Abstract:[Background] 1,2,4-butanetriol (BT), a chiral polyhydric alcohol, is an important chemical intermediate in organic synthesis. Among the biosynthesis routes, four-step enzymatic reaction with xylose as raw material is popular with scholars. However, Escherichia coli shows poor robustness, with low tolerance to some inhibitors in the fermentation broth and carbon catabolite repression. In recent years, the robust yeast has attracted the interest of researchers, particularly the Candida tropicalis which harbors the xylose transporter.[Objective] To develop the metabolic pathway from xylose to BT in C. tropicalis.[Methods] The xylose reductase gene GRE3 in C. tropicalis was knocked out to block the innate xylose metabolic pathway. The genes encoding D-xylose dehydrogenase (xylB) and D-xylonate dehydratase (xylD) from Caulobacter crescentus and the gene encoding the branched-chain α-keto acid decarboxylase (kdcA) from Lactococcus lactis were cloned into C. tropicalis 207, to yield the recombinant C. tropicalis BT. Then, the ability of the recombinant C. tropicalis to synthesize BT from xylose was investigated and the rate-limiting step was identified. The production of BT was improved by increasing the copy number of key genes xylD and kdcA.[Results] Under the conditions of 30℃, 200 r/min, inoculum of 1%, and 30 g/L xylose, the yield of BT of the recombinant bacteria reached 1.2 g/L. In a 5 L fermenter, the yield was up to 3.7 g/L. [Conclusion] We developed a BT metabolic pathway in C. tropicalis with xylose as substrate, and a strain with high BT yield was constructed by increasing the copy number of key genes on the genome. The study lays a foundation for further improving the production of BT in C. tropicalis.

Abstract:[Background] Phages are an important part of microbial community, but the classification and presence of them in traditional baijiu fermentation are still unclear. [Objective] By exploring prophage regions in the genomes of Bacillus licheniformis in public databases and in sauce-flavor baijiu fermentation, we intend to clarify the classification and infection of prophages in key functional strains in traditional sauce-flavor baijiu fermentation. [Methods] With culture-independent (bacterial whole genome analysis) and culture-dependent (strain screening and specific PCR) techniques, the classification and presence of prophages in B. licheniformis from different natural habitats and sauce-flavor baijiu fermentation were analyzed. [Results] The whole genome analysis of bacteria detected 165 prophages in genomes of 30 B. licheniformis strains from different habitats, of which 63.6% (105/165) had intact sequences. A total of 97.1% of the prophages in the B. licheniformis belonged to Siphoviridae, and 2.9% Myoviridae. The functions of 53.0% genes of the intact prophages were unknown. Seven integrated prophage regions were detected in sauce-flavor baijiu-derived B. licheniformis MT-B06, of which 57.1% (4/7) were intact prophage regions. Co-infection of multiple prophages was detected in B. licheniformis from sauce-flavor baijiu fermentation. There was horizontal gene transfer of CotD family spore coat protein gene on the bacterial genome to the prophage. In the 26 sauce-flavor baijiu-derived B. licheniformis strains, 69.2% (18/26) were detected positive of prophage-encoded major capsid protein gene, and 100.0% (26/26) positive of prophage-encoded CotD family spore coat protein gene. [Conclusion] High level of prophage integration was found in B. licheniformis from different natural habitats and sauce-flavor baijiu fermentation. Horizontal gene transfer of CotD family spore coat protein gene from host to the prophage of B. licheniformis in sauce-flavor baijiu fermentation was widely observed. This study is the first time to explore the classification and presence of bacteriophages in traditional baijiu fermentation, which helps to deepen the understanding of bacteriophage-host interaction in the fermenting microbial community.

Abstract:[Background] Dark septate endophyte (DSE) widely colonize plant roots in cadmium- contaminated habitats, and can enhance host plants' uptake of mineral nutrients and cadmium tolerance. [Objective] The response of DSE Exophiala pisciphila to mineral nutrient and low molecular weight organic acid secretion was studied.[Methods] Liquid culture method was used to explore the effects of different concentrations (0, 25, 50, 100, 200, 400 mg/L) of cadmium on DSE mycelial growth, mineral elements (nitrogen, phosphorus, potassium, sulfur, magnesium, iron, calcium) and cadmium content and oxalic acid secretion.[Results] With the increase of cadmium concentration, the mycelium biomass dropped by 22.8%-90.6%, and the content of nitrogen, potassium and iron in mycelia decreased by 26.0%-52.8%, 53.8%-92.9% and 12.8%-34.3%, respectively. The phosphorus, magnesium and calcium content increased by 15.4%-111.4%, 20.4%-31.4% and 35.1%-62.5%, respectively, and the sulfur content increased by 25.1% under 100 mg/L cadmium. Cadmium stress also caused a decrease in the pH value of the culture medium, and the oxalic acid concentration and the oxalic acid secretion per unit mycelium elevated remarkably. Correlation analysis showed that the cadmium content in mycelia was negatively correlated with that of sulfur and potassium (P<0.05, P<0.01), while positively correlated with oxalic acid secretion (P<0.01).[Conclusion] Cadmium stress can significantly inhibit the growth of DSE, change its absorption of mineral elements, and promote the secretion of oxalic acid.

Abstract:[Background] Soil microbial diversity has become the research frontier and hot spot in soil science, and science-based soil sampling is the prerequisite for accurate research result. [Objective] To explore the influence of sampling strategy on the detection result of soil nematode-trapping fungi (NTF) and to seek for scientific soil sampling method.[Methods] Soils were collected from different number of sampling sites (3, 5, 9 and 13) which were indifferent arrangements (straight line, V-shaped, W-shaped, triangle-like, and plum blossom-like) in 3 habitats and they were either mixed or unmixed for detection. NTF were purified by single spore isolation. The species were identified based on morphological observation and molecular techniques, and the difference in species diversity and species number of NTF among soils collected with different methods was analyzed. [Results] In the case of single-site sampling, separate purification, and integrated analysis, the α diversity and the number of detected strains rose with the increase in the integrated sampling sites. The difference in the integrated data of 5 and 9 sites was small and the detection results were relatively stable. In the instance of mixing of soil from different sites and then purification, the mixing of soil from more than 5 sites was not conducive to the detection of NTF. [Conclusion] Soil sampling strategy affects pure culture results of NTF. In the case of single-site sampling, separate purification, and integrated data analysis, the 5-site or 9-site sampling is optimal. As for the strategy of soil mixing first and then purification, the 5-site sampling is relatively scientific and feasible.

