Abstract:Abstract:We provide an overview of proposals applied and projects funded by the division of microbiology，department of life sciences，National Natural Science Foundation of China in 2013,. The traits and problems in different sub-disciplines were also analyzed，which provides reference for Chinese researchers to apply funding in microbiology next year.

Abstract:Abstract:Shikimic acid(SA) is an important intermediate in the synthesis of aromatic amino acids and has emerged as a key chiral starting material for the synthesis of antiviral drug oseltamivir phosphate(Tamiflu)．Microbial production of SA has a variety of advantages，and E．coli is commonly applied in large-scale fermentation and industrial production．Metabolic engineering is one of the main technical methods to construct the industrialized high-yield shikimic acid producing strains．Phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) is the major active transport system involved in the glucose internalization and phosphorylation in E．coli in which it affects the use ratio of PEP in cell．Modification and transformation of the PTS system could regulate the flow of intracellular metabolism and reduce the waste of PEP caused by the PTS system，in the same time，a more ideal shikimic acid producing strain can be constructed combined with the specific modifications of metabolic pathways．It was reported that in the 10 L system，shikimic acid yield reached up to 0.36 mol/mol，with concentration up to 84 g/L．This paper takes a brief overview of the metabolic engineering in the shikimic acid pathway and the transformation of the glucose transporter system，summarizes some latest researches and developments in recent years.

Abstract:Abstract:［Objective］To know food contamination and genetic diversity of Campylobacter jejuni in four provinces of South China，and to provide data for C. jejuni-associated foodborne disease prevention and control.［Methods］According to the national standard and the most probable number(MPN) method，we detected the contamination of C. jejuni from 558 food samples including vegetables，meat product，cooked food，seafood，frozen food，dairy product and edible fungi during 2011 and 2012．The isolates were used to detect 12 virulence-associated genes with PCR methods and construct ERICPCR fingerprints.［Results］Fourteen positive samples were determined from 558 samples，and all positive samples come from meat product samples．The average value of MPN of positive samples was 8.77 MPN/g．Virulence-associated gene analysis reveals that more than 50% of the C.jejuni isolates had at least 9 virulence genes．Interestingly，virB11 gene was not found and the genes of pldA and wlaN were 14.30% in all isolates．Total of 15 C．jejuni isolates could be divided into 10 genotypes belonging to 3 clusters by ERIC-PCR fingerprints.［Conclusion］Meat product was the main source of C. jejuni food contamination in four provinces of South China．More control measures must be taken to avoid C．jejuni contamination.

Abstract:Abstract:［Objective］In order to redirect carbon flows into aromatic amino acids biosynthesis pathway and further improve the production of L-tryptophan in Corynebacterium pekinense PD-67，two schemes were implemented.First，the supply of phosphoenolpyruvate (PEP)，one of precursors of L-tryptophan biosynthesis，was increased. Second，the feedback inhibition of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase(DS)，a key enzyme in the aromatic amino acids biosynthesis，was relieved and the activity of DS was increased.［Methods］The phosphoenolpyruvate synthase gene (pps) was cloned from C． pekinense PD-67 chromosome by PCR and inserted into expression vector to construct a recombinant plasmid pXPPS; the aroG gene encoding DS isozymes was cloned from Escherichia coli chromosome by PCR and the mutation of Leu175Asp was introduced by site-directed mutagenesis using sequence-overlap extension PCR．The mutated gene named as aroGfbr was cloned to expression vector to construct a recombinant plasmid pXA; and the recombinant plasmid pXAPS co-expressing pps and aroGfbr was constructed．The three recombinant plasmids were transformed into PD-67 to generate the engineering strains PD-67 /pXPS，PD-67/pXA and PD-67/pXAPS，respectively．The fermentation characteristics of the three engineering strains were investigated.［Results］The expression of pps and aroGfbr was confirmed by enzyme activity assays．The deregulation of feedback inhibition of AroGfbr was confirmed by determining DS activity in the presence of three aromatic amino acids．The overexpression of pps and aroGfbr resulted in an increase of L-tryptophan biosynthesis by12. 1% and 26. 8%，respectively，while the co-expression of two genes increased the production of L-tryptophan by 35.9% in the engineering strain PD-67/pXAPS． ［Conclusion］ Both of the overexpressions of the pps gene and aroGfbr gene can increase L-tryptophan biosynthesis，while the production was further improved by the co-expression of the two genes．

