microbial lipases are major sources of commercial ones，which have been extensively used in a wide variety of industrial fields，such as foods，beverages，lipids，detergents，feeds，textiles，leathers，advanced materials，fine chemicals，medicines，cosmetics，papermaking，pollution treatment，and bioenergy． Compared with fungal lipases，bacterial lipases have more types of reactions and exhibit higher activity and better stability in aqueous or organic phases． Amongst bacterial lipases，the most excellent ones are those originating from the genus Pseudomonas． So far，the conventional strategies，such as traditional breeding，optimization of medium and fermentation conditions，cannot fundamentally solve the problem of low production of bacterial lipases． Construction of genetically engineered strains to efficiently overexpress their own lipases is an effective solution． But it must base on clarifying molecular regulation mechanism of lipase gene expression and further finding out key regulators． In this article，we reviewed the progress in expression regulation of bacterial lipase genes from the aspects of direct regulators，quorum sensing system，Gac/Rsm signal transduction system，regulators controlling the Gac/Rsm system，and other regulators． To provide a useful reference for the construction of genetically engineered strains，we also discussed a research prospect in this field based on our ongoing research.

Abstract:Abstract:［Objective］The work was aimed at selecting osmo-regulated prompters possessing excellent performance for further research of the industrial yeast Candida glycerinogenes.［Methods］Promoters PCgPGI，PCgTPI，PCgZWF，PCgSTL1，PCgSTL2 and PCgSTL3 were amplified by PCR and their bioinformatics analysis of stress response elements (STREs) were conducted． We constructed integrative plasmids containing 5. 8S rDNA，a fluorescence protein gene gfp and a promoter PCgPGI，PCgTPI，PCgZWF，PCgSTL1，PCgSTL2 or PCgSTL3．The promoters’activities and osmoregulations were compared according to the results of fluorescence and qRT-PCR.［Results］PCgSTL3 had more STREs，higher transcription level，lager gfp expression and it was more sensitive to stress.［Conclusion］PCgSTL3 is an excellent induced promoter responding to hyperosmotic stress． Controlled expression of target genes can be realized using PCgSTL3 in the industrial yeast.

Abstract:Abstract:［Objective］The aim is to reveal the role of PFKFB3 in 11'-deoxyverticillin A(C42)-induced autophagy and apoptosis.［Methods］Electron and fluorescence microscopy，immunoblotting，MTS assay，siRNA interference and real time PCR were used.［Results］C42 could induce multiple cell death in HeLa cells． Knockdown of either Beclin 1 or LC3，two important autophagic genes，increased both PARP-1 cleavage and cell viability loss． Although high dose of C42 triggered more cell viability loss，yet，it failed to augment autophagic flux． While PFKFB3 inhibitors attenuated C42-induced autophagy，the overexpression of PFKFB3 increased the induced autophagic flux.［Conclusion］PFKFB3 is involved in C42 induced-autophagy，which blunts the caspase-dependent apoptotic process.

Abstract:Abstract:［Objective］To obtain the cryptic lanthipeptide from Streptomyces clavuligerus by semi-in vitro biosynthesis that is a novel method for mining lanthipeptides resource from Streptomyces.［Methods］ The core peptide of cryptic lanthipeptide was modified in E.coli by nisin modification system，and purified by affinity chromatography and High Performance Liquid Chromatography (HPLC). After the leader peptide was removed，the core peptide was obtained and its dehydration and cyclic structure were analyzed by MALDI-TOF MS and tandem MS.［Results］A novel lanthipeptide named CLA 124 with 4-fold dehydration 2 thioether bridges and one disulfide bridge was produced.［Conclusion］Cryptic lanthipeptides from Streptomyces could be produced by semi-in vitro biosynthesis.