Abstract:[Background] The natural forest dominated by Picea purpurea plays an important role in maintaining ecological security in the upstream of Taohe river. It is still unclear how altitude influences the bacterial diversity and nutrients in rhizosphere and non-rhizosphere soil of P. purpurea, and how the altitude, soil nutrients, and bacterial diversity interact with each other. [Objective] To explore the community structures of bacteria in rhizosphere and non-rhizosphere soil of P. purpurea and the influencing factors.[Methods] Illumina Miseq was employed for sequence analysis of the rhizosphere and non-rhizosphere bacteria in the natural P. purpurea forest at different elevations in the upper reaches of Taohe river. The changes of soil physical and chemical factors and bacterial diversity with altitude were analyzed. Correlation and redundancy analysis was performed to elucidate the effect of some environmental factors on bacterial community. [Results] The nutrients of rhizosphere and non-rhizosphere soil samples of P. purpurea increased first and then decreased with the rise of altitude. The intergroup difference was significant for rhizosphere soil nutrients (P<0.05) but insignificant for non-rhizosphere soil nutrients (P>0.05). The rhizosphere species diversity index (H), evenness index (E), richness index (Chao1/ACE), and number of operational taxonomic units (OTU) demonstrated a unimodal trend with the increase in altitude. The non-rhizosphere bacteria diversity showed a bimodal variation trend with the rise of elevation. Bacteria diversity was in close correlation with soil nutrients, particularly in positive correlation with organic matter, total nitrogen, and available nitrogen (P<0.05) but in negative correlation with pH and available phosphorus (P>0.05). The community structures of bacteria in the forest at different elevations were highly consistent. A total of 7 159 bacterial OTUs were identified from 30 samples, which belonged to 37 phyla. The dominant bacteria phyla were Actinobacteria, Proteobacteria, Acidobacteria, and Chloroflexi, respectively. The response of different bacteria phyla to soil nutrients was different, and organic matter, total nitrogen, and available nitrogen were in positive correlation with Proteobacteria (P<0.05). [Conclusion] Composition and diversity of rhizosphere and non-rhizosphere soil bacteria of P. purpurea are significantly influenced by soil physical and chemical factors. The driving effects of environmental factors such as altitude and hydrothermal conditions on plants and soil are important reasons for the stable bacterial community structure. This study is expected to help gain a clearer insight into the variation of soil bacteria diversity of P. purpurea and the driving mechanism, which is expected to provide a reference for natural forest restoration in the upstream of Taohe river.

Abstract:[Background] With the industrialization, heavy metals have been under the major environmental pollutant category. Microbial remediation has become a popular method for heavy metal removal in recent years. Thus, screening and developing related efficient microbial strains is of important practical significance. [Objective] To screen the strain with heavy metal-absorbing function and plant growth-promoting (PGP) activity, so as to provide microbial resources for both bioremediation and plant growth promotion. [Methods] The selective medium was used to screen the lead-tolerant strain from sludge. The strain was identified based on morphological observation, physiological and biochemical determination, and 16S rRNA gene sequence analysis. Through single-factor experiment, the effects of different culture conditions on the growth of the strain were analyzed. Atomic absorption spectrometry, colorimetry, and plate confrontation assay were employed to analyze the lead-absorbing efficiency, inorganic phosphorus-solubilizing ability, secretion of indole-3-acetic acid (IAA), and the ability of antagonizing Fusarium. [Results] A strain with high lead-adsorbing efficiency (over 90% for 150 mg/L Pb²⁺), which was identified as Bacillus cereus and named SEM-15, was screened out. It can solubilize inorganic phosphorus, secrete IAA, and antagonize Fusarium. Moreover, it had strong adaptability, as it survived at pH 10.0. Therefore, the strain has great potential in removing lead, promoting plant growth, and preventing diseases. [Conclusion] SEM-15, with lead-adsorbing function and PGP activity, is of great value in remediation of heavy metal-contaminated soil and plant growth.

Abstract:[Background] Phenolic compounds are among the main water pollutants in the environment. The Mn(III) -organic acid chelate mediated by versatile peroxidase (VP) is considered to have great potential in the degradation of phenolic organic pollutants owing to the high redox potential. [Objective] To explore the degradation of phenolic compounds by VP-mediated Mn(III)-organic acid chelate and thereby to provide a new mindset and method for the biodegradation of phenolic compounds. [Methods] The active VP from Pleurotus eryngii (PeVP) was obtained by renaturation of inclusion body in vitro. We characterized the enzymatic properties of PeVP and optimized the reaction conditions of Mn(III) chelate to explore the ability of the complex system to degrade the phenolic compounds. [Results] The optimum conditions for renaturation of PeVP were as follows:The inclusion body was refolded in pH 9.5 solution containing 0.5 mol/L urea, 0.5 mmol/L oxidized glutathione (GSSG), 0.1 mmol/L dithiothreitol (DTT), 0.1 mmol/L ethylenediamine tetraacetic acid (EDTA), 5 mmol/L CaCl₂, 10% glycerol, and 5 µmmol/L hematin at 4℃ for 24 h, and then incubated with 5 µmol/L hematin for 12 h. The optimal reaction conditions of PeVP-mediated Mn(III)-organic acid chelate were 75 mmol/L malic acid buffer (pH 4.5), 6 mmol/L Mn²⁺, and 0.2 mmol/L H₂O₂. Under the above conditions, the catalytic activity of the chelate system on phenolic compounds 2,2ʹ-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol (DMP), guaiacol, and syringaldazine was detected, and it was found that the oxidation activity of Mn(III) chelate system at pH 4.5 was 1.5-7.5 times that of PeVP alone. Moreover, the average degradation rates of phenol, hydroquinone, resorcinol, and p-nitrophenol by Mn(III)-malic acid chelate system were 10.91, 10.69, 6.50, 5.71 mg/(L·h), respectively. Therefore, the Mn(III)-organic acid complex might capture the electrons of phenolic substrate to form phenolic free radicals and lead to the fracture of benzene ring with the decrease of bond energy, thus achieving the oxidative degradation of phenolic compounds. [Conclusion] At pH 4.5, Mn(III)-malic acid chelate system mediated by PeVP shows strong ability to oxidize phenolic substrates, which is a promising solution for the biodegradation of phenolic organic pollutants.