Abstract:Abstract:［Objective］ To identify biomarkers associated with germination and virulence of Beauveria bassiana．［Methods］Spore germination rate and virulence of seven B． bassiana isolates against Euproctis pseudoconspersa were determined，and an LC-MS-based metabolomic analysis was applied to identify the biomarkers from mycelia and conidial extracts associated with spore germination and virulence.［Results］ The metabolites of carnitine，hercynine，acetylcarnitine，α，α-trehalose; Octa-Me，arg-arg-gln，phosphatidylethanolamine(PE(18:2/0:0))，phosphotidylcholine (PC(18:3/0:0)) and PC(18:2/0:0)) were higher in the mycelia of highly virulent isolates than those less virulent strains．Conidia of isolates with a high germination rate were characterized by containing higher levels of 2，3-dimethylmaleate，acetylcarnitine，propionyl-carnitine and PC(18:2/0:0)．Histamine，2，5-pyrrolidinedicarboxylic acid;Diamide，carnitine，acetylcarnitine，propionyl-carnitine，butyrylcarnitine，PE(18:2/0:0)，PC(16:1/0:0) and PC(18:3/0:0) were higher in the conidia of highly virulent isolates．Furthermore，relative content comparison of insecticidal cyclopeptides，such as beauverolides，beauvericins and bassianolide in mycelia showed that the content of a single peptide was not highly related to fungal virulence． However，the contents of 9 peptides were found higher in the highly virulent isolate Bb1898，suggesting that they might exert synergetic effects against insect hosts． ［Conclusion］ The common biomarkers related to fungal virulence and germination are acyl carnitine and phospholipid which may play roles in maintaining appressorium turgor pressure and providing energy for penetrating the host cuticle．

Abstract:Abstract:［Objective］This study aimed to broaden the substrate spectrum of Ralstonia eutropha W50 to use D-xylose，which can produce poly-β-hydroxybutyrates(PHB) at a high level.［Methods］The D-xylose transporter gene xylE from Escherichia coli K-12 W3110 was cloned by PCR technique and integrated into the R．eutropha W50 chromosome．The recombinant strain W50-E was obtained．The D-xylose catabolic genes xylAB from E. coli K-12 W3110 and the promotor of PHA synthase gene phaC1 from R．eutropha H16 were cloned into pBBR1MCS to construct a recombinant plasmid．The plasmid was transformed into R．eutropha W50 and W50-E to generate the recombinant strains W50-AB and W50-EAB respectively．The characteristics of D-xylose utilization by W50-AB and W50-EAB were investigated.［Results］The expression of xylA and xylB genes in R．eutropha W50 was confirmed by enzyme assay．The recombinant strain W50-AB could grow on 0.1 mol/L D-xylose with the maximum specific growth rate of 0.025 h－1，but no growth and D-xylose consumption were observed when cultivated on 0.01 mol/L D-xylose．The recombinant strain W50-EAB exhibited a faster growth than W50-AB on 0.1 mol/L D-xylose，with the maximum specific growth rate of 0.035 h－1．Furthermore，it exhibited a slow but defined growth and D-xylose consumption on 0.01 mol/L D-xylose．The PHB content assay showed that both recombinant strains accumulated a small amount of PHB，with a proportion of 15.07±1.01% and 15.07±1.64% on the basis of dry cell weight respectively，by using D-xylose(0.1 mol/L) as substrate．And their final D-xylose-PHB conversion rates were 0.0920 g·g－1 and 0.0838 g·g－1 respectively，which were much lower than their glucose-PHB conversion rates(＞0.22 g·g－1)．However，the recombinant strains W50-AB and W50-EAB exhibited better fermentation performance and more PHB accumulation when using glucose(0.01 mol/L) and D-xylose(0.09 mol/L) mixed sugars as fermentative substrate.［Conclusion］The recombinant strain W50-AB can metabolize D-xylose by the expression of xylAB genes，and the further expression of xylE gene is able to improve its D-xylose consumption rate．Meanwhile，the two recombinant strains can accumulate a small amount of PHB by using D-xylose as the sole carbon source．

