Abstract:With the advent of the era of big data, how to transform the omics data into easy-to-understand and visualized knowledge is one of the important challenges in bioinformatics. Recently, machine learning techniques had been utilized to analyze the complicated, high-dimensional microbiome data to address the complex mechanisms of human diseases. Here, we firstly summarized microbiome data procession approaches and the most commonly used machine learning algorithms, such as support vector machine (SVM), random forest (RF), and artificial neural networks (ANN). Then, the workflow of machine learning studies was described, and the application of ML algorithms in predicting host phenotypes based on microbiome data was evaluated. Finally, the model construction and validation of machine learning algorithms were demonstrated by using saliva microbiome data to predict oral malodour as an example, and R/Python code for practical data analysis was provided (https://github.com/LiLabZSU/microbioML).

Abstract:Polysaccharides are the main natural bioactive components of edible and medicinal fungi to regulate the immune function of the body from multi-level and multi-target. Early studies focus on the anti-tumor mechanism of edible and medicinal fungi polysaccharides by enhancing the immunity of the body to inhibit tumor cells. However, recent studies show that these polysaccharides can regulate the tumor microenvironment, restore the ability of monitoring tumor and tumor microenvironment, enhance tumor-antigen-specific immune responses ability of the body to the tumor microenvironment, so as to give to inhibit and kill tumor. Our previous studies also show that edible and medicinal fungi polysaccharides can positively increase the number of immune cells in the peripheral blood of tumor mice, promote the infiltration of immune cells into the tumor microenvironment, help the body recognize and kill tumor cells, regulate the tumor microenvironment. Here, we summarize the biological activity of edible and medicinal fungal polysaccharides as immunomodulators in inhibiting tumor immune escape, combined with the tumor microenvironment to discuss their relationship with tumor immunity, mechanism of action, and their role in tumor treatment, to provide new ideas for edible and medicinal fungal polysaccharide immunotherapy.

Abstract:The type VI secretion system (T6SS) is a versatile bacterial weapon, which is widespread in Gram-negative bacteria and used to act against both eukaryotic and prokaryotic cells. Pseudomonas aeruginosa is an opportunistic pathogen that is resistant to a variety of antibiotics and can cause acute and chronic infections in humans. P. aeruginosa encodes three separate T6SSs, namely H1-, H2-, and H3-T6SS, which play key roles in virulence and environmental adaptation by mediating inter-bacterial competition, biofilm formation, metal uptake, and interaction with eukaryotic host cells. This article reviews the latest progress in assembly, secretion, function, and regulation mechanisms of T6SS in P. aeruginosa, aiming to provide reference for the study of T6SS and provide guidance for the prevention and treatment of P. aeruginosa infection.

Abstract:Nosemosis is an infectious disease caused by Nosema ceranae, which infects honey bees globally. Nosema ceranae infection led to premature aging, decreased nursing and productivity, as well as fecundity. Under extreme circumstances, the infection caused colony collapses. In this study, we summarized the etiology, epidemiology and the control methods of Nosema ceranae in the last ten years. This study aims to improve the understanding of Nosema ceranae biology and provide new insights for the parasite control.

Abstract:Ammonia-oxidizing archaea is one of the most abundant microbial groups driving the nitrogen cycling on Earth. Especially, in the deep sea, its relative abundance can reach 20%-40% of the total prokaryotes. However, the lack of deep-sea ammonia-oxidizing archaea isolates hindered our comprehensive understanding of their physiology and ecological contribution. In this paper, we analyzed the relationship between characteristics of deep-sea environments and the adaptability of microorganisms, focusing on the potential survival strategies and metabolic preferences of deep-sea ammonia-oxidizing archaea. This knowledge will assist us to design suitable cultivation techniques on them. Moreover, the ammonia-oxidizing archaea habituated in the deep sea apparently distanced from those in soil or surface ocean, in terms of phylogeny as well as physiology. Therefore, we are about to reconsider the global oceanic nitrogen budget estimation.

Abstract:The Newcastle disease virus (NDV) M protein is a non-glycosylated membrane-associated protein, which locates on the inner surface of NDV envelope and constitutes the bridge between the viral envelope and the nucleocapsid. Previous studies have shown that the NDV M protein is a nucleocytoplasmic shuttling protein, which plays essential roles in inhibiting host gene transcription and protein synthesis, and also facilitating viral assembly and budding. Up to now, numerous studies have mainly focused on viral F, HN, V proteins and RNP complex to reveal the relationship between the virulence and replication of NDV. However, in recent years, several studies using the reverse genetics techniques have indicated that the M protein is also associated with the virulence and replication of NDV. Therefore, this summary reviews the structural features of M protein, and the relationship between M protein and NDV virulence and replication, which will provide a new theoretical reference for further investigating the functions of NDV M protein.