Abstract:[Background] High concentrations of nicotine waste are generated during the production and processing of tobacco, causing serious environmental pollution. [Objective] The purpose of this study is to screen out the microbial strain capable of degrading nicotine, and analyze the metabolic pathways of the strain in nicotine degradation. [Methods] We employed the conventional method for strain isolation and identified the target strain based on morphological characteristics and molecular evidence. The fermentation conditions including nicotine concentration, temperature, and pH were optimized by single factor tests with degradation rate as the indicator. The main metabolites of the strain in nicotine degradation were detected by gas chromatography-mass spectrometry. [Results] Arthrobacter sp. D4 was isolated, which could utilize nicotine as the sole carbon and nitrogen source. The optimal conditions of the strain for degrading nicotine were 30.0℃, pH 7.0, and nicotine concentration of 1 g/L, under which the degradation rate reached 90% within 18 h. The growth of the strain was significantly inhibited when the concentration of nicotine in the culture medium was greater than or equal to 4 g/L. Subsequent experiments revealed that the strain produced new end-products N-methylpyrrolidone and cotinine and the intermediate product myosmine, which were different from the reported degradation pathway of Arthrobacter sp. [Conclusion] In this study, an Arthrobacter sp. strain characterized by fast nicotine degradation was isolated and identified. Moreover, this strain may have a new nicotine degradation pathway.

Abstract:[Background] Elucidation of antibiotic biosynthetic pathways can help improve the yields of target compounds and create new compounds with more effectiveness. In-frame deletion of genes is a routine method in the study of natural product biosynthesis. By analyzing the intermediate products accumulated by mutant strains, we can deduce the synthetic pathways of natural products and the functions of involving genes. The biosynthetic gene clusters for natural products are generally more than 20 kb in size, which make the in-frame deletion of each gene time-consuming and labor-intensive. Therefore, it is of great significance to optimize the method for in-frame deletion of genes derived from Streptomyces. [Objective] On the basis of the principle of PCR-targeting, we redesigned a method for gene in-frame deletion in cosmids from Streptomyces gene library and established a technical system for rapid and high-precision in-frame deletion of Streptomyces genes in Escherichia coli. [Methods] We used the ampicillin resistance gene bla as a marker for the screening of PCR-targeting DNA fragments and replaced the in vivo Flp/FRT system by the in vitro Pac I digestion and ligation system to mediate the construction of in-frame deletion vectors.[Results] Using this method, we completed the in-frame deletion of 14 genes in the mildiomycin biosynthetic gene cluster within 6 days. [Conclusion] Compared with the traditional PCR-targeting method, the established method demonstrates improved efficiency of constructing in-frame deletion vectors. The rarity of the Pac I recognition sequence in the Streptomyces genome makes this method universal for constructing in-frame deletion vectors of essential genes in antibiotic biosynthesis gene clusters.

Abstract:[Background] China, one of the world's largest agricultural economy, has seen the large-scale use of pesticides. However, the overuse of pesticides, which has led to high detection rate, has threatened the environment and human health.[Objective] Alkaline phosphatase (ALP) can degrade organophosphorus pesticides. Therefore, we optimized the extraction of ALP from Lactobacillus rhamnosus Z23 (LGG Z23) and explored the mechanism underlying the degradation of organophosphorus pesticides. [Methods] Single factor test and orthogonal test were used to optimize the extraction. The enzyme activity was determined by detecting the amount of p-nitrophenol released. Fractional precipitation and chromatography were employed for the purification of ALP. The degradation rate of organophosphorus pesticides was determined based on the inhibition of acetylcholinesterase. [Results] The optimum conditions for extracting ALP from LGG Z23 were as follows:disruption of cells for 15 min at 450 W, material-liquid ratio (mass to volume ratio) of 1:6, and pH 10.0. Under the conditions, the activity of ALP was (4.95±0.26) U/mL, 2.11 times higher than that before optimization. The degradation rate of 6 organophosphorus pesticides was in the order of DDVP (95.79%±0.01%)>methyl parathion (90.69%±0.03%)>chlorpyrifos (88.90%±0.02%)>trichlorfon (86.07%±0.03%)>malathion (85.31%±0.02%)>dimethoate (83.18%±0.03%). Among them, the activity of degrading DDVP and methyl parathion was the highest (over 90%), and the difference was significant (P<0.05). [Conclusion] The result lays a theoretical basis and provides data for the application of ALP from LGG Z23.

Abstract:[Background] Sclerotinia ginseng, one of the major pathogen attacking ginseng, seriously affects the yield of ginseng. [Objective] To explore the mechanism of ethyl acetate extract of the endophytic bacteria Endomelanconiopsis microspora from Taraxacum albiflos Kirschner & Štepanek in inhibiting S. ginseng. [Methods] The inhibitory effect was verified by the test of mycelial growth and spore germination of S. ginseng. The morphological changes of mycelia were observed under a microscope. The cell membrane permeability was detected by changes in conductivity and nucleic acid content, and the membrane lipid peroxidation was measured by changes in malondialdehyde (MDA) content and superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. [Results] The ethyl acetate extract from E. microspora significantly inhibited the mycelial growth of S. ginseng with the minimum inhibitory concentration (MIC) of 3.75 mg/mL and the inhibition rate of 76.22% after 6 days of culture. The ethyl acetate extract significantly inhibited the spore germination of S. ginseng. The 15.00 mg/mL ethyl acetate extract demonstrated the optimal inhibitory effect with the inhibition rate of 90.69%. The ethyl acetate extract influenced mycelial morphology, increased cell membrane permeability of S. ginseng, and resulted in extravasation of mycelial inclusions. Compared with the control group, the treatment with 7.50 mg/mL ethyl acetate extract for 10 h increased the conductivity and nucleic acid content by 30.11% and 62.85%, respectively. At the same time, the ethyl acetate extract significantly increased MDA content and SOD, POD and CAT activities of S. ginseng. The MDA content and SOD, POD and CAT activities in the 7.50mg/mL treatment group first increased, peaked at the time point of 12 h, and then decreased. [Conclusion] The ethyl acetate extract from the endophytic bacteria E. microspora changed the cell membrane permeability of S. ginseng, aggravated the membrane lipid peroxidation, and destroyed the cell membrane integrity, thus leading to the loss of cell inclusions and inhibiting the spore germination and mycelial growth.

