Abstract:[Objective] This study aimed to identify the ORM1 gene in Pichia pastoris, and to illustrate the effect of its deletion on cell growth, endoplasmic reticulum stress response, cell calcium homeostasis and reactive oxygen species accumulation. [Methods] Sequence alignment and analysis were done by the relevant bioinformatic software. The orm1Δ mutant was constructed by PCR-mediated gene disruption, and the reconstituted strain orm1Δ+ORM1 was constructed by transforming orm1Δ with the pIB1-ORM1 plasmid. The growth rates of the strains were detected using both liquid and the solid media. Gene expression related to the unfolded protein response, calcium homeostasis and antioxidant system was detected via real-time PCR. The activity of antioxidant enzymes, including catalase and superoxide dismutase, and the content of reduced glutathione were measured by relevant kits. [Results] The P. pastoris ORM1 gene sharing high homology with S. cerevisiae ORM1 and ORM2, was identified in P. pastoris genome database. Deletion of ORM1 caused growth defect, increased sensitivity to endoplasmic reticulum stress caused by tunicamycin, activation of unfolded protein response, disturbance of calcium homeostasis, reactive oxygen species accumulation, and activation of the antioxidant system. [Conclusion] Because unfolded protein response, calcium homeostasis, and production of reactive oxygen species are all correlated with ER function, P. pastoris Orm1 protein plays critical roles in cell growth and maintenance of ER functions.

Abstract:[Objective] To study the high density fermentation process for beta-fructofuranosidase production by recombinant Escherichia coli BL21(DE3)/pET22b-β-ffase. [Methods] we compared the influence of the dissolved oxygen feedback control and the exponential fed-batch culture on the β-FFase production of the Escherichia coli BL21(DE3)/pET22b-β-ffase fermentation, and the specific growth rate and induction time were optimized. [Results] This study confirmed the specific growth rate during the dual-stage specific growth rate controlled exponential fed-batch culture. Specific growth rate were controlled at 0.20 h?1 and 0.13 h?1 in pre-induction phase and late phase after induction respectively, the express system can be induced at mid-logarithmic phase. Finally, cell dry weight reached up to 51 g/L, the peak of activity attained at 1.79×105 U/L, the unit cell enzyme activity leaped to 3 510 U/g, and the enzyme production rate reached up to 3.58×104 U/(L·h). The biomass, the unit cell enzyme activity and the enzyme production rate have been strikingly improved, which were 1.8-fold, 1.7-fold and 3.0-fold compared with values of pre-exponential feeding culture without optimization. [Conclusion] Dual-stage specific growth rate controlled exponential fed-batch culture significantly contributes to improve the enzyme β-fructofuranosidase production, and also lays the foundation for the further industrialization of the enzyme.

Abstract:[Objective] The aim of our study is to improve Coprinus cinereus peroxidase (CIP) for textile dyes decolorization. [Methods] We synthesized CIPmt4, a CIP gene with Pichia pastori codon bias and 4 amino acid substitutions (I49S, V53A, M166F and M242I), using our gene synthesis and site-specific mutagenesis platform. Then, we carried out random mutagenesis using CIPmt4 as template, after three rounds of error-prone PCR and high-throughput screening by assaying enzymatic activity toward 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid ABTS), we obtained a mutant (CIPmt5) with improved enzymatic activity. Furthermore, we built the 3-dimensional model of CIPmt5 and wild type CIP using Swiss-model software, analyzed their thermostability using molecular dynamics simulation and investigated the decolorization ability of CIPmt5 and wild-type CIP for 7 textile dyes (Congo red, Amino black, Methyl orange, Methylene blue, Aniline blue, Bromophenol blue and Crystal violet) subsequently. [Results] Sequence analysis revealed that 5 amino acid substitutions (I49S, V53A, T121A, M166F and Y272F) were accumulated in CIPmt5. Compared to the wild-type enzyme, the specific activity of CIPmt5 toward ABTS was increased to 2.01-fold (24.44 U/mg), and its optimal temperature and pH were changed from 25 °C and 5.0 to 45 °C and 6.5 of the wild-type enzyme, respectively. The optimal decolorization pH of CIPmt5 shifted toward neutral to alkaline pH except for methylene blue. [Conclusion] The new variant obtained through directed evolution shows its potential in industrial application in textile dyes decolorization.