Abstract:[Objective] This research aims to investigate the diversity of microbial community structure in different seagrass growth periods, and to analyze the internal factors that lead to the differences in microbial community structure.[Methods] The rhizosphere sediments of seagrass and unvegetated areas surface sediments were collected. High throughput sequencing (Illumina MiSeq Platform) was used to analyze the microbial community characteristics of rhizosphere and unvegetated areas at different seagrass growth stages.[Results] The difference in microbial community structure was driven by the growth period of seagrass and whether seagrass was colonized. At the maturation stage of seagrass, Propionium was significantly enriched in the roots of Z. marina and Z. japonica, with a relative abundance of 11.58% and 14.26%, respectively. In the seedling stage of seagrass, Desulfobulbaceae was enriched at the seagrass roots (Z. marina:2.299%, Z. japonica:4.092%). The relative abundance of Sulfurovum was higher in the rhizosphere during the decline stage of seagrass (Z. marina:5.624%, Z. japonica:3.749%). In addition, the growth period of seagrass had the greatest explanatory power for the differences in microbial community structure (R²=0.20335, P=0.002). The results of PICRUSt2 function prediction showed that all functional genes showed the same trend in different growth stages of seagrass, but the abundance showed the result of seedling stage > mature stage > decline stage.[Conclusion] The microbial community structure of seagrass bed sediments in Swan Lake presented different diversity characteristics at different growth stages of seagrass, with obvious rhizosphere effect and no significant difference in the rhizosphere microbial community of different species of seagrass, which was not species-specific.

Abstract:[Objective] Here we studied the degrading properties of different food-derived Aspergillus oryzae strains to beta-cypermethrin (β-CP) and 3-phenoxybenzoic acid (3-PBA), so as to lay a theoretical foundation as well as provide potential microbial food cultures resources for pesticides reduction towards agricultural by-products and fermented foods.[Methods] Identified by morphology, ITS sequencing and production of aflatoxin B1 (AFB1), fifteen A. oryzae strains were screened from fermented foods. A. oryzae RIB40 (collection number:ATCC 42149) and A. oryzae M4 (collection number:CGMCC 11645), together with isolated strains, were determined for β-CP and 3-PBA degrading characteristics via HPLC-UV, GC-MS as well as LC-MS.[Results] After 5 days of shaking culture in potato dextrose broth (PD), degradation rates of 50 mg/L β-CP by seventeen strains varied from 19.33% to 50.29% and 50 mg/L 3-PBA from 45.59% to 99.67%, with 3-PBA been detected during β-CP degradation. Cultured in mineral basal medium (MM) for 5 days, strains failed to grow and no removal of β-CP or 3-PBA was observed. In contrast, seventeen strains could grow in glucose enrichment medium (GM) shaking culture, and 2 days later, conversion or degradation rates of 100 mg/L 3-PBA ranged from 69.28% to 99.58%, of which systems detected 3-phenoxybenzyl alcohol (3-PBlc) and hydroxy-3-phenoxybenzoic acid (HO-3-PBA).[Conclusion] A. oryzae strains exhibit the common nature of co-metabolizing β-CP and its intermediate 3-PBA, with high-efficiency 3-PBA degradation performance. Moreover, A. oryzae strains can briefly transform 3-PBA into 3-phenoxybenzyl alcohol (3-PBlc) which has lower toxicity at initial stage. Meanwhile, 3-PBA is hydroxylated to yield HO-3-PBA that shows better water-solubility, subsequently into further metabolism.

Abstract:[Objective] Marine planktonic archaea are the key drivers of biogeochemical cycling, but the knowledge about their horizontal spatial distribution characteristics in the coastal waters has not been well understood. In this study, we used the Meishan Island (closely connected to the land) as an example to study the horizontal distribution patterns of planktonic archaea in the transition zone between land and sea.[Methods] 16S rRNA gene amplicon sequencing was used to reveal the spatial distribution characteristics of planktonic archaea in terms of the distribution of dominant taxa, community composition variation, and the co-occurrence pattern of taxa in the lagoon (called Meishan Bay) and adjacent seawaters around Meishan Island in spring.[Results] The results showed that relative abundance of planktonic archaea in the whole prokaryotic community ranged from 0.6% to 26.5%, and the archaeal abundance in seaward waters was significantly higher than that in the lagoon. Planktonic archaeal communities were dominated by Thaumarchaeota Marine Group (MG) I and Euryarchaeota MGII. The composition of MGI taxa was relatively simple, while MGII taxa had relatively high phylogenetic diversity. The spatial distribution of archaeal communities was shaped by homogenizing dispersal, environmental selection, and undominated processes (including ecological drift), and environmental selection was mainly driven by suspended particle, nitrate, dissolved oxygen, water temperature and ammonium. The network analysis showed that MGI taxa commonly had negative correlation with Rhodobacteraceae taxa, while MGII taxa commonly had positive correlation with heterotrophic bacteria affiliated to the clades SAR11, SAR116, and SAR86.[Conclusion] This study preliminary revealed the spatial distribution characteristics and drivers of planktonic archaeal communities in the coastal waters around Meishan Island in the spring, expanding the understanding of the distribution patterns of archaea in the transitional zone between the land and ocean.