Abstract:Abstract:［Objective］ To study the repair mechanisms of frozen sublethally damaged Staphylococcus aurous cells.［Methods］We resuscitated frozen sublethally damaged S．aureus at 37 ℃ for different time within 3 h． Meanwhile，we compared the morphological changes of the frozen sublethally damaged cells after 1 h of resuscitation using transmission electron microscopy assay (TEM). The expressions of the transcriptional attenuator MsrR (msrR)，iron (Fe3+) ABC transporter ATP-binding protein (fhuC)，and cytochrome b (cytB) genes were quantitatively analyzed by real-time fluorescence quantitative PCR (Real-time PCR) method． The content of cells outside leakage，active oxygen (ROS)，and superoxide dismutase (SOD) activity were also determined by ultraviolet spectrophotometry.［Results］More than 99% of the frozen sublethally damaged S． aureus repaired after 3 h． The resuscitated cells expressed an equal resistance to high concentration of NaCl. Real-time PCR results showed that the msrR and fhuC genes expressions were down-regulated，whereas the cytB gene expression was up-regulated significantly． The frozen sublethally damaged S． aureus cellar surface ultrastructure significant changed during resuscitation． The cell surface became compact and sturdy from smooth and transparent． The cell leakage rate of ultraviolet absorption material gradually decreased． Meanwhile，the intracellular ROS level declined along with the decrease of SOD activity． ［Conclusion］Frozen sublethally damaged cells may regain the capability of resistance to high salt stress by repairing cell membrane integrity，reducing the content of ROS through gene regulation，inhibiting the toxicity of active oxygen to the cells． Meanwhile，the regulation of metabolism related genes (cytB) provides the energy for the requirement of cells，therefore，the frozen sublethally damaged cells were repaired finally.

Abstract:Abstract:［Objective］In order to develop a safe，nontoxic and efficient biological antistaling agent and to decrease the incidence of rot diseases caused by the Penicillium italicum and Penicillium digitatum in orange fruit storage，［Methods］the present experiment was carried out with Pythium oligandrum broth (POB) produced by our self- isolated strain (P．oligandrum CQ2010) to study the toxicity to animal． Thereafter，mycelium growth and spore germination of both P．digitatum and P． italicum and control effect of rot disease in orange storage were compared after treated by liquid culture medium (control)，POB，prochloraz (PC)，and PC+POB.［Results］Gastric lavage with large amount POB did not influence mouse weight． The animals also showed no abnormality in appearance，behaviors and pathology changes in heart，liver，kidney，lung and intestine． POB decreased the hyphal growth by 70.24%－93.74% and spore germination by 44.91%－87.82% (24 h after POB addition) of these two pathogenic fungi． Disease incidence of orange fruit following P.italicum inoculation changed in the sequence: CK＞POB＞PC＞PC+POB and the control efficacy behaved otherwise．In commercial simulation storage，the disease incidence of orange fruit caused by P．digitatum and P．italicum was above 50% of the total．The fruit rot rate was 26.40% (CK)，15.03% (POB)，16.61% (PC) and 4.21% (PC+POB)．There were no significant differences in fruit quality under different treatments.［Conclusion］POB was safe to animal and could decrease rot disease incidence caused by P. italicum and P. digitatum in orange storage whereby producing a positive interaction with prochloraz and controlling rot diseases caused by these two fungi.

Abstract:Abstract:［Objective］Several key genes (thlA，bcs-operon/crt-bcd1-etfB2-fixB2-hbd and adhE) in butanol pathway from Clostridium saccharobutylicum DSM13864 were cloned，and a butanol-producing Escherichia coli strain was successfully constructed．［Methods］Using genome of Clostridium saccharobutylicum DSM13864 as template，the key genes in butanol synthesis pathway were amplified，the recombinant plasmids pETDuet-bcs and pRSFDuet-thlA-adhE were constructed． Then the resultant plasmids were transformed into E.coli JM109(DE3) to obtain E． coli BUT1 for butanol production，under the semi-anaerobic condition．Effects of different mediums on butanol production were studied． ［Results］The recombinant E.coli was capable of producing butanol (25.4 mg/L) under semi-anaerobic fermentation．After optimization on the fermentation medium，butanol titer reached 34.1 mg/L．［Conclusion］Butanol production by recombinant E． coli harboring exogenous butanol-producing pathway from Clostridium saccharobutylicum provides a feasible solution to overcome the hurdles in traditional butanol production approach by Clostridia.