Abstract:Abstract:［Objective］The gene diversity of the bacterial 48 family glycoside hydrolase(GH48) in rumen environment was studied and new gene resources for efficient cellulose degradation were provided．［Methods］A pair of gh48 degenerate primers was designed through sequences alignment of the gh48 gene sequences from ruminal Ruminococcus．The total DNA and RNA were extracted from two rumen samples and cDNA was synthetized through reverse transcription from total RNA． Four gh48 gene clone libraries were constructed and analyzed．［Results］In total 455 gh48 gene sequences were obtained from the 4clone libraries． Sequence similarity among the 455 gene sequences varies between 58.65% and 100%．They fell into 66 species with the sequence similarity ≥89%，and divided into 5 different clusters．OTU65 in cluster Crepresents an abundant gh48 gene which in both DNA and cDNA clones libraries，accounting for 36.4% and 19.5% respectively．Our studyrevealsrich gene diversity of the 48 family glycoside hydrolase and provided new gene resources for cellulose degradation.

Abstract:Abstract:［Objective］ The purpose of this research was to apply Aspergillus aculeatus solid fermentation extracts to convert stevioside and rebaudioside C，followed by identifying and purifying the new conversion product．［Methods］The product was identified by high performance liquid chromatography，chromatography-mass spectrometry and Infrared spectrum．The new product and rebaudioside A exited in the supernatant were purified by alcohol and macroporous resin．［Results］The Aspergillus aculeatus enzyme extracts could convert the stevioside and rebaudioside C to the new product within 10 hours． The conversion rate was 98.0% in 24 hours．The conversion product existed in deposit was identified as steviol．The purity and recovery percent of steviol were 95.2% and 84.0% respectively．Because stevioside could occur to deposit，the rebaudioside A existed in supernatant was purified easily． We used the resin chromatography to purify RA and the recovery could reach 80.5%．［Conclusion］Aspergillus aculeatus enzyme extracts could convert stevioside efficiently and specifically，and we could obtain rebaudioside A and steviol at the same time．

Abstract:Abstract:［Objective］The objective of this study is to understand the contamination of human enteric viruses in economic shellfish along the Chinese coast，an important issue of ensuring the seafood safety.［Methods］We established the specific，sensitive and high-throughput gene chip technology，to investigate the contamination of economic shellfish by enteric viruses across a large geographical region of China.［Results］The percentage of positive samples for each virus was as follows: Hepatitis A Virus 4.3%，norovirus 14.8%，rotavirus 6.2%，astrovirus 5.6%，and adenovirus 9.9%．In these five viruses，norovirus was contaminated in the first place．The results detected by gene chip were highly consistent with that of polymerase chain reaction ( PCR)．The economic shellfishes in shellfish-growing areas along the coastal cities were all contaminated with enteric viruses at different levels． However，there was no significant correlation between any two cities．In the selected 6 economic shellfishes，oyster had the highest positive rate of enteric viruses，followed by blood clam.［Conclusion］The contamination of shellfish with human enteric viruses was common across the main coastal cities of China，indicating a potential public health threat from seafood.