Abstract:[Objective] With the development of synthetic biology, great progress has been made in targeted therapy by designing and synthesizing complex, multifunctional gene circuits in bacteria. Although using bacteria as a therapeutic delivery system to selectively release effective therapeutic components in vivo has great advantages, how to make bacteria secrete functional proteins effectively and play a role under low metabolic load remains a challenge.[Methods] To solve this problem, this study provided a new strategy. In this strategy, protein germicide and filamentous phage encoding genes widely existed in bacteria were used as biobricks, and through the rearrangement and assembly of these endogenous biobricks of P. aeruginosa, an engineered bacterium capable of lysis and release of functional proteins under specific conditions was constructed. In order to evaluate whether the biobricks constructed in the engineered bacterium could work, the extracellular polysaccharides hydrolase PelA and PslG were selected as functional proteins to construct the engineered bacterium PAO1102. The destruction and prevention effects of PAO1102 on P. aeruginosa biofilm were tested by the biofilm destruction experiment, biofilm inhibition experiment and antibiotic resistance experiment.[Results] Compared with the control group, the treatment of PAO1102 could not only significantly destroy the mature biofilm and inhibit the biofilm formation, but also significantly enhance the sensitivity of the bacteria in the biofilm to tobramycin. Moreover, these functions of PAO1102 were mainly dependent on the release of functional proteins by phage Pf4 induced cell lysis.[Conclusion] The engineered bacterium could be used as a microbial tool for the targeted delivery of proteins. In the follow-up study, different functional genes will be expressed in the engineered bacterium according to different needs, and the targeted delivery of functional proteins will be realized, to perform different biological functions.

Abstract:[Objective] This study aimed to analyze the succession of the microbial community and the changes of physicochemical metabolites during the fermentation of Chinese traditional broad bean paste, moreover, to explore the core functional microbiota that affects the flavor.[Methods] We used high-throughput sequencing to analyze the microbial community structure and succession, together, detected the physicochemical metabolites during the fermentation process. The correlations between the microbial community and physicochemical metabolites were also analyzed. Finally, core species were isolated from broad bean paste with clearly evaluated functional characteristics.[Results] The community structure changed significantly in the early stage of fermentation, and gradually stabilized in the mid-late stage. The dominant bacteria were Staphylococcus, Bacillus and Weisiella, among which Staphylococcus showed an upward trend during the whole fermentation, while Bacillus and Weisiella both showed a downward trend. The fungal community structure was relatively simple and stable, with average abundance of Aspergillus accounted for more than 97% of the total fungal community, and Zygosaccharomyces increased during the mid-late stage and then declined. Correlation analysis and in vitro functional validation showed that functional microbes (Aspergillus oryzae, Bacillus subtilis, Staphylococcus gallinarum, Weisiella confusa and Zygosaccharomyces rouxii) played different key roles in different fermentation stages.[Conclusion] In the early stage of fermentation, Aspergillus oryzae and Bacillus subtilis secreted enzymes to degrade macromolecular substances. Aspergillus oryzae, Staphylococcus gallinarum and Weisiella confusa resulted in acidification and amino acid production of broad bean paste, while salt-tolerant Zygosaccharomyces rouxii were essential for the formation of flavor substances in mid-late stage of fermentation.

Abstract:[Objective] ZntR is a metal regulatory protein, which can catalyze the transcriptional activation of zinc efflux genes, thus controlling the intracellular Zn(II) from toxic. However, the effect of zntR on bacterial biological function remains unclear.[Methods] In this study, we constructed Aeromonas hydrophila (A.h) ∆zntR deletion strain and ∆zntR::zntR rescued strain to evaluate the biofilm formation ability, hemolytic activity, motility ability, the characters of responding to metal ion stress and other physiological phenotypes.[Results] The results showed that zntR deletion strain was sensitive to zinc and chromium stress and tolerant to cobalt ion stress, meanwhile, the biofilm formation ability was decreased and the movement ability was enhanced. These phenotypes could be recovered in the rescue strains. Furthermore, the differential protein expression between wild-type strain and zntR deletion strain was compared by DIA based quantitative proteomics. Results showed that zntR may also be involved in the regulation of two-component system, bacterial chemotaxis and other metabolic pathways.[Conclusion] The above research can provide theoretical basis for further study on the regulation mechanism of ZntR transcription factors in bacterial physiological function.