Abstract:Abstract:［Objective］The aim of this study was to screen acid-producing strains from the broth of psychrotolerant biogas fermentation and evaluate the acid-producing character of them.［Methods］Acid-producing strains were isolated by a medium with methyl red at 4 ℃ in Petri dishes and identified by morphology observation and 16S rRNA sequencing．Moreover，the ability of hydrolysis of starch，fermentation of carbohydrates，liquefaction of gelatin and production of catalase were studied．［Results］Two acid-producing strains (FJ-8 and FJ-15) were isolated． The result of the 16S rRNA phylogenetic tree shows that FJ-8 and FJ-15 belong to Pseudomonas sp． and Shewanella sp.，respectively． Both FJ-8 and FJ-15 could hydrolyze starch，liquidize gelatin and produce catalase． The optimum temperature for acid-producing of FJ-8 and FJ-15 is 15 ℃ and 20 ℃，respectively． After 10 days cultivation at 4 ℃，the concentration of acetic acid was 792 mg/L and 966 mg/L of FJ-8 and FJ-15 ，respectively.［Conclusion］The selected strains，FJ-8 and FJ-15，have the potential to produce acids at low temperature.

Abstract:Abstract:［Objective］We expressed a novel alkaline-adapted beta-mannanase gene and characterized the enzyme for potential industrial applications.［Methods］We obtained a mannanase gene (named manB) from Bacillus pumilus Nsic-2 and expressed the gene manB in Escherichia coli and Bacillus subtilis． Furthermore，we characterized the enzyme.［Results］The gene manB had an open reading frame of 1104 bp that encoded a polypeptide of 367-amino-acid betamannanase (ManB)．The protein sequence showed the highest identity with the beta-mannanase from B．pumilus CCAM080065． We expressed the gene manB in E.coli BL21 (DE3) with the enzyme activity of 11021. 3 U/mL．Compared with other mannanases，ManB showed higher stability under alkaline conditions and was stable at pH6.0－9.0．The specific activity of purified ManB was 4191±107 U/mg．The Km and Vmax values of purified ManBwere 35.7 mg/mL and 14.9 μmol/(mL·min)，respectively．Meanwhile，we achieved recombinant protein secretion expression in B．subtilis WB800N.［Conclusion］ We achieved heterologous expression of the gene manB and characterized its enzyme．The alkaline-adapted ManBshowed potential value in industrial applications due to its pH stability.

Abstract:Abstract:［Objective］ This study aimed to monitor targeted protein expression levels and changes in intermediate metabolites in the primary metabolic pathways of Escherichia coli to obtain basic data and to develop testing methods that can be used in metabolic engineering．［Methods］We used the Skyline software to design a label-free method (multiple reaction monitoring) for relative quantitative monitoring of proteins from primary metabolic pathways (i.e.，glycolytic pathway，pentose phosphate pathway，mixed acid fermentation，tricarboxylic acid cycle，and fatty acid synthesis pathway)．We used the same mass spectrometry platform (Triple Quad 4500) for liquid chromatography-tandem mass spectrometry (multiple reaction monitoring) analysis of targeted intermediate metabolites during absolute quantitative monitoring.［Results］Protein expression in the primary metabolic pathways of E．coli showed four different phenomena in the different growth periods (exponential phase，stationary phase，and decline phase)． Expression levels of a single protein cannot provide accurate information regarding the status of these pathways． More proteins in the pentose phosphate pathway，mixed acid fermentation，and the tricarboxylic acid cycle showed the highest expression in the decline phase，but accumulation of several targeted intermediate metabolites (ATP，ADP，AMP，NAD+，NADH，NADP +，NADPH， CoA，and acetyl-CoA) in the stationary phase and decline phase correspondingly decreased compared to the levels in the exponential phase (in addition to acetyl-CoA).［Conclusion］ The detection methods used in this study can help determine the basic conditions of E．coli metabolism in vivo.