Abstract:[Background] Fusarium wilt of banana is a devastating disease in banana production. Biological control is an effective means to prevent the occurrence of this disease. In previous studies, Streptomyces misionensis TF78, a biocontrol strain, was isolated from the rhizosphere soil of healthy bananas, which showed good control effect on fusarium wilt of potted bananas. However, the biocontrol potential of S. misionensis TF78 against fusarium wilt of banana in the field and its impact on soil microbial environment are still unclear.[Objective] The paper was designed to evaluate the control effect of S. misionensis TF78 on fusarium wilt of banana in the field and determine its influence on the microbial communities in the rhizosphere soil of bananas. [Methods] The control effect of the biocontrol strain on fusarium wilt of banana was measured in two banana plantations. Microbial diversity and abundance of 12 rhizosphere soil samples from the inoculation group and the blank control group were analyzed by amplicon sequencing. [Results] The field control efficiency of S. misionensis TF78 on the two banana plantations were 55.30% and 45.32%. The slope for rarefaction curve of the inoculation group was larger than that of the blank control group, and Kickxellomycota was significantly enriched, while the abundances of Chloroflexi, Acidobacteria and Bryobacter were reduced. The biocontrol strain had no marked effect on the relative abundances of the dominant Proteobacteria, Firmicutes, Actinobacteria, Gemmatimonadetes, Trichoderma, Sphingomonas, Stenotrophomonas and Bacillus in the soil. [Conclusion] S. misionensis TF78 shaped the unfavorable soil environment to the survival of Fusarium oxysporum f. sp. cubense, and reduced the occurrence of fusarium wilt of banana in the field. Additionally, the strain failed to affect most of the dominant microbial populations with important ecological and bacteriostatic functions in the soil. The results lay a foundation for further development and application of S. misionensis TF78.

Abstract:[Background] Alternaria alternate-induced black spot, among the major diseases of Korla pear during storage, lowers the quality of the fruit, thus causing huge economic loss. [Objective] To screen strains with strong inhibitory effect on A. alternata, explore the underlying mechanism, and thus to lay a theoretical basis for postharvest preservation of Korla pear.[Methods] The rhizosphere soil samples of healthy Korla pear plants were collected and bacteria were isolated and purified by serial dilution and plating. The antagonistic strains were screened by plate confrontation assay and cylinder plate method. The preliminary identification of antagonistic bacteria was based on morphological characteristics and 16S rRNA gene sequence analysis. The antifungal spectrum of antagonistic bacteria was determined by plate confrontation assay. The extracellular enzyme species of antagonistic bacteria were detected by specific plate. The enzyme activity was quantified with 3,5-dinitrosalicylic acid colorimetric method and Folin-phenol method. Spore germination test and scanning electron microscopy (SEM) were employed to detect the antagonistic effect of the bacteria on A. alternaria. [Results] JE53 and JE56 with strong activity against A. alternaria were screened out from 152 isolates, with inhibition zone diameters of 22.49 mm and 22.40 mm, respectively. According to phylogeny analysis, they were identified as Paenibacillus polymyxa (JE53, GenBank accession No. OL966428) and Bacillus subtilis (JE56, GenBank accession No. OL966429), separately. The results of antifungal spectrum showed that JE53 and JE56 had different degree of inhibition on 9 plant pathogens such as Rhizoctonia solani. They produced four kinds of extracellular enzymes. The supernatant of the fermentation broth could significantly suppress the spore germination of A. alternaria, with inhibition rates of 70.30% and 88.87%, respectively. SEM showed that the suppressed hyphae were rough and distorted.[Conclusion] JE53 and JE56 inhibit A. alternaria, which have the potential of further development and application.

Abstract:[Background] The indigenous Saccharomyces cerevisiae NX11424 in Ningxia enjoys a reputation in fermentation, as it endows the Cabernet Sauvignon wine with strong fruit aroma. [Objective] To investigate the fruit aroma characteristics of Cabernet Sauvignon wine fermented with NX11424. [Methods] Cabernet Sauvignon was used for wine fermentation. Three fermentation conditions were designed:spontaneous fermentation, inoculation with NX11424 after grape sterilization, direct inoculation with NX11424. 26S rDNA D1/D2 sequencing was used to analyze and identify the yeast species during wine fermentation. The aroma components of Cabernet Sauvignon wine fermented under different conditions were determined by head space-solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). [Results] The physicochemical indexes of wine fermented under the three conditions showed no significant difference. The isolated yeasts belong to 3 species in 2 genera:Hanseniaspora uvarum, S. cerevisiae, and S. boulardii. All the 3 species were detected in spontaneous fermentation and only H. uvarum and S. cerevisiae in fermentation with NX11424. A total of 69 kinds of aroma components were identified respectively in the wine fermented under the three conditions:28 esters, 25 alcohols, 5 organic acids, 2 terpenes, and 9 others. There were no significant differences in species of these aroma components among different treatments but significant differences in the content of them. Cluster analysis showed that these 69 kinds of aroma components were clustered into 3 clades. Clade Ⅰ contained 9 components such as citronellol and propanol, among which 7 had high content in the wine fermented by inoculation with NX11424 after grape sterilization. The 31 components in clade Ⅱ such as ethyl hexanoate and ethyl palmitate had high content in the wine fermented by in direct inoculation with NX11424. Clade Ⅲ had 29 components such as ethyl butanoate and ethyl lactate, among which 27 had high content in the wine under spontaneous fermentation. Although the yeast diversity under direct inoculation fermentation was lower than that under spontaneous fermentation, the wine fermented by direct inoculation showed high content of esters and strong fruit aroma. [Conclusion] The interaction between NX11424 and other indigenous yeast during wine fermentation could improve wine quality. This study lays a solid foundation for using S. cerevisiae NX11424 to improve wine quality of Cabernet Sauvignon wine.