Abstract:[Objective] To describe and compare the structure of intestinal microbiota between patients with non-small cell lung cancer and benign lung disease and explore the influence of specific microbial flora in the lung cancer.[Methods] We collected 63 cases of fecal samples which including 39 non-small cell lung cancer patients and 24 benign lung disease patients and use high-throughput second-generation sequencing technology platform to analyze the V3-V4 region of 16S rDNA gene in fecal samples of bacteria to explore the overall structural characteristic of intestinal microbiota.[Results] Lachnospira, Ruminococcus, Faecalibacterium, Roseburia, Fusicatenibacter are significantly enriched in lung adenocarcinoma patients. But in lung squamous cell carcinoma and benign lesions there is no obvious difference. And Haemophilus, Prevotella, Streptococcus, Citrobacter are significantly enriched in the intestines of patients with benign diseases.[Conclusions] Non-small cell lung cancer and benign diseases have different gut microbiome.

Abstract:The effect of a single microorganism in degrading rice straw is not apparent. The microbial flora, combined with multiple microorganisms, can effectively degrade rice straw, which is currently the primary choice for straw waste degradation.[Objective] To explore the interspecies coordination mechanism, the microbial flora can transform rice straw more efficiently than a single strain and provide a theoretical reference for improving the biodegradation process of straw materials.[Methods] A microbial population combination with a degradation effect better than monoculture was constructed artificially through the combination of strains and the weight loss of rice straw as an indicator. The experimental strains were identified by molecular biology methods. Combined with cellulase activity and GC-MS analysis of fermentation products revealed the interspecies coordination mechanism of microbial populations during rice straw degradation.[Results] Strain B (Bacillus cereus) cultured at 30℃ for 8 days, the degradation rate of rice straw was 50.9%. The combination of strain B and strain D₂ (Bacillus amyloliquefaciens), W₁ (Ochrobactrum intermedium), and G₁ (Bacillus licheniformis) constituted 4 strains of BD₂W₁G₁. For the composite flora, rice straw's degradation rate by culturing at 30℃ for 8 days is 73.3%, 22.4% higher than that of single strain B. The fermentation product analysis showed that strain B produced a large amount of acid, phenol, and other substances in a single culture. B+D₂ co-culture, the degradation rate of rice straw is 64.6%, the acid in the fermentation broth is relatively reduced by 87.4%, and the phenol is reduced by 61.9% (pentadecanoic acid, n-hexadecanoic acid, 2,4 Di-tert-butylphenol, and 6-tert-butyl p-cresol is significantly reduced). When B+D₂+W₁ is co-cultured, the fermentation broth phenols are further reduced by 15.7% (6-tert-butyl-p-cresol is significantly reduced), and rice straw is degraded by 71.0%. When B+D₂+W₁+G₁ is co-cultured, phenols and acids continue to decrease by 10.7%, and rice straw is degraded by 73.3%.[Conclusion] Pentadecanoic acid, n-hexadecanoic acid, 2,4-di-tert-butylphenol and 6-tert-butyl-p-cresol, and other acids phenols can inhibit the efficiency of strain B in decomposing rice straw. Strain D₂, W₁, and G₁ can reduce the content of acids and phenols, including the four substances (A total of 82.9% reduction in acids and 88.2% reduction in phenols), improve the decomposition efficiency (A total of 22.4% increase in decomposition effect). The composition of rice straw is complex, and the bacteria with different functions are combined into a compound inoculum, which reduces feedback inhibition and forms metabolic complementarity, which can effectively improve the biodegradation effect of rice straw.

Abstract:[Objective] Bacillus subtilis can induce efficient intestinal mucosal immune responses, but the detailed mechanism for dendritic cell activation remains to be defined.[Methods] Mouse intestinal epithelial CMT93 cells were stimulated with different concentra amyloliquefaciens tions of Bacillus subtilis and the cytokine expression was measure by quantitative RT-PCR and ELISA. After incubation with the cytokine-containing cell culture medium, activation of mouse bone marrow-derived dendrites (BMDC) was analyzed by flow cytometry and the role of IL-33 in BMDC activation was confirmed by RNA interference.[Results] Bacillus subtilis could significantly stimulate CMT93 cells to secret IL-6, IL-33 and IFN-g, and induce the IL-33 selection in a dose-dependent manner. The cytokine-containing cell medium could significantly activate BMDC. After interfering IL-33 gene expression and stimulation with Bacillus subtilis, however, the ability of CMT93 cell medium for BMDC activation was significantly reduced.[Conclusion] These data suggest that IL-33 from Bacillus subtilis-stimulated mouse intestinal epithelial cells plays an important role in the dendritic cell activation.