Abstract:Abstract:［Objective］To study crystallization and X-Ｒay diffraction of ectoine hydroxylase from Bacillus pseudofirmus OF4.［Methods］We cloned the gene BpectD from B．pseudofirmus OF4 with a His6 tag and overexpressed it in E．coli BL21(DE3)． Then，we purified protein BpEctD by Ni2 + -chelating affinity and size-exclusion chromatography．After that，crystals were grown by the sitting-drop vapour-diffusion method at 289 K and diffracted at 100K using an in-house X-ray source．［Results］Protein BpEctD was expressed and purified successfully． We obtained well diffracting crystals of about 360 μm×240 μm×60 μm in size using a solution consisting of 0.2 mol/L magnesium chloride hexahydrate，0.1 mol /L bis-tris pH6.5，25% (W/V) polyethylene glycol 3，350 at a protein concentration of 6.5 mg/mL，and collected X-ray diffraction data to 2.40  resolution in the anorthic space group P1，with unit-cell parameters a=45.18，b=58.87，c=68.81，α=77.48°，β=86.03°，γ=66.97°．The asymmetric unit contains two molecules of BpEctD with a Mattews coefficient of about 2.443/Da and a solvent content of 49.53%．［Conclusion］ According to the X-ray diffraction data，the three-dimensional structure of BpEctD from B．pseudofirmus OF4 soon will be analyzed，and it will provide insights into the biochemical properties of ectoine hydroxylase.

Abstract:Abstract:［Objective］We aimed to select a stable lactic acid bacteria community from switchgrass silage，that was efficient in lactic acid production.［Methods］We obtained the community by continuous restricted subcultivation in MRS broth，and analysed the composition diversity and stability of the community by 16S rRNA gene-based pyrosequencing and Denaturing Gradient Gel Electrophoresis (DGGE)，respectively．In addition，we studied the effect of different nitrogen sources on growth and lactic acid production of the community，through adding different concentrations of yeast extraction，different nitrogen sources［yeast extract，peptone，urea and (NH4)2SO4］and different proportions of (NH4 )2SO4 and yeast extract leveled with elemental nitrogen 1.8 g/L.［Results］The microbial composition of SGL became stable from the 8th generation according to the results of DGGE．The pH value of the MRS inoculated with SGL dropped to 3.7，and the concentration of lactic acid reached 26 g/L after 24 h cultivation．The result of the pyrosequencing showed that the major composition of SGL were Lactobacillus nantensis (78.78%)，Lactobacillus plantarum (7.92%)，Lactobacillus pantheris (5.27%)，Bacillus coagulans (4.41%) and Lactococcus lactics (3.31%)．The best supplementation of yeast extraction for SGL was 20 g/L．When the elemental nitrogen ratio of (NH4)2SO4 to yeast extract was 1:4，the growth and lactic acid production were no significant difference with 0:5 (P＜0.05).［Conclusion］SGL had a great potential of application，as an efficient inoculant for ensilage or lactic acid production． This study would offer theoretical basis for cultivate and application of SGL in production.