Abstract:[Objective] To discover the diversity of endophytic fungi and establish its germplasm bank in mangrove plants of the Beibu Gulf of Guangxi, and provide the theoretical basis for using the endophytic fungi biotechnology to promote agricultural sustainable development. [Methods] Mangrove tissue samples were collected from Beibu Gulf, the fungi were isolated by surface disinfection method and screened by determining whether the isolates were pathogenic to the host plants, and the endophytic fungi were classified and identified according to the morphological characteristics and molecular biological analyses. [Results] The results showed that 1 764 isolates were obtained from 60 mangrove plant tissue samples, and 41 endophytic fungi were screened by pathogenicity test. The isolation rate of endophytic fungi was 2.3%. Among them, 15 endophytic fungi were isolated from the host plant Rhizophora stylosa, accounting for 36.6% of the total strains with the highest proportion. The results showed that these endophytic fungi were clustered into seven branches both in ITS-NJ and NS-NJ phylogenetic trees, belonging to 8 families/orders. Mycosphaerella, Devriesia, Pseudosercospora, Cladosporium and Pleosporales are the dominant fungi of mangrove in Guangxi. [Conclusion] There is a wide variety of fungal species in mangrove plants of Beibu Gulf, Guangxi.

Abstract:[Objective] To analyse methanogenic community and the pathway of biogenic methane in the formation water from coalbed methane (CBM) wells in the Qinshui Basin. [Methods] Based on the methyl coenzyme-M reductase (mcrA) gene, the diversity of methanogens from different CBM wells formation water were investigated by 454 pyrosequencing and aligning mcrA sequences from NCBI functional genes library. [Results] The high-throughput sequencing indicated that the numbers of OTUs (Operational taxonomic units) were 64 to 157 in five detected methanogens communities, only 22 OTUs were identical in all samples, which accounted for 14% to 34% in each sample. According to the alignment results of high-throughput sequencing data and mcrA library, four genera were detected in five formation water, including Methanobacterium, Methanomicrobium, Methanolobus and Methanospirillum, the dominant genus was Methanobacterium. Phylogenetic analysis indicated that the majority of unidentified genera were closely related to Methanobacterium, Methanomicrobium, Methanococcus and Methanoculleus. Although the percentage of methanogens was different in five sample, the detected genera were about the same. The hydrogenotrophic methanogenesis was main pathway of methane formation in the Qinshui Basin. [Conclusion] The methanogens species were obvious different in different CBM blocks of the Qinshui Basin. However, methanogenesis pathways were similar and had no correlation with the geographical location and reservoir conditions.