Abstract:[Objective] This study aims to obtain a novel thermostable mannanase to satisfy industrial application.[Methods] Sequence analysis revealed that the Mtman1 encodes for 409 amino acids (AAs), including a signal peptide and a glycoside hydrolase (GH) 5 catalytic domain. Recombinant MtMAN1 heterologously expressed in Pichia pastoris showed an optimal pH and temperature of 6.0 and 70℃, respectively. The K_m and V_maxvalues of MtMAN1 were 4.28±0.73 mg/mL and 203.9±14.61 μmol/s·mg against locust bean gum, respectively.[Results] The MtMAN1 was stable under 60℃ incubation for 1 h. After treatment at 80℃, 90℃ and 100℃ for 10 min, the enzyme maintained (68.23±7.47)%, (56.01±5.69)% and (14.91±2.92)% residual activities, respectively. Interestingly, its secondary structures and maximum wavelength remained as unchanged, suggesting relatively stable conformational structure. Additionally, the MtMAN1 was considerably resilient to Fe²⁺ (<0.1 mmol/L), Cu²⁺ (<0.1 mmol/L), Ca²⁺ (<0.5 mmol/L), Mg²⁺ (<0.1 mmol/L) or Zn²⁺ (<0.1 mmol/L).[Conclusion] MtMAN1 is a hyperthermostable β-mannanase and could be a candidate for handling industry tanks at high temperatures, such as feed pelleting.

Abstract:[Objective] L-homophenylalanine (L-HPA) is a key intermediate for many pharmaceuticals and pesticides. As chemical synthesis of L-HPA was complicated and environmentally unfriendly, this study aimed to develop an efficient and environmentally friendly route for the biosynthesis of L-HPA.[Methods] A multi-enzyme cascade reaction for L-HPA production from L-glycine and phenylacetaldehyde was constructed by modular assembly method.[Results] Firstly, the multi-enzyme cascade reaction, which consisted of threonine aldolases (TA), threonine deaminase (TD), phenylalanine dehydrogenase (PheDH), and formate dehydrogenase (FDH), was constructed by literature mining. Then, according to the introduction and deletion of chiral -OH/-NH₂ groups, the whole cascade reaction was divided into two parts, in which the basic parts included TA and TD and the extender parts included PheDH and FDH. Next, the two modules were assembled and optimized with different plasmids, enhancing the L-HPA production up to 208.6 mg/L in the optimal engineered strain BL21-C-M1-R-M2. Finally, the whole cell catalytic system was optimized to improve the L-HPA production up to 1226.6 mg/L and the molar conversion of phenylacetaldehyde reached 34.2%.[Conclusion] This environmental-friendly and efficient process represented a promising strategy for large-scale L-HPA production in the future.

Abstract:[Objective] This study aimed to investigate the effect of modified mouse host defense peptide CRAMP on the mature biofilm of Pseudomonas aeruginosa PAO1 srtain based on RNA-Seq technology.[Methods] Crystal violet method was used to detect the biofilm biomass, and laser confocal scanning microscope (CLSM) was used to observe the morphological changes of biofilms. The gene expression differences at transcription level between biofilms treated with CRAMP and the non-peptide control were analyzed by the PE150 sequencing strategy using Illumina second-generation high-throughput sequencing platform. The content of alginate in PAO1 biofilms was determined by the 1,3-Dihydroxynaphthalene method.[Results] The biofilm biomass was significantly reduced by modified peptide CRAMP at the range of 0.98-62.50 μg/mL. CLSM showed that CRAMP could significantly reduce the total fluorescence intensity of biofilms. The results showed that 12636700 clean-read pairs were obtained by transcriptome sequencing. A total of 1582 differential genes were identified, including 800 genes that were up-regulated, and 782 genes were down-regulated. The GO functional enrichment analysis showed that 1226 genes were compared to the GO functional analysis database. Among these differentially expressed genes, they were related to molecular function, biological process, and cell composition. Enrichment analysis of the KEGG pathway showed that 603 differentially expressed genes were compared with 96 metabolic pathways in KEGG. There are various kinds of amino acid metabolism pathways, fatty acid metabolism pathway, tricarboxylic acid cycle, biofilm regulatory system, and so on. It was found that modified peptide CRAMP may act on PAO1 c-di-GMP system, enhance bacterial motility and biofilm dispersion, and be related to the quorum sensing (QS) system and alginate synthesis. Finally, it was verified that the modified peptide CRAMP significantly reduced the content of alginate in PAO1 biofilms.[Conclusion] The modified peptide CRAMP has an impair effect on PAO1 mature biofilms and can lead to the decrease of alginate content in mature biofilm. Transcriptomics analysis implied that CRAMP might exert its effectiveness by reducing the level of PAO1 c-di-GMP, and the mechanism remains to be further explored.

