Abstract:CRISPR-Cas is a defense system ubiquitous in bacteria and archaea. It has been successfully applied in genome editing in a variety of organisms. At present, CRISPR-Cas9 and CRISPR-Cas12a are the most widely used genome editing tools. However, the large protein sizes of Cas9 and Cas12a (more than 1 000 amino acids (aa)) hinder their delivery. TnpB and IscB (about 400 aa) encoded by the transposon family are considered ancestors of Cas12 and Cas9, respectively, whereas their functions are revealed just recently. They are named as obligate mobile element-guided activity (OMEGA), with the associated RNA named ωRNA. Since then, the OMEGA system has become one of the research hotspots in genome editing. OMEGA systems are diverse, with wide distribution in all the three domains of life. The in-depth research on the OMEGA system will aid in the development of new genome editing tools that are streamlined, efficient, and safe. Here, we reviewed the discovery history, structural characteristics, mechanisms of cleavage, and genome editing applications of OMEGA systems, aiming to lay a foundation for the development and optimization of genome editing tools.

Abstract:Depression is a prevalent affective disorder exerting negative impacts on an individual’s emotional and cognitive functions and is commonly complicated with other diseases. As depression is affecting increasingly younger populations, it poses a serious challenge to the mental health initiatives in China. In recent years, the relationship between depressive symptoms and gut microbiota has garnered increasing attention, fueled by the deepening research on gut microbiota. Lactulose, a medication aimed at promoting gut health and known for its potential benefits on emotional and cognitive functions, has sparked interests regarding its clinical application in ameliorating depressive symptoms. This article reviews the clinical research findings on the efficacy of lactulose in alleviating depressive symptoms and outlines the potential mechanisms through which lactulose may mitigate these symptoms. Furthermore, this article offers a perspective on the mitigation of depressive symptoms through the gut-brain axis.

Abstract:Type 2 diabetes mellitus (T2DM) stands as a chronic metabolic disorder posing a challenge to global public health, owing to its widespread prevalence. The intricate interplay between gut microbiota and the onset and progression of T2DM, along with the potential therapeutic benefits of modulating gut microbiota, has emerged as a focal point in contemporary research. Recent studies have underscored the capacity of traditional Chinese medicine to ameliorate T2DM by inducing alterations in gut microbiota. Nevertheless, the precise mechanisms underlying the pharmacological actions of traditional Chinese medicine via gut microbiota regulation remain elusive. The diverse bioactive compounds in traditional Chinese medicine play pivotal roles in eliciting its pharmacological effects. This article systematically reviews the advancements in the research concerning the modulation of gut microbiota for T2DM intervention by a spectrum of bioactive components in traditional Chinese medicine, encompassing polysaccharides, alkaloids, flavonoids, saponins, and other compounds. The objective of this review is to furnish a comprehensive theoretical framework supporting the preventive and therapeutic potential of traditional Chinese medicine in T2DM management, thereby significantly contributing to the modernization of traditional Chinese medicine.

Abstract:Agar is one of the important components in the cell wall of red algae. The biodegradation of agar affects marine ecological processes, such as nutrient recycling, succession of large seaweed communities, heavy metal pollution, and carbon sequestration. In addition, the degradation products of agar demonstrate great application potential in aquaculture, agriculture, medicine, health products, bioenergy, etc. Therefore, the biodegradation of agar and its ecological and application values have become research hotspots in recent years. This article reviews the research progress in the significance of agar degradation, microbial agarases, and agar metabolic pathways, providing theoretical support for the research on the ecological effects and comprehensive utilization of the agar from red algae.

Abstract:Levilactobacillus brevis is a common species of lactic acid bacteria mainly detected on the surface of plant stems and leaves and in pickles, dairy products, and intestines. With excellent physiological functions, L. brevis is a potential probiotic species. With the rise of genomics, it is of great significance to reveal the genetic characteristics and functional gene properties of L. brevis at the gene level for application of this bacterium. This paper reviews the genetic background and major functional genes of L. brevis, aiming to lay a theoretical foundation for the application of L. brevis.

Abstract:Streptococcus pneumoniae causes serious diseases such as pneumoniae and meningitis in humans. Capsular polysaccharides (CPSs) surrounding bacteria are not only key virulence factors but also major antigens. Therefore, CPSs have been prepared into polysaccharide vaccines and polysaccharide conjugate vaccines, which have greatly reduced the infection of pneumococci. CPSs are formed by polymerization of oligosaccharide repeating units which generally have 2−8 monosaccharides. CPSs present complex structures with diverse antigenic epitopes, being the basis of bacterial serotyping. Currently, 107 serotypes of S. pneumoniae have been identified. Each serotype has a unique CPS structure, a stable genetic basis, and specific serological characteristics. The diversity and constant changes of CPS structures explain the difficulty in the eradication of pneumococci. This review summarizes the known chemical structures of 95 CPSs and discusses the genetic basis, biosynthesis mechanism, and purification methods of CPSs. This review aims to enrich the knowledge about CPS diversity and provide a reference for probing into the functions and evolution of CPSs as well as for preparing polysaccharide vaccines.

Abstract:Glucose-1-phosphate is a key precursor for starch biosynthesis of photoautotrophs. Phosphoglucomutases (PGMs) belonging to the phosphohexomutase family have a high conserved characteristic and perform the interconversion between glucose-6-phosphate and glucose-1-phosphate to regulate the starch biosynthesis. Compared with the higher plants, microalgae possess unique photosynthetic systems. Additionally, some microalgae strains can utilize organic carbon sources to produce valuable biomass by heterotrophic or mixotrophic cultivation, which might endow PGMs with specific structural features and biological functions in starch metabolism to regulate the levels of carbon fixation by photosynthesis, carbohydrate metabolism, and other pathways in microalgae. This article summarizes the molecular characteristics, functions, and activity regulation of PGMs for microalgae. Moreover, this article elucidates the potential mechanisms by which PGMs regulate microalgae starch synthesis to influence intracellular protein and lipid metabolic pathways. This review lays a theoretical foundation for microalgae carbon sequestration and the value-added utilization of microalgae resources, contributing to the achievement of China’s “dual-carbon” goals.