Abstract:[Objective] In order to find a safe and stable method to produce collagenase in vitro, we expressed the Bacillus cereus collagenase colR75E in Pichia pastoris. [Methods] With the Bacillus cereus genomic DNA as template, we successfully amplified the collagenase colR75E DNA fragment by PCR and cloned it into pPICZαA plasmid. The pPICZαA/colR75E recombinant plasmid was lineared with Sac I, and then the lineared plasmid was transformed into Pichia pastoris X-33 competent cell in order to integrate the inducible AOX1 promoter controlled colR75E fragment into Pichia pastoris X-33 genomic DNA. The successfully integrated Pichia pastoris X-33 strains was cultured and induced by methanol addition. To acquire the highest production, the optimized conditions for ColR75E collagenase expression in Pichia pastoris X-33 were investigated here. After induction, we purified recombinant ColR75E collagenase in supernatant sequentially by ammonium sulfate precipitation, desalting and affinity capture. Finally, the recombinant collagenase ColR75E was analyzed by catalytic activity assay, SDS-PAGE, zymography, type I collagen proteolysis and substrate specificity assay. [Results] The highest level of collagenase ColR75E induction was gained under pH 6.0 for 72 hours incubation by 2.5% methanol. As expected, the molecular weight of the recombinant collagenase is nearly 110 kD to ColR75E. The results of zymography and type I collagen degradation analysis uncovered that the recombinant collagenase ColR75E had an excellent collagen proteolysis activity. Its specific activity after purification reached to nearly 4.977 U/mg under standard conditions. The recombinant collagenase ColR75E exhibited specific proteolysis to type I collagen, but not to BSA, Casein or Lysozyme. [Conclusion] Pichia eukaryotic expression system is suitable for the expression of Bacillus cereus collagenase ColR75E, which supplied a good basement both for its subsequent theoretic research and industrial exploitation.

Abstract:[Objective] The study aims to isolate and identify the pathogen from Saxifraga stolonifera anthracnose. [Methods] The pathogen were isolated by tissue associated anthracnose and confirmed through Koch’s postulates, then the strains were identified through morphologic characteristics and multi-locus (ACT, CAL, CHS-1, GAPDH, ITS, TUB2) phylogeny analysis. [Results] Two strains, isolated from the leaves of S. stolonifera, were numbered as LPSU 20120244 and LPSU 20120251. Conidium of both two strains was hyaline, aseptate, straight, cylindrical. Conidium size of LPSU 20120244 was (11?25) μm×(5?9) μm, LPSU 20120251, (15?25) μm×(5?7) μm. Strain LPSU 20120244 and LPSU 20120251 was clustered with Colletotrichum karstii Y.L. Yang, Zuo Y. Liu, K.D. Hyde & L. Cai and C. jiangxiense F. Liu & L. Cai, respectively, and the bootstrap support of both two clades were 100%. [Conclusion] Based on morphologic characteristics and multi-locus phylogeny analysis, the two strains represent 2 Colletotrichum species, Colletotrichum karstii (strain LPSU 20120244) and C. jiangxiense (strain LPSU 20120251).

Abstract:[Objective] Current study was designed to determine the functional characterization and expression pattern of C4-dicarboxylate binding protein coding gene dctP in nitrogen fixing bacterium Pseudomonas stutzeri A1501. [Methods] We constructed the nonpolar dctP mutant. The nitrogenase activity and growth curve between the wild type and mutants were assayed in minimal medium supplemented with different C4-dicarboxylates (succinate, malate or fumarate) as a sole carbon source were measured. The dctP-lacZ fusion vector was constructed and transformed to wild type A1501, dctB mutant, rpoN mutant and ntrBC mutant, respectively. The β-galactosidase activity was detected to analyze the expression of dctP gene in recombinant strains grown with different C4-dicarboxylates as a sole carbon source. [Results] It was found that dctP mutant lost the ability to utilise C4-dicarboxylates and its nitrogenase activity was much lower than the wild type. The analysis of β-galactosidase activity indicated that the succinate, malate or fumarate induced expression of the dctP gene. In comparison with wild type, the expression level of dctP gene was significantly decreased in the deletion mutants for dctB, rpoN or ntrBC. [Conclusion] The DctP protein played an important role in the C4-dicarboxylate utilization of P. stutzeri A1501. C4-dicarboxylates (succinate, malate or fumarate) induced the expression of dctP gene. The DctP transcription was RpoN-dependent and under the control of DctB and NtrBC.