Abstract:[Objective] To study the relationship between Klebsiella and multireplicon resistance plasmids, and to analyze the mechanism of multireplicon plasmid carrier in response to antibiotic pressure.[Methods] We collected fifty-six isolates of Klebsiella from different wild animals and environments during 2018 to 2020. We analyzed the multidrug resistance (MDR) phenotype by microbroth dilution method, sequenced and analyzed the representative isolates by bacterial genome-wide association study (BGWAS).[Results] Our results revealed that the isolates from non-human sources showed a more extensive drug resistance, and especially stronger resistance to ampicillin (reach 80.36%). In particular, the isolates from Malay pangolin were highly resistant to cephalosporins, chloramphenicol, levofloxacin and sulfamethoxazole. Genomic analysis showed that the resistance plasmids carried by these isolates were related to carry many antibiotic resistance genes. Further analysis of 69 plasmids demonstrated that 28 plasmids were multireplicon plasmids, mainly carrying beta-lactamase genes such as bla_CTX-M-15, bla_CTX-M-14, bla_CTX-M-55, bla_OXA-1 and bla_TEM-1. According to the analysis of plasmids carried by different isolates, Klebsiella pneumoniae may be an important host of multireplicon plasmid. Plasmid skeleton and structure analysis showed that multireplicon plasmid was formed by the fusion of two or more single plasmids, which made the isolates strong adaptability to the antibiotic environment, and increased the ability of drug-resistant isolates to spread around the world year by year.[Conclusion] The phenotype of multidrug-resistant bacteria is related to the carrying multireplicon plasmid.Multireplicon plasmid has a stronger ability to carry resistance genes than non-multireplicon plasmid, which may be an important mechanism for bacteria response to adapt to stronger antibiotic pressure environment. This phenomenon is of great significance for exploring the transmission and diffusion mechanism of bacterial resistance genes in the future.

Abstract:[Objective] From the perspective of the soil fungal community, we explored the effect of forage rape in winter wheat-summer forage rape multiple cropping model as green manure returning to field on soil fertility.[Methods] We used the conventional winter wheat-summer maize multiple cropping model as the control(CK), and used Illumina Miseq high-throughput sequencing technology, to investigate the effect of the different sowing rates of rapeseed (S:small seeding rate, 7.5 kg/hm²; M:medium seeding rate, 15 kg/hm²; L:large seeding rate, 22.5 kg/hm²) and returning to the field (D1:September 10; D2:September 20; D3:September 30) on the abundance and community structure of soil fungal.[Results] Our results showed that compared with the control, with the increase of rape seeding rate, the returning biomass increased significantly, the wheat yield increased firstly and then decreased. We could get the highest wheat yield when rape was planted at 15 kg/hm² and returned on September 20. In addition, the amount of sowing increased, and the time of returning to the field should be advanced. Forage rape as green manure returning to field significantly influenced soil fungal Operational Taxonomic Unit numbers and richness indices (ACE and Chao1). Also, we find that in terms of fungal community composition, the dominant flora at the phylum level mainly includes Ascomycota, Basidiomycota, Zygomycota, and Chytridiomycota. At the level of class classification, the proportion of dominant fungi in forage rape returning to the field increased compared with the control. The function prediction results show that saprotroph is the main trophic mode of soil fungi, accounting for 41.32%-50.72%; and the facultative functional group increases to 30.05%-40.10% after returning to the field. At the same time, the abundance of beneficial biocontrol fungi such as Mortierella, Cephalosporium, Chaetomium and Penicillium was significantly increased.[Conclusion] In wheat arid areas of South Shanxi Province, popularizing the forage rape return to the field was beneficial to improve the soil fungal community structure. Under the condition of this experiment, the best method was to plant rapeseed with a medium sowing rate and return to field on September 20.