Abstract:Abstract:［Objective］To study the diversity of cultivable actinobacteria in Xinghu wetland and screen actinobacteria with a pharmaceutical potential for producing biologically active secondary metabolites.［Methods］We studied the diversity of actinobacteria isolated from Xinghu wetland by using different selective isolation media and methods. The high bioactive actinobacteria were identi ed and further investigated for the presence of polyketide synthases (PKS-I，PKS-II)，nonribosomal peptide synthetases (NRPS)，3-amino-5-hydroxybenzoic acid synthases (AHBA) and 3-hydroxy-3-methylglutaryl Coenzyme A (HMG CoA) sequences by speci c ampli cation．［Results］More than 300 actinobacteria were isolated，and 135 isolates were selected on the basis of their morphologies on different media and were further characterized by 16S rRNA gene sequencing． The isolates belonged to 7 orders，10 families，13 genera，Streptomyces was the most frequently isolated genus，followed by the genera Micromonospora and Nocardia． Twenty-four isolates showed high activity against Staphylococcus aureus and Escherichia coli，but there no strain displaying antagonistic activity against Salmonella sp．High frequencies of positive PCR amplication were obtained for PKS-I (16.7%，4 /24)，PKS-II (62.5%，15/24)，NＲPS (16.7%，4/24)，HMG CoA (29.2%，7/24) and AHBA (12.5%，3/24) biosynthetic systems． High Performance Liquid Chromatography showed that strain XD7，XD114，XD128 produce lots of secondary metabolites.［Conclusion］This study indicated that actinobacteria isolated from Xinghu wetland are abundant and have potentially benecial and diverse bioactivities which should be pursued for their biotechnical promise.

Abstract:Abstract:［Objective］To study the effect of substrates variation on the electricity production of microbial fuel cell (MFC) and anodic microbial communities．［Methods］An MFC was started up and operated by feeding in turn with lactate，then with propionate and finally with lactate． The anodic microbial communities were monitored by using cultureindependent microbial molecular ecological techniques．［Results］The switch of substrates markedly affected the power efficiency of MFC. It required relatively long time to recover the electricity generation capability as the substrate was switched． The substrates switch also changed the microbial community structure． Anaeromusa spp.，Pseudomonas spp．and Thiobacillus thioparus were dominated with lactate fed because they were enriched in the presence of lactate． When propionate was supplied as sole substrate，Dechloromonas spp. and Comamonas testosterone were selected． The electricityproducing bacteria，Geobacter spp． ，were enriched by acetate from either lactate or propionate degradation． Hence，Geobacter spp． was an overlapping microbial population in the presence of the two different substrates.［Conclusion］A well correspondence between substrate and anodic microbial community was observed in MFC with switching substrates. To reduce the effect of substrate fluctuation on the MFC electricity production，more complex organic substrate should be provided as broader nutrients which could improve the functional overlap of populations and MFC stability．

Abstract:Abstract:［Objective］To construct a phosphate starvation derepressed expression vector for functional integration and expression of exogenous genes in Rhodosporidium toruloides.［Methods］Sodium: inorganic phosphate symporter promoter pPHO89 and heat shock protein terminator tHSP were predicted by bioinformatics assay．DNA fragments of the two elements were amplified from the cDNA of the oleaginous yeast R. toruloides NP11． Then the lyceric acid kinase promoter pPGK and terminator tNOS in vector pZPK-pPGK-hyg-tNOS were replaced by pPHO89 and tHSP，respectively，using restriction free (RF) cloning method． Hygromycin phosphotransferase gene hyg was retained and as a report gene． The resulting vector pZPK-pPHO89-hyg-tHSP was transformed into R． toruloides using agrobacterium-mediated transformation (ATMT). The function of the pPHO89 promoter and the tHSP terminator can be confirmed by the transformant harboring the hygromycin resistance． Furthermore，we developed new inducible vectors containing two expression cassettes driven by the constitutive pPGK promoter and inducible pPHO89 promoter． ［Results］pPHO89 was regulated by phosphate and activated when the recombinant R. toruloides strains were grown under phosphate-limited condition.［Conclusion］pPHO89 is an ideal promoter in terms of regulation by phosphate，comparative response strength，simplicity，and cost effectiveness． These ATMT vectors can provide useful tools for rational engineering of oleaginous yeast to produce fatty acid derived biofuels and biochemicals.