Abstract:[Objective] To screen effective biocontrol agents from healthy banana rhizosphere in a diseased field and further study the inhibition mechanism. [Methods] Double-deck plates and fermentation antagonism study were used as primary- and secondary-screening for antagonists. Observing strains were identified based on physiological and biochemical tests, 16S rRNA gene sequencing and specific gene amplification. Crude extracts of fermentation were observed by using acid precipitate method and then added to the fermentation of pathogen for 5 days. Concentration of protein, malondialdehyde (MDA), ergosterol and pectinase activities were analyzed based on colorimetric method and HPLC to find out the effect of antagonists crude extracts on the growth of pathogen. [Results] Two antagonists, named as H-2 and H-7, were observed and identified as Bacillus subtilis (GenBank: KX791428) and Bacillus amyloliquefaciens (GenBank: KX791430), respectively. Fusarium wilt of banana was suppressed by these two strains and the biocontrol efficacies were 59.1% and 53.0%, respectively, based on the greenhouse condition. Concentration of MDA was significantly increased to 0.55 μmol/L and 0.48 μmol/L by treating the pathogen hyphae with suspension of H-2 and H-7, respectively. Meanwhile, concentration of protein, ergosterol and pectinase activities were significantly decreased in both H-2 and H-7 treatments and the lower index values were observed by H-2 by showing as 0.15 mg/g, 1.31 mg/g and 0.008 7 U/mL, respectively, which were significantly lower than those of CK (0.25 mg/g, 1.96 mg/g and 0.035 U/mL). [Conclusion] In conclusion, two antagonists screened from healthy banana rhizosphere soil were able to inhibit the growth of pathogen by increasing the lipid peroxidation of pathogen hyphae and decreasing the synthesis of metabolism products, which provide theoretical basis on the biocontrol application of two antagonists.

Abstract:[Objective] To verify that Escherichia coli Nissle 1917 also exists in swine as a natural isolate and could be isolated from swine faeces. The method for identifying the Escherichia coli Nissle 1917 using in situ hybridization was established. [Methods] All 135 pieces of fresh faeces from healthy weaned piglets were collected to prepare DNA template samples. And the DNA template samples were screened and tested by PCR with five pairs of specific primers targeting the chromosomal genes encoding the major ?mbrial subunit FimA (type 1 ?mbriae) and FocA (F1C ?mbriae), and the two cryptic plasmids pMUT1 and pMUT2, and mean while the human E. coli Nissle 1917 was chosen as the positive control. The 427 bp-long plasmid fragment named pMUT2(a) was purified from the gel and made into DNA probe using digoxigenin random primer labeling. [Results] The PCR result showed that the expected fragments of the five specific primers target were amplified from the two of 135 DNA template samples and the potential presence of Nissle 1917 in pigs was initially confirmed. Using the method of in situ hybridization with prepared pMUT2(a) probe, two positive strains were screened from the two positive faecal samples. Two positive clones were finally confirmed as positive strains of Nissle 1917 through further experiments of serological test, PCR and sequencing. [Conclusion] The isolation and identification of swine-originated probiotics Nissle 1917 laid a foundation for further study and application of excellent animal originated probiotic.

Abstract:[Objective] To determine the cause of death for Taiwan Loach and screen sensitive drugs, a dominant bacteria strain Zy01 was isolated from of sick Taiwan Loach. This study will provide reference for further prevention and treatment of Aeromomas veronii in Taiwan Loach. [Methods] The pathogenic bacteria were isolated and purified from ulcerated muscle of Taiwan Loach. And the identification of phenotypic information of the strain Zy01 was conducted, including the biochemical identification and 16S rRNA gene sequence determination. The tests of artificial infection by using strain Zy01 to infect Taiwan Loach. Antimicrobial susceptibility test was conducted by K-B methods. [Results] Strain Zy01 was the pathogens of Taiwan Loach, the LC50 of the isolated was counted to 2.0×106 cfu/mL. According to morphological and biochemical characteristics as well as the result of 16S rRNA gene sequence analysis, the isolated strain Zy01 was A. veronii. Strain Zy01 was susceptible to ciprofloxacin, norfloxacin, azithromycin and gentamicin and other 10 kinds of antibiotics. Meanwhile, it showed resistance to oxacillin, penicillin, amoxicillin and other 9 kinds of antibiotics. [Conclusion] The isolated bacterial strain Zy01 was pathogenic to Taiwan Loach, and prevented by administering drugs such as gentamicin and neomycin in fisheries farming.