Abstract:[Objective] Allelopathic effects of cyanobacterial volatile organic compounds (VOCs) on other algae promote cyanobacteria becoming the dominant species in eutrophicated waters. The aim of the present study is to uncover the allelopathic lethal mechanism of cyanobacterial VOCs by using α-ionone.[Methods] In the treatment with α-ionone, the cell growth of Chlamydomonas reinhardtii was investigated, and the photosynthetic abilities, caspase-like activities and DNA ladders were investigated at lethal concentration.[Results] When C. reinhardtii cells were treated with α-ionone at 0.05 and 0.1 mmol/L, the cell growth was significantly inhibited, and 38.3% of the cells were killed by 0.1 mmol/L α-ionone. However, all the cells were killed by 0.2 mmol/L α-ionone. During the cell death, the photosynthetic pigments gradually degraded with prolonging the treatment time, and Fv/Fm gradually declined and even disappeared, indicating that the cell death is not a necrosis. Meanwhile, the activities of caspase-9-like and caspase-3-like increased remarkably. In the treatment with 0.2 mmol/L α-ionone for 1 h, DNA showed ladders and then gradually degraded to the fragments of 100-250 bp.[Conclusion] This suggests that cyanobacterial VOCs play the allelopathic role by inducing programmed cell death.

Abstract:[Objective] Pyruvate and acetyl-CoA were efficiently supplied by modifying citrate synthase (CS), pyruvate dehydrogenase complex (PDHC) or ATP-citrate lyase (ACL), thus increasing the L-leucine production in C. glutamicum.[Methods] Firstly, the effects of CS and PDHC activity on L-leucine production were studied using C. glutamicum as chassis cells, and then the effects of co-modifying CS and PDHC as well as introduction of ACL from C. tepidum on L-leucine production were investigated.[Results] The strain with low activity of CS (i.e, C. glutamicum XL-3 P_dapA-R2gltA) was beneficial to L-leucine production, and the yield of L-leucine was 17.5±0.6 g/L. However, changing the activity of PDHC is not conducive to L-leucine production. In addition, the strain with expression of CS under the weak promoter P_dapA-R2 and expression of PDHC under the medium promoter P_gapA (i.e., XL-4) produced 20.2±1.7 g/L of L-leucine. In addition, the yield of by-products in strain XL-4 was significantly decreased. Moreover, the ACL of Chlorobium tepidum was introduced into the recombinant strain XL-4, resulting in the low cell growth and L-leucine production. However, the production of L-leucine of recombinant strain with introduction of ACL in original strain XL-3 (i.e., XL-5) was significantly increased because of the efficient supply of pyruvate and acetyl-CoA. Recombinant strain XL-5 produced 18.5±1.2 g/L of L-leucine, which was 14.2% higher than that of the original strain XL-3.[Conclusion] Pyruvate and acetyl-CoA has been effectively in strain XL-4 by modifying citrate synthase (CS) and pyruvate dehydrogenase complex (PDHC), thus increasing the L-leucine production. Therefoere, the results of this study have important reference value for the following application of metabolic engineering to improve branched-chain amino acid producing strains.

Abstract:[Objective] To achieve the high-efficiency synthesis of lactose to tagatose in one-step, we cloned β-galactosidase gene (lacZ) and arabinose isomerase gene (araA) from Escherichia coli K-12 genome and co-expressed in E. coli BL21(DE3).[Methods] The araA and lacZ genes were amplified from the E. coli K-12 genome by PCR. The two genes with SD-AS sequence as a linker were cloned into the expression vector pET28a-1 to get the recombinant plasmid pET28a-araA-lacZ, which was transformed into the competent cells of E. coli BL21(DE3) to obtain E. coli BL21/pET28a-araA-lacZ. The synthesis conditions of tagatose by whole cells of E. coli BL21/pET28a-araA-lacZ were optimized and the process was scaled up.[Results] The araA and lacZ genes were efficiently co-expressed in E. coli BL21 simultaneously. The optimal conditions for the synthesis of tagatose by the whole cells of E. coli BL21/pET28a-araA-lacZ were determined:pH 8.0, 50℃, 5 mmol/L Mn²⁺, 0.5 mol/L borate and 0.1% SDS as permeabilizing agent. Under these optimal conditions, the highest yield of tagatose was 24.03±2.03 g/L with a molar conversion rate of 45.67% using 100 g/L lactose as substrate. The yield of tagatose was increased with the increasment of the substrate lactose concentration. The yield of tagatose was 83.81±1.38 g/L with 500 g/L lactose as substrate.[Conclusion] The genes lacZ and araA coding two target enzymes were co-expressed in E. coli BL21 to realize the efficient synthesis of high valued rare sugar tagatose from the cheap substrate lactose in one step. This research has laid a good research foundation for the preparation of low-energy functional sugars by biological methods.