Abstract:[Background] Isoamyl alcohol is the main higher alcohol synthesized by yeast via the catabolic Ehrlich pathway or by anabolic amino acid metabolism during Chinese spirit fermentation, and the content of isoamyl alcohol influences the drinkability of Chinese spirit. [Objective] To analyze and compare the ability of yeasts isolated from fermented grains to synthesize isoamyl alcohol and reveal the synthetic pathways of isoamyl alcohol in the yeasts.[Methods] Isoamyl alcohol-synthesizing yeast strains were isolated from fermented grains of strong-aroma Chinese spirit, and their capability of synthesizing isoamyl alcohol at different growth phases was compared. Moreover, the synthetic pathways of isoamyl alcohol in these strains were detected based on the metabolism of its precursors. [Results] The capability of synthesizing isoamyl alcohol by the five yeast strains isolated from the fermented grains was in the order of Naumovozyma castellii JP3-1>Saccharomyces cerevisiae JP3>Pichia fermentans JP22>P. kudriavzevii JP1>Naumovozyma dairenensis CBS421. Overall, isoamyl alcohol was synthesized mainly at the logarithmic phase, and it was also synthesized at the stationary phase of N. castellii JP3-1, P. fermentans JP22, and N. dairenensis CBS421. In addition, it was synthesized via the Harris pathway in the whole growth periods of S. cerevisiae JP3, N. castellii JP3-1, and N. dairenensis CBS421 and via the Ehrlich pathway in the whole growth period of P. kudriavzevii JP1. At the logarithmic phase, the isoamyl alcohol-synthesizing ability of P. fermentans JP22 through Harris pathway was identical to that through the Ehrlich pathway. However, P. fermentans JP22 at the stationary phase mainly synthesized isoamyl alcohol via the Harris pathway. [Conclusion] This study revealed the relationship of the synthesis ability and pathways of isoamyl alcohol in five yeast strains isolated from fermented grains with their growth phase. The result lays a theoretical basis for analyzing the mechanism of isoamyl alcohol synthesis and accumulation in the fermentation process of strong-aroma Chinese spirit and the precisely regulating isoamyl alcohol synthesis during the fermentation of strong-aroma Chinese spirit.

Abstract:[Background] Lactococcus lactis is a representative strain in the food industry. The molecular mechanism of L. lactis responding to environmental factors and the metabolic regulation through the two-component system is of great importance to the fermented food and the probiotics industries. [Objective] This study aimed to explore the effect of two-component system of L. lactis on aerobic respiration metabolism, and was expected to supply a new direction for adaptive metabolism in lactic acid bacteria. [Methods] The domainof sensor histidine kinases and response regulatory factor as well as the function of the two-component system in L. lactis was systematically analyzed using bioinformatics methods. The correlations between two-component system and aerobic respiration were calculated and further verified by transcriptional expression and untargeted metabolomics. [Results] The protein-protein interaction network of the representative L. lactis strain NZ9000 showed that the key connection point between the two-component system and pyruvate metabolism in the network was pyruvate-ferredoxin oxidoreductase (nifJ). At different growth stages, the transcriptional expression of the two-componentsystem in L. lactis NZ9000 changed significantly in the lag period. Compared with the anaerobic culture, the transcriptional expression of the two-component system in L. lactis NZ9000 cultivated under aerobic culture and aerobic respiratory culture was down-regulated. The two-component system participated in oxidative stress and heme stress in lactic acid bacteria. [Conclusion] To identify the two-component system and metabolic pathway of Lactococcus lactis involved in aerobic respiration. It can improve the survival rate and competitiveness of starters and probiotics.

Abstract:[Background] Salmonella is an important food-borne zoonotic pathogen that causes a variety of food-borne diseases among humans and animals. [Objective] This research was to investigate the prevalence, antimicrobial resistance and virulence gene of salmonellosis in ducks in Yunnan province. [Methods] A total of 169 liver samples of dead ducks from various regions in Yunnan province were collected for Salmonella isolation, and the isolated strains were subjected to serotyping, drug sensitivity test, and screening for related drug resistance genes and virulence genes. [Results] Forty-eight Salmonella strains were isolated, with the isolation rate being 28.40%. Three serotypes were discovered, of which Salmonella enteritidis was the dominant one. The isolates had a 100% resistance to penicillin G, lincomycin, clindamycin and rifampicin. Each was resistant to at least 6 kinds of 3 categories of antibiotics, and there was one strain resisting as more as 14 kinds, resulting in 22 drug resistance spectra. A total of 5 resistance genes were detected, of which the detection rates of bla_TEM and tetB were 27.08% and 22.92%, respectively, while those of tetA, sul2 and EBC were low. Ten virulence genes were detected, and the detection rates of SPI-1 (avrA), SPI-3 (mgtC), SPI-4 (siiD), SPI-5 (sopB) and bcfC were all 100%, whereas those of SPI-2 ssaQ, spvB, spvC, pefA and stn reached above 60%, with cdtB undetected. [Conclusion] S. enteritidis was the dominant serotype in ducks in Yunnan province, with severe drug resistance and multi-drug resistance, and the mechanism of drug resistance was complex. The coincidence rate of resistance gene and phenotype was low, while the detection rate of virulence gene was high. These results provide a reference for the prevention, control and eradication of salmonellosis in ducks in Yunnan province.