Abstract:[Objective] This paper studied the biotransformation of the catalytic substrate dehydroepiandrosterone (DHEA) generated 7α,15α-diOH-DHEA by Gibberella intermedia C1 in the ionic liquid-water biphasic system. [Methods] The comparison of substrate conversion rates and product yields in five different ILs/water two-phase systems was carried out. And then, the optimization of bioconversion process in ionic liquid-water biphasic system was investigated to establish a suitable biphasic system with G. intermedia C1. [Results] The optimal conversion condition in ionic liquid-water biphasic system was as follows: 6 g/L DHEA and [Emim][EtSO4] (VILs:Vwater =0.8) were simultaneously added after 12 h fermentation of G. intermedia C1. In addition, the amplification experiments were carried out in 5 L fermentor. At 60 h fermentation of G. intermedia C1, the concentration of 7α,15α-diOH-DHEA increased to 5.03±0.21 g/L and the product molar yield reached 75.5%. [Conclusion] The optimal conversion condition in ionic liquid-water biphasic system was determined and the amplification experiments were carried out on the 5 L fermentor, which laid a solid foundation for industrial application further.

Abstract:[Objective] To determine the effect of atmospheric pressure plasma jet on the inactivation of Pseudomonas aeruginosa, and to explore the sterilization mechanism of atmospheric pressure plasma jet. [Methods] The bactericidal effect of atmospheric pressure plasma jet was determined by plate counting method. Treated cells were examined by fluorescence microscope and transmission electron microscope. Soluble proteins were quantified and analyzed by SDS-PAGE electrophoresis. [Results] More than 99.9% P. aeruginosa cells were killed after the plasma treatment for 5 min. The cell wall and membrane systems of P. aeruginosa was broken as evidenced by leakage of cellular contents of proteins. Amounts of proteins was detected in supernatant after treatment for 2 min. [Conclusion] Atmospheric pressure plasma jet is able to destroy the cell integrity, which results in leakage of cellular contents and eventually kills P. aeruginosa effectively.

Abstract:[Objective] To study the inhibition of staphylococcus aureus (S. aureus) biofilm by antimicrobial peptide 17BIPHE2 used alone or combined with antibiotics. [Methods] Congo red plate test and crystal violet staining test were used to assess the ability of biofilm formation by S. aureus. Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of S. aureus were tested by broth microdilution method and agar plate method. The bacteria adhension was observed by S. aureus adhesion test and biofilm formation was analyzed by biofilm formation test. They were treated by 17BIPHE2 alone or combined with antibiotics. The removal of mature biofilm was observed by scanning electron microscope (SEM) after the treatment of 17BIPHE2 alone or combined with antibiotics. [Results] When MIC of 17BIPHE2 was 8 μmol/L, 1/2×MIC could inhibit the growth of planktonic bacteria effectively. In the adhension stage, the inhibition rate was 40% by 17BIPHE2 treated only. In the biofilm formation stage, the inhibition rate was 35%. Further, the rates for both stages decreased if combined with antibiotics. Antimicrobial peptide 17BIPHE2 could promote biofilm disintegration in the mature stage at 1/4×MIC concentration. Biofilm disintegration and bacterial adhesion could decrease at 1×MIC concentration. Furthermore, the combination of vancomycin promoted disintegration of bacterial biofilm as well as lots of leakage. [Conclusion] Antimicrobial peptides 17BIPHE2 was able to inhibit biofilm effectively, especially combined with antibiotics, could promote the inhibition more effectively. This article provides a new idea for treatment of S. aureus biofilm related infection.