Abstract:[Objective] Identifying and correcting the overestimation on contamination of metagenome-assembly genomes (MAGs) caused by the broken marker genes.[Methods] The impact of broken genes on quality assessment of genome was first analyzed using the simulated genomes from randomly fragmented the complete genome of isolates. We designed a corrected pipeline that identifying the broken genes pairs from the same "source" gene according to the taxonomic annotation against the nr database. Then the genome contamination was corrected by removing the redundant marker genes.[Results] The phenomenon that the genome contamination is positively correlated with the genome fragmentation degree was observed in both simulated genomes and MAGs obtained by genome binning. We designed a corrected pipeline based on the idea of identifying broken genes from the same "source" gene and the results based on the simulated genomes showed the contamination can be adjusted to complete genome level. Testing on 760 MAGs with contamination from gut and soil samples, we observed a reduction in contamination for nearly half of the MAGs, with 43 of them dropping to 0.[Conclusion] Our pipeline can correct the overestimated contamination of genome caused by broken genes to some extent and improve the availability of MAGs. The pipeline is expected to apply to the genome quality assessment of the increasing number of MAGs.

Abstract:[Objective] This experiment studies the genetic characteristics of Lactobacillus plantarum from different sources and its genetic diversity in different environments, and explores the similarities and differences in the adaptability of two strains of L. plantarum A8 and P9 in the animal intestine and plant surfaces, so as to provide a theoretical basis for the development of excellent strains.[Methods] This study analyzed the genomes of L. plantarum A8 and L. plantarum P9 isolated from the Animal intestines and plant surface. The research uses Next Generation Sequencing (NGS), based on the Illumina NovaSeq sequencing platform, and using the third-generation single-molecule sequencing technology, based on the PacBio Sequel sequencing platform to sequence L. plantarum A8 and L. plantarum P9. Carbohydrate-active enzymes (CAZy), Koyto encyclopedia of genes and genomes (KEGG), and Clusters of orthologous genes (COG) databases are used to annotate the genome function; CGView software is used to draw the genome circle map of the strain. At the same time, comparative genomics was used to compare with other published L. plantarum genomes.[Results] The research shows that the L. plantarum A8 and L. plantarum P9 genomes were different in genome size, the phylogenetic tree showed that the two strains were in the same branch with L. plantarum from other sources, and L. plantarum P9 was the closest to L. plantarum WLPL04 from breast milk, while L. plantarum A8 was the closest to L. paraplantarum DSM 10667. According to gene family analysis, there are 2643 common genes in the two strains, including some anti-stress proteins, such as heat shock proteins and cold shock proteins. There are 321 and 336 unique genes in plantarum A8 and P9, respectively. The unique genes in L. plantarum A8 are mainly involved in DNA replication, ABC transport system, PTS system, sulfonate transport system, amino acid biosynthesis and other metabolic pathways; The unique genes of L. plantarum P9 are mainly involved in carbohydrate transport and metabolism, such as rpiA gene, lacZ gene, fruA gene and so on.[Conclusion] By analyzing the genomic information of L. plantarum with comparative genomics method, it was found that L. plantarum from animal intestine has better amino acid transport ability, and the L. plantarum strain attached to the surface of the plant has good carbohydrate utilization ability, which provides a theoretical basis for the development and utilization of probiotics.

Abstract:[Objective] Coevolution and mutualism have existed between endophytic fungi and tea plants for a long time. They play an important protective role in the ecosystem of biotic and abiotic stress. Their community structure composition is relatively stable and has certain changes under the influence of external factors, but the effect of biotic stress on endophytic fungi community structure of tea leaves is still lack of systematic research. Therefore, it is necessary to study the diversity of endophytic fungi community structure under biotic stress.[Methods] We sequenced the ITS1 region of ITS rRNA genes of endophytic fungi in tea leaves of the diseased group and healthy group by high-throughput sequencing technology, and the microbiota composition and diversity were compared and analyzed.[Results] The results showed that the diseased group had lower endophytic fungal diversity and microbial community richness than those of the healthy group. At the phylum level, Ascomycota was dominant in both of them. At the genus level, Colletotrichum and Pseudopestalotiopsis were dominant in diseased group samples, Cladosporium was dominant in healthy group samples. In addition, there were significant differences in community structure composition of endophytic fungi between the two groups. The relative abundance of Pseudopestalotiopsis, Colletotrichum and Arthrinium in diseased group was significantly higher than that of the healthy group. However, the relative abundance of Mortierella, Aspergillus, Plectosphaerella, Lectera, Botryotrichum, Penicillium, Gibberella, Chaetomium, Lulwoana and Verticillium in diseased group were significantly lower than that of the healthy group.[Conclusion] The occurrence of tea grey blight changed the community structure of endophytic fungi in tea leaves and made a few species grow preferentially. These results provide insights into the effect of fungal diseases on the endophytic fungal community structure of tea leaves, and also lay the foundation for the pathogenic mechanism of the pathogen and provide the theoretical basis for the prevention and control of tea plant diseases.