Abstract:[Background] Pyrola incarnata Fisch (P. incarnata) has been widely used in the fields of food and medicine owing to the abundant chemical components with a variety of pharmacological activities. However, no report on the secondary metabolites of its endophytic fungi is available. [Objective] To isolate and identify the secondary metabolites of endophytic Penicillium solitum from P. incarnata and screen out the ones with anti-neuroinflammatory activity. [Methods] They were isolated and purified by column chromatography and preparative high performance liquid chromatography (HPLC). The structures were identified by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). Moreover, the anti-neuroinflammatory activity was verified in LPS-induced inflammatory BV-2 cells by reverse docking. [Results] A total of 12 compounds were isolated and identified:cyclopenin (1), dehydrocyclopeptin (2), viridicatin (3), p-hydroxybenzoic acid (4), indoleacetic acid (5), methyl compactin (6), cyclopeptine (7), cyclopenol (8), protocatechuic acid (9), viridicatol (10), N-[2-(3-indolyl)ethyl]malonamicacid (11), solitumidine A (12). Molecular docking showed that compound 11 had remarkable binding activity to p38 mitogen-activated protein kinase (p38) and extracellular regulated protein kinase 1 (ERK1) in mitogen-activated protein kinase (MAPK) signaling pathway which was associated with inflammation. In the experiment on BV-2 cells, compound 11 (10, 20 μmol/L) significantly improved the survival rate of BV-2 cells. Moreover, it inhibited the release of inflammatory factors (tumor necrosis factor-α, interleukin-1β, and interleukin-6) and nitric oxide (NO) in BV-2 cells induced by LPS. [Conclusion] Compound 11 is a new natural product with potential anti-neuroinflammatory activities, and the mechanism is the likelihood that it inhibits the expression of p38 and ERK proteins in MAPK signal pathway, which needs further investigation.

Abstract:[Background] Loranthus tanakae has rich flavonoids with good pharmacological activities, whereas the studies of the flavonoids are limited because of the shortage of plant resources. [Objective] To isolate and identify the flavonoid-producing endophytes from L. tanakae and study their antioxidant and antibacterial activities in vitro. [Methods] The flavonoid-producing endophytes were isolated from the twigs of L. tanakae and screened by color reaction and thin-layer chromatography (TLC). The total flavonoid content was determined via NaNO₂-Al(NO₃)₃ colorimetry. The biological activities of flavonoid-producing endophytes were preliminarily evaluated by experiments of scavenging free radicals and inhibiting bacteria in vitro. [Results] A total of four flavonoid-producing endophytes, including three fungal strains and one bacterial strain, were screened out and identified as Botryosphaeria sp. (ZC020), Phoma sp. (ZZ105), Nemania sp. (ZS042), and Pseudomonas sp. (ZC026) based on morphological and molecular characteristics. ZC020 and ZS042 had higher total flavonoid content, which was (44.58±0.72) mg/L and (31.98±0.18) mg/L (P<0.05, n=3), respectively. Notably, ZS042 produced the same flavonoids as L. tanakae. ZC026 and ZS042 exhibited excellent antioxidant activity, which showed the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rates of 72.85%±0.66% and 57.01%±0.89% and the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging rates of 85.36%±0.75% and 88.17%±0.15%, respectively (P<0.05, n=3). ZZ105 had inhibitory effects on both Gram-positive bacteria and Gram-negative bacteria. [Conclusion] Four flavonoid-producing endophytes were isolated from L. tanakae and one of them was a bacterial strain. This work provides a new resource for the production of flavonoids by L. tanakae and a reference for developing and conserving other rare and precious medicinal plants and materials.

Abstract:[Background] Bacterial biofilm is a major factor causing bacterial resistance and recurrent nosocomial infections. But up to now there have been no effective anti-biofilm drugs. Rhizosphere microorganisms of some plants in high-cold, special environment can produce large quantities of active components which can improve the host immunity, and are thought to be a medicinal resource with great anti-biofilm potential. [Objective] To understand the rhizosphere microbial diversity of Astragalus yunnanensis and A. tatsienensis var. incanus growing in Baima Snow Mountain, and to screen cultivable strains with antibacterial and anti-biofilm activities. [Methods] In this study, metagenomics and traditional culture-dependent methods were employed to explore the rhizosphere microbial diversity of A. yunnanensis and A. tatsienensis var. incanus from Baima Snow Mountain in Deqin County, Diqing Tibetan Autonomous Prefecture, Yunnan Province. The microplate assay was conducted for determining the antibacterial and anti-biofilm activity of these cultivable microbes. [Results] Metagenomic sequencing indicated that the rhizosphere microorganisms of A. yunnanensis samples belonged to 6 phyla, 7 classes, 8 orders, 8 families, 9 genera and 10 species, and Thermus was the dominant microflora. The rhizosphere microbes of A. tatsienensis var. incanus samples belonged to 6 phyla, 8 classes, 10 orders, 11 families, 14 genera and 15 species, and Bradyrhizobium was the dominant microflora. A total of 145 cultivable strains were obtained by pure cultivation, including 112 bacteria and 33 fungi. Among them, 59 bacterial strains of 16 genera and 35 species and 19 fungal strains of 4 genera and 5 species were isolated from the rhizosphere soil of A. yunnanensis, with Streptomyces, Pseudomonas and Aspergillus being predominant. The other 53 bacterial isolates of 16 genera and 29 species and 14 fungal isolates of 3 genera and 4 species were derived from the rhizosphere soil of A. tatsienensis var. incanus, among which Bacillus, Stenotrophomonas and Aspergillus were the most abundant genera. For the screened microbes with antibacterial and anti-biofilm activity, 51 bacterial strains and 7 fungal strains were chosen at different species levels as representatives to explore their potential as medicinal resources of natural antibiotics. The crude ethyl acetate extract of the fermentation broth of 5 bacteria and 1 fungus were demonstrated to have moderate to strong activity against Gram-positive pathogens, and 4 strains of them showed anti-biofilm activity against methicillin-resistant Staphylococcus aureus (MRSA). Finally, two microbial strains Streptomyces fulvissimus KTA1 and Aspergillus fumigatus YNF5 were considered as the promising bioactive strains. [Conclusion] It is reported for the first time that there is abundant species community composition in the rhizosphere microorganisms of Astragalus plants distributed in northwestern Yunnan province. These cultivable microorganisms could be regarded as a great medicinal source for the exploitation of natural antibiotics. This study was significant for utilization and protection of characteristic plants-associated microorganisms in high-cold, special environment of northwestern Yunnan.