Abstract:Microbiome describes the microbial community and its -omics that reside in a specific niche, and almost all the biological processes in nature are conducted by certain microbiome. Along with the development and cost effectiveness of DNA sequencing technique, microbiome has become a hot research area. After synthetic biology, synthetic microbial community based on the microbiome will increase our ability to harness the power of microbial communities in biotechnology and to manipulate such communities to promote human health, agriculture, industry and environment. This paper reviews the research progress and application of microbiome in various fields from a viewpoint of ‘from microbiome to synthetic microbial community’, and provides ideas from the theoretical research to application of microbiome.

Abstract:Deep-sea hydrothermal fields are rich in iron, zinc, copper and other metal-bearing minerals (sulfides, oxides, carbonates, etc) and inorganic gases (H2S, H2, CO2, CH4), which are unique ecosystems. Microbes, the base of the food chain, can control or induce the mineralization and thus influence biogeochemical cycling in deep-sea hydrothermal fields. Different types of microbial functional groups are involved in biomineralization processes, such as sulfur-oxidizing microbe, metal-oxidizing microbe, sulfur-reducing microbe, and metal-reducing microbe. Their phylogenetic diversity, molecular mechanisms for biomineralization and the prospect of microbial biomineralization were mentioned in the paper.

Abstract:Penicillin binding proteins (PBPs) are a class of membrane proteins that are widely present on the surface of bacteria. They are the main target of β-lactam antibiotics. In the process of the bacterial cell wall peptidoglycan synthesis, PBPs play key roles of glycosyl transferase, peptidyl transferase and D-alanyl-D-alanine carboxypeptidase and thus are indispensable for bacterial growth. Different bacteria contain various PBPs. Changes of protein structure, increase of quantity, decrease of susceptibility to antibiotics and production of new PBPs are important reasons resulting in drug-resistance to β-lactam antibiotics. With wide use of different antibacterial agents, the problem of bacterial resistance is becoming increasingly serious. Therefore, research on PBPs has been carried out in recent years around the world. In this paper, the classification, structure and function of PBPs, their relationship with bacterial drug-resistance and detection methods are summarized. This paper also indicates future research directions.

Abstract:Nematophagous fungi are an important group of soil microoganisms, which serve as natural enemies of nematodes and play important roles in maintaining nematode population dynamics in natural environments. Nematophagous fungi can attack and kill nematodes through producing specialized capturing devices, or toxins. Serine proteases are important virulence factors involved in the pathogenicity of nematophagous fungi infect against nematodes. Recently, pathogenicity-related serine proteases from different nematophagous fungi have been extensively characterized, especially, greater progress have been made in their crystal structure and molecular evolution. Here, the biochemical properties and functions of the pathogenicity-related serine proteases in nematophagous fungi were summarized, and the latest progress about their crystal structure, catalytic mechanism and molecular evolution were reviewed.

Abstract:Viruses play an important role in ecological systems and widely distribute in various environments, including hypersaline environments. The study of viruses in hypersaline environments has become a new hotspot in the field of extremophiles. To date, around 90 viruses out of more than 100 haloviruses have been described for extremely halophilic archaea, while only 14 viruses are known to infect bacterial halophiles. This article reviews the morphological properties, response to salinity and genomics of 14 bacteriophages isolated from hypersaline environments, and also analyzes their morphological diversity, survival strategies and original and evolutionary information in genomes. It reveals that the Caudovirales are the most abundant viruses in hypersaline environments. These bacteriophages are euryhaline and their adsorption and replication were affected by salinity. They may have common ancestry with those from other environments. Although after nearly 30 years research, only 14 bacteriophages were isolated from hypersaline environments. Hence, isolation and purification of halophages is one of the important works in the future, and studies combining with culture-independent technology to elucidate their diversity and ecological functions are the future developmental direction.