Abstract:[Objective] We characterized the uterine microbiota in healthy felines and felines with pyometra, aiming to reveal the effect of pyometra on the uterine microbiota of felines and explore the potential pathogens causing pyometra. [Methods] High-throughput sequencing of the full-length 16S rRNA gene was employed to determine and compare the uterine microbiota in healthy felines and felines with pyometra. The key strains were isolated and identified by the culture method. [Results] The dominant bacterial genera in the uterus of healthy felines were Acinetobacter, Pseudomonas, Sphingomonas, and Weissella. The dominant bacterial genus and species in the uterus of felines with pyometra were Escherichia-Shigella and Escherichia coli, respectively. Functional prediction showed that pathways such as protein export, amino acid-related enzymes, protein processing in endoplasmic reticulum, and aminoacyl tRNA biosynthesis in the pyometra group were significantly reduced. The results of isolation and identification showed that the prevalent bacterial species in the uterus of felines with pyometra was E. coli. The isolates all belonged to the phylogroup B2 and were mostly tested positive for hylA, fimH, iroN, cnf1, papC, kpsMTII, and iutA. [Conclusion] We compared the uterine microbiota in healthy felines and felines with pyometra. The dominant bacteria in the uterus of healthy felines were mostly non-pathogenic, while those in the uterus of felines with pyometra changed significantly, with E. coli being dominant and carrying multiple virulence genes. The findings provide a theoretical basis for treating pyometra in felines.

Abstract:[Objective] To compare the metabolism and transcription between the probiotic Escherichia coli Nissle 1917 (EcN) and the model strains, thus providing a reference for the engineering and promoting the application of the food-safe strain EcN. [Methods] The genome and transcriptome were compared between EcN and model strains BL21(DE3) and W3110 by software, and plasmids were constructed to verify the differences. EcN-derived microcin was expressed in BL21(DE3) and the antibacterial effect of microcin was verified. [Results] A total of 904 differentially coding genes were identified. The differences in carbon source absorption and utilization of different strains were verified by experiments with different carbon sources as substrates. The expression of the promoter P_flic confirmed the differences in transcription among different strains. The recombinant strain of microcin showed an increase of 30.3% in the inhibition rate after 12 h of culture. [Conclusion] This study clarifies the metabolic characteristics of EcN and confirms the differences in transcription between EcN and model strains. Moreover, this study provides ideas for the development of microcin as a narrow-spectrum therapeutic drug to inhibit intestinal pathogens and reduce intestinal bacterial blooms.

Abstract:[Objective] To provide candidate strains and effective strategies for the control of mulberry fruit sclerotiniose, we screened out the endophytic bacteria with biocontrol potential for mulberry fruit sclerotiniose from a resistant mulberry cultivar. [Methods] The endophytic bacteria antagonistic to mulberry fruit sclerotiniose were isolated from mulberry plants by the tissue culture and confrontation culture methods. The antagonistic strain was identified based on morphological features, physiological and biochemical characteristics, and the phylogenetic relationship based on 16S rRNA gene sequences. The antimicrobial spectrum and control efficiency to detached mulberry fruits were determined to evaluate the application potential of the antagonistic strain. Furthermore, we observed the inhibitory effect of the fermentation supernatant of the strain on the mycelial growth of the pathogen, measured the variations in glycogen and reactive oxygen species accumulation of the pathogen treated with the antagonistic strain, and determined the expression of pathogen-related genes after treatment with the antagonistic strain to decipher the antagonistic mechanism of this strain. [Results] An endophytic bacterial strain C1R32 with strong and stable antagonistic activity on Sclerotinia sclerotiorum PZ-2 (the pathogen of mulberry fruit sclerotiniose) was isolated from a healthy mulberry branch. C1R32 showed similar morphological features and physiological and biochemical characteristics with Bacillus. The phylogenetic analysis based on 16S rRNA gene sequences revealed that C1R32 was located in the same clade with B. subtilis. Therefore, strain C1R32 was identified as B. subtilis. B. subtilis C1R32 had antagonistic activities against a variety of phytopathogens including S. sclerotiorum. The suspension and fermentation supernatant of B. subtilis C1R32 showed the control effects of 52.94% and 46.43%, respectively, on sclerotiniose of detached mulberry fruits. The cell-free fermentation supernatant of B. subtilis C1R32 caused the hypha swelling and distorting, cell wall breaking, and cytoplasm leakage of S. sclerotiorum PZ-2. Moreover, B. subtilis C1R32 inhibited S. sclerotiorum PZ-2 by reducing glycogen accumulation, promoting reactive oxygen species burst, and influencing the expression of genes associated with antioxidant activity. [Conclusion] We isolated an endophytic B. subtilis strain capable of controlling mulberry fruit sclerotiniose from a resistant mulberry cultivar and preliminarily explored its antagonistic mechanism, providing potential strain resources for the biocontrol of mulberry fruit sclerotiniose.

Abstract:[Objective] Outer membrane vesicles (OMVs) are spherical bilayer membrane structures secreted by Gram-negative and some Gram-positive bacteria. OMVs contain abundant surface antigens and are of great research significance in vaccine development. However, the presence of lipopolysaccharides (LPS), which is the primary