Abstract:[Background] Hypoxia-inducible factor 1-alpha (HIF-1α) is a key factor in response to cellular hypoxia and plays an important role in erythropoiesis, angiogenesis, energy metabolism and regulation of host immune metabolism.[Objective] To investigate the effects of HIF-1α/Bcl-2-adenovirus E1B 19-kDa interacting protein 3 (BNIP3) signaling pathway on the BCG infection-induced autophagy of macrophage RAW 264.7 cells. [Methods] Small interfering RNA of HIF-1α, siHIF-1α, was constructed, and after the transfection of siHIF-1α and/or Bacillus Calmette-Guerin (BCG) infection of RAW 264.7 cells, the autophagy rate of the cells was detected by flow cytometer. Western blotting or immunofluorescence technique was employed to determine the protein levels of HIF-1α, BNIP3, LC3, Beclin 1, Rheb and mTOR.[Results] BCG infection up-regulated the expression of LC3 and HIF-1α in RAW 264.7 cells. The transfection of siHIF-1α down-regulated the levels of HIF-1α, BNIP3, LC3, and Beclin 1 and decreased the autophagy rate of the macrophages after BCG infection. Moreover, siHIF-1α promoted the expression of Rheb and p-mTOR.[Conclusion] Our results indicated that the knockdown of HIF-1α inhibited the HIF-1α/BNIP3 signaling pathway, thereby activating the mTOR pathway and inhibiting autophagy in RAW 264.7 cells after BCG infection.

Abstract:[Background] Two species were discovered during the investigation of macrofungal resources in Hancongling Red Leaf Valley, Dunhua city, Yanbian Korean Autonomous Prefecture, Jilin province, China. [Objective] To determine the taxonomic status of the two species. [Methods] The samples of macrofungi were collected, and their morphology was observed and described in detail. After DNA extraction, the rDNA ITS sequence of each species was determined, and phylogenetic trees were constructed based on maximum likelihood method and Bayesian inference. [Results] The two species were respectively identified as Tulosesus callinus and Russula lakhanpalii, with the morphology consistent with the original description. The phylogenetic analysis confirmed the identification results based on morphology. [Conclusion] R. lakhanpalii is firstly reported in China and T. callinus is a new record species in Jilin province.

Abstract:[Background] Tobacco black shank, induced by Phytophthora nicotianae, is one of the major soil-borne diseases that cause serious stem and root rot in flue-cured tobacco production. [Objective] Screening of strong antagonistic strains is the basis of biological control. [Methods] In this study, the dual culture assay was used to screen Closnostachys strains against P. nicotianae. Sixty-five strains of Closnostachys belonging to eight species were tested. According to their abilities of sporulation on colony of P. nicotianae and hyphal coilings produced surrounding hyphae, sporangiophores, and sporangia of the pathogen, the antagonism of Closnostachys was classified into four degrees:strong, moderate, weak, and absent. [Results] A total of 6 tested strains had strong antagonistic ability, 27 moderate antagonistic ability, 22 weak antagonistic ability, and 10 no antagonistic ability. The inhibition rates of Closnostachys strains tested to P. nicotianae were 20.0%-86.7%. [Conclusion] The strains tested with strong antagonistic ability were C. rosea 7901 and 11361, C. byssicola 5072, 6729, and 7507, and C. grammicospora 6730. Their potential as biocontrol resources needs further pot experiment confirmation.

Abstract:[Background] Most of the phosphorus in the soil exists in the form of insoluble phosphate, which is unavailable to crops, and traditional chemical fertilizer causes environmental pollution. [Objective] To tackle soil phosphorus deficiency and develop a safe and efficient microbial fertilizer. [Methods] An efficient phosphate-solubilizing bacterial strain was screened out from the soil behind the library of Wuhan University of Science and Technology and identified by morphological observation, determination of physiological and biochemical indexes, and 16S rRNA gene sequencing. The phosphate-solubilizing conditions were optimized based on NBRIP medium, and the phosphorus-solubilizing mechanism was explored by high performance liquid chromatography. [Results] The strain was identified as Burkholderia gladioli. Among the 20 amino acids, D-methionine was prominent in promoting the growth and phosphate solubilization of the strain, improving the growth and solubilized phosphate by 19.09% and 16.16%, respectively. Sodium formate inhibited the growth and phosphorus dissolution of the strain, with the inhibition rate of 39.08% and 10.66%, respectively. The strain dissolved phosphate in the environment by secreting organic acids such as glucuronic acid and D-L-malic acid. The fertilizer prepared with the strain could significantly promote the growth of pepper seedlings. [Conclusion] B. gladioli secretes organic acids to dissolve phosphate in the soil, which lays a theoretical basis for the preparation and application of biological fertilizer.

Abstract:Lactic acid bacteria are a representative probiotic in human intestine that affect host lipid metabolism, which is closely related with the production of bile salt hydrolase (EC3.5.1.24, BSH) and the conjugate conversion of polyunsaturated fatty acids (PUFAs). The production of BSH and conjugated fatty acids is influenced by strain differences, flora distribution, and dietary differences. This paper focuses on the mechanism of BSH and conjugated fatty acids in regulating the host lipid metabolism, in order to provide reference for further research. BSH can degrade bile acids (BAs) secreted by liver and reduce the absorption of lipids. The degradation products of BAs, deoxycholic acid and lithocholic acid, can promote cholesterol transport and conversion to BAs through signaling pathways such as farnesoid X receptor, small heterodimer partner and liver X receptor. Additionally, BSH can also inhibit lipid synthesis and promote lipid decomposition by down-regulating sterol regulatory element binding protein 1c, up-regulating 5ʹ-AMP activated protein kinase and peroxisome proliferator-activated receptor α (PPARα). PUFAs can be converted into conjugated fatty acids by lactic acid bacteria, such as conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA). CLA/CLNA boosts the production of leptin in the body, suppresses appetite and promotes energy consumption. Moreover, CLA/CLNA can promote the oxidative decomposition of human lipids by activating PPARα. Lactic acid bacteria modulate the host lipid metabolism through the above-mentioned multiple pathways, which is of great significance for the in-depth understanding of its regulatory mechanism and clinical application.