Abstract:The excessive accumulation of organic pollutants in the environment forms great threat to ecosystem and human health. In recent years, many researches have constantly showed prominent effects of the plant-microorganism combination on the degradation of environmental organic pollutants. It has been widely accepted that the plant-microorganism combination generally exists inside the plant and in the rhizosphere, which provides precondition to degrading the organic pollutants and lays foundation for practical application of environmental remediation. In this review, we summarized the research progress based on three aspects of the plant-microorganism combination, including plant-endophyte, plant-mycorrhiza and plant-rhizospheric microorganism. We analyzed the function of the plant-microorganism combination during the process of organic pollutants biodegradation and discussed the degradation mechanisms. However, there are still challenges to the study of the plant-microorganism combination degradation of environmental organic pollutants, the underlying mechanisms and ecological effects are unclear yet. Therefore, every effort should be made to further clarify the degradation mechanisms, as well as to enhance practical application, thus contributing to the ecosystem management and sustainable development.

Abstract:The sesame flavor liquor is one of the two flavor-typed products through innovative and creative investigation after the founding of new China. This type of liquor is famous for its unique favor combined with thick, sauce and fragrant ones generated in manufacturing process. However, the flavor and aroma mechanism of sesame flavor liquor is not yet clear. In this paper, we review from the brewing process of sesame flavor liquor, its microbial flora, the types and characteristics of flavored components.

Abstract:Bacillus is an important microbial resource forming endospore to resist stress. Taxonomy research is vital to explore and utilize functional Bacillus strains. Based on the taxono-genomic method, the classification status of Bacillus is becoming more and more precise. Untill to June 2016, 813 Bacillus-like species have been reported worldwide, belonging to 74 genera within 5 Families including Bacillaceae, Alicyclobacillaceae, Paenibacillaceae, Planococcaceae, and Sporolactobacillaceae. The first new species of Bacillus-like bacteria was published by Chinese scientists in 2004. Up to now, 176 Bacillus-like new species have been recognized, which were isolated from China. In this review, we describe the present taxonomy method for Bacillus and their application in different fields, aiming to provide relevant information for peers.

Abstract:As the soil environment is increasingly deteriorating, the potential use of ACC deaminase in improving plant tolerance and bioremediation has received significant attention in recent years. Our aim is to objectively analyze the current development of ACC deaminase studies worldwide, thus to promote the development of related research work and their utilization in bioremediation of contaminated soils in China. Based on Web of science database, the countries, institutions, authors, research fields, journals and key words of ACC deaminase literatures published during 1991?2016 are quantitatively and qualitatively analyzed. The ACC deaminase publications have witnessed a continued increase in recent years all over the world. India, Canada and Pakistan are in a leading position in the ACC deaminase research field. Both of the number and influence of the ACC deaminase publications of Canada rank the first. The ACC deaminase study in Canada has focused and strong research strength. The ACC deaminase research in China initialized late. Although the number of published paper of China ranks the fourth place worldwide, the influence is low. The worldwide research area of ACC deaminase mainly focused on agricultural production and environmental remediation. The research area in China mainly concentrated on microbial ecology, plant-microbe interaction and phytoremediation, which are worth focusing and tracking in the future. The research strength of ACC deaminase in Canada ranks the first all over the world. This research field in China started late, but has been developing rapidly during the recent five years. The hot topics of attention are also unique and forward-looking. Nevertheless, more efforts are still needed to publish high level papers, at the same time strengthening the cooperation with high level research institutes, thus to drive the overall improvement of the research strength of ACC deaminase in China.

Abstract:Since protein glycosylation was found in prokaryotes, multiple O-glycosylation mechanisms in the bacteria from varied genera have been reported. According to the requirement of O-oligosaccharyltransferase (OTase), the O-glycosylation in prokaryotes was divided into OTase-independent and OTase-dependent. Furthermore, the two glycosylation mechanisms were elucidated in detail. The insight into various O-glycosylation mechanisms in prokaryotes would facilitate the application of these pathways to explore targeted glycoproteins.