Abstract:Abstract:［Objective］We determinated the virulence factors of Vibrio alginolyticus strains isolated from the environment by multiplex PCRs and animal experiments，in order to compare the differences between the highly virulent strain and attenuated virulent strain，and to explore the virulent mechanism of V. alginolyticus in mammals.［Methods］ The virulence-related genes of V. alginolyticus were investigated by multiplex PCRs．Hemolysin and pathogenic proteins were detected using Kanagawa phenomenon tests and enzyme activity tests．In vivo pathogenetic tests of V．alginolyticus were done through orogastric and intraperitoneal Kunming mousel.［Results］Amylase and lecithinase activities were observed in 100% of the strains，whereas lipase and gelatinase activities were found in only 70% and urease activity was not detected. In Kanagawa phenomenon tests 60% of the strains gave positive results． The related virulence genes such as toxR，Collagenase，tlh，FlaA，ompW，AspA and fur were distributed among 10 strains of V．alginolyticus collected，with the exception of toxS，trh，tdh and UreR．Among those 10 strains，VA009 has shown a strong pathogenesis to the mouse，which caused fluid accumulation and led the mortality rate as high as 80% within 7 days by intraperitoneal infection.［Conclusion］This study indicates that there is a great difference in pathogenicity among V. alginolyticus strains to mouse．The cell toxicity of V．alginolyticus made more contribution than extracellular secretion，while the extracellular secretion of V. parahaemolyticus played a major role in its toxicity. The virulence gene profiles were consistent between the highly virulent and attenuated virulent strains，indicating that V． alginolyticus might have a different virulence system and different pathogenic mechanism compared with V． parahaemolyticus．

Abstract:Abstract:［Objective］To construct an expression vector for alkaliphilic Bacillus sp．N16-5．［Methods］Bacillus subtilis expression vector pHCMC04 was used as a backbone．Its xylose-inducible promoter cassette was replaced by the constitutive promoters P43 (from B．subtilis) and PEF(from Bacillus sp． N165)，separately，resulting in two expression vectors pABN165P43 and pABN165PEF．Green fluorescent protein gene gfp was linked to the two vectors as a reporter gene．Fluorescence microscope and multifunctional fluorescent reader were used to test the expression efficiency of the system．［Results］Green fluorescence was visualized in Bacillus sp．N16-5 with pABN165PEF-gfp or pABN165P43-gfp．Quantitative data analysis revealed that fluorescence was first detected around the 7th hour．The fluorescence intensity increased rapidly from the 7th hour to the 12th hour and reached the maximum at about the 12th hour.［Conclusion］Two expression vectors for Bacillus sp．N16-5 have been constructed，allowing expression of exogenous protein in alkaliphilic Bacillus sp．N165．

Abstract:Abstract:The error-prone PCR is one of the main methods for in vitro gene mutagenesis，usually through adding Mn2+，increasing Mg2+ and dCTP/dTTP concentration．［Objective and Methods］In this study，both the antifungal protein gene Ace-AMP1 from Allium cepa and the Bt toxin gene cry1A (c) from Bacillus thuringiensis were subjected to PCR mutagenesis through reducing the dATP concentration，but without adding Mn2 + or adjusting other PCR components．［Results］The result showed that the rates of base mutation and sequence variation were increased along with the decrease of dATP concentrations． When dTTP/dCTP/dGTP:dATP equaled 20∶1－40∶1，the rate of base mutation was between 1.4% and 1.8%，and the rate of sequence variation was between 77.8%and 100%．［Discussion and Conclusion］This method is simple and practical，and enables the process optimization of several mutagenic factors in conventional errorprone PCR．Moreover，as the resulting base mutations were mainly A·T→G·C transition，the present method provides a new way to improve the GC content of gene by in vitro mutagenesis．The mutagenesis method of simply reducing single dNTP concentration could improve AT or GC content of the target gene，it is an expansion of error-prone PCR.

Abstract:Abstract:［Objective］At transcriptional level，the inhibitory effects of formic acid was investigated on Candida shehatae，a model yeast strain capable of fermenting xylose to ethanol． Thereby，the target genes were regulated by formic acid and the transcript profiles were discovered．［Methods］ On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose，the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search．These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid．［Results］By quantitative analysis of 42 candidate genes，we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid，respectively.［Conclusion］With regard to gene transcripts regulated by formic acid in C．shehatae，the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2)，acetyl-CoA synthetase (ACS)，ribose-5-phosphate isomerase (RKI)，transaldolase (TAL)，phosphogluconate dehydrogenase (GND1)，transketolase (TKL)，glucose-6-phosphate dehydrogenase (ZWF1)，xylose reductase (XYL1)，pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD)，glucokinase (GLK)，malate dehydrogenase (MDH)，6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH)．