Abstract:Legionella pneumophila (L. pneumophila) is a distinct model for pathogen-host interaction research. Its unique secretion systems as well as the structures and functions of their substrate effectors are the research hotspots in the field of pathogenic microorganism. Type Ⅱ secretion system (T2SS) plays a major role in promoting bacterial survival in the environment and in human hosts, and Legionella secretion pathway (Lsp) is a typical T2SS in Gram-negative pathogens. This review briefly summarizes the research progress of L. pneumophila T2SS and its substrate effectors, emphasizing on their structures and functions, so as to provide in-depth understanding of the function and mechanism of T2SS in Gram-negative pathogens.

Abstract:Farmland soil pollution is becoming serious worldwide. As common soil pollutants, heavy metals, pesticides, and microplastics have posed a threat to farmland ecosystem and food security. Bacterial biofilm (BF), the multi-component aggregate on the surface of bacteria, has proved to have high value in environmental protection in recent years. This paper introduces the composition and functions of BF and summarizes the applications and mechanism of bacteria and BF in remediation of heavy metal- and organic-polluted soil in recent years. Moreover, the potential of BF for remediation of polluted soil is summarized. This study is expected to gain a clearer insight into the key role of BF and guide the exploration of potentials of BF in environmental protection.

Abstract:Recent rapid developments of synthetic biology and genome sequencing technology opened new possibilities for the discovery and development of microbial natural products. However, a majority of natural products are difficult to access because many microorganisms cannot be cultured, or are difficult to be genetically manipulated. Heterologous expression of biosynthetic gene clusters (BGCs) for natural products in well-characterized hosts provides an alternative way for the discovery of natural products or improvement of their production. However, the success of this strategy hinges upon the cloning and refactoring and/or optimization of large gene clusters. CRISPR/Cas system greatly boosts the development of cloning and refactoring large gene clusters, and promotes the discovery of natural products from microorganisms. In this review, we summarized the recent advances in strategies for gene cluster cloning and refactoring based on CRISPR/Cas system, and discussed the application of related technologies in the discovery and development of natural products and their significance.

Abstract:Dimethylsulfoniopropionate (DMSP) is one of the most important organic sulfur compounds in the world. It participates in the global sulfur cycle, signal transmission and potential regulation of global climate. The production of DMSP can reach up to 10⁹ tons every year, with the main producers being marine phytoplankton and macroalgae. In recent years, it has been reported that some marine bacteria can also produce DMSP, which is a previously overlooked source of DMSP. Up to date, three DMSP synthesis pathways have been identified:methylation pathway, transamination pathway and decarboxylation pathway. Five key enzymes involved in DMSP synthesis were identified. On the basis of the research results in recent years, this study reviewed the key enzymes involved in DMSP synthesis, and provided outlook for future research.

Abstract:Persisters, a subgroup of bacteria that can survive high dosage of antibiotics, make it more difficult to treat infections with antibiotics and threaten human life and health. How to control and remove persisters has become a research hotspot at the moment. In this paper, we summarize the characteristics and harms of persisters, analyze the formation mechanism of them, and sum up methods for controlling and removing this subgroup. This review is expected to guide the control of persisters and the treatment of persistent infection.

Abstract:Renal calculus, a common urinary disorder in adults, influences the physiological function of the kidney and leads to urinary tract infection, thus threatening human health. Renal calculi are surrounded by a diversified microbial community, and the changes of microorganisms in the intestinal tract and urinary system may cause the occurrence and development of renal calculus. Particularly, Bifidobacterium, Lactobacillus, and Enterobacteriaceae are closely related to the occurrence of this disease. This review focused on the role of microorganisms in intestinal tract and urinary system in the formation of renal calculi. In addition, we also introduced the kidney-gut axis, influence of intestinal microorganisms on renal calculus from the production of short-chain fatty acids, the change of oxalate, and the occurrence of inflammation, and the prevention and treatment of this renal disease.

Abstract:The Gram-negative Pseudomonas syringae causes diverse plant diseases. It injects effector proteins into host cells via type III secretion system (T3SS), thereby suppressing the immune system of the host and colonizing the host. RhpR/S, the main regulator of T3SS, directly regulates hrpR/S and other virulence-related pathways in response to changes in environmental signals. The expression of the T3SS genes is also affected by other regulatory factors, including sigma factor HrpL, two-component system GacA/S, Lon protease, second messenger molecules, and environmental signals. This paper aims to brief the composition and functions of T3SS in P. syringae and review the regulatory mechanism of T3SS in the bacterial species, which is expected to serve as a reference for in-depth research on the pathogenic mechanisms of P. syringae.

Abstract:Medicinal plants are important raw materials for Chinese medicine industry. The yield and quality of medicinal plants are affected by diverse factors such as heredity and growth environment. Plant microbiome has attracted increasing attention for its positive role in the growth, development, and secondary metabolites accumulation of host plants and the related studies have been reviewed in detail except the works around the microbiomes of medicinal plants. In this paper, we summarized the microbiome diversity of medicinal plants and their effects on the secondary metabolites of medicinal plants since 2010. Furthermore, we discussed the potential application of microbiome in the quality improvement and ecological cultivation of medicinal plants.

Abstract:To improve the comprehensive quality of students and adapt to the rapidly development of food microbiological examination and constantly changing food microbiological examination standards, the curriculum reform of Food Microbiological Examination guided by post competence was carried out in our university. The teaching reform was carried out from these four aspects, namely, curriculum objectives, curriculum content, teaching methods and assessment methods. Except for the cultivation of explicit competence based on professional knowledge and skills, more and more attentions were paid to the improvement of implicit competencies, such as the ability of autonomous learning, solving practical problems, seeking truth from facts, and bearing hardships. Through three years of teaching practice, it was confirmed that the teaching reform of Food Microbiology Examination effectively improved professional quality, comprehensive quality and post competence of students, thereby providing references for the teaching reform of food quality and safety.