Abstract:The implementation of the experimental research module into microbiology experiment teaching improves undergraduate students’ motivation and engagement with inquiry-based learning. In this experimental research module “screening of amylase producing bacteria, optimization of fermentation conditions, bacterial identification, and strain improvement”, students will focus on studies of amylase-producing bacteria, and will be assigned with 5 experimental units that include preparation of culture medium, collection of soil samples and isolation of amylase-producing bacteria, optimization of fermentation conditions of the isolated amylase-producing bacterium, morphologic and molecular identification of bacterial strain, and strain improvement through ultraviolet mutagenesis. This experimental research module will guide students to experience an entire inquiry-based learning process by consistently planning and conducting research projects, through which students will learn and eventually master fundamental microbial experimental skills. This module is student-centered, implemented with “fixed plus mobile” mode, to ensure its integrity and consistency. To improve students’ scientific literacy and basic microbiological techniques, the examination is composed of two parts: scientific paper writing and onsite skills test. Overall, this experimental research module inspires students’ enthusiasm and motivation in learning microbiology; meanwhile it improves the teaching quality of microbiology curriculums.

Abstract:[Objective] Deep-sea derived shutter vector pSW2 has been constructed previously. In this study, a cold inducible expression vector was constructed based on pSW2 to provide useful explant materials and toolbox for the construction of an engineered strain that can degrade environmental pollutants. [Methods] Mutation experiments were performed to identify essential gene in the vector, and green fluorescent protein (GFP) was used as reporter gene to test the expression efficiency of mutated vectors. Finally, the cold inducible vector pSW4 was constructed by adding purification tags and multiple cloning sites, and by replacing of the promoter region. [Results] The mutation experiment indicated that the GFP intensity was significantly increased by deletion of fpsB gene, which encodes the ssDNA binding protein. The expression efficiency of pSW4 was higher than that of pSW2 (10.7-fold at 4 °C) by using GFP as reporter gene. The sps01203 gene in the deep-sea bacterium Shewanella psychrophila WP2 was successfully expressed using Shewanella piezotolerans WP3 as expression host and pSW4 as expression vector. Subsequent assay indicated that the protein possesses nuclease activity. Specifically, Mg2+ is essential for the ssDNA substrate, whereas Mg2+ or Mn2+ is required for the dsDNA substrate. [Conclusion] A cold-induced expression vector pSW4 has been successfully constructed, and its applicability has been verified. Thus, our study will contribute to the further applied research of bioremediation bacterium.

Abstract:[Objective] The response surface methodology was used to optimize thermotolerant Pseudoxanthomonas oxidation effect. [Methods] We use Plackett-Burman method to select key factors for the sulfur oxidation. The steepest ascent method has been used to determine the optimal range of values and confirm the central axis. Box-Behnken experimental design was used to confirm the optimal concentration of the key factors. The concentration of sulfur was determined by classic barium sulfate turbidity method. [Results] Beef extract, malt sugar and Mg2+ were the key factors affecting the sulfur oxidation performance. The results of the response surface methodology showed that the interaction effect between beef extract and Mg2+ had the most effects on sulfate conversion rate. The optimization results were: malt sugar (%)=0.07, beef extract (%)=0.11, Mg2+=0.04, the sulfate conversion rate reached peak value. The F-value of the model was 52.60 (P<0.000 1) and correlation coefficient (R2)=0.980 2 which indicated the model terms were significant and showed good degree of fitting. The simulation composting experiments results showed that the concentration of sulfate in compost increased with Pseudoxanthomonas addition. [Conclusion] This model could be used to analyze and calculate the optimal formula of thermotolerant Pseudoxanthomonas culture medium. The sulfur conversion rate increased from 36.89% to over 80% after optimization. Addition of Pseudoxanthomonas in compost could increase sulfate concentration, compared with the suspension culture under basic fermentation conditions, which was of good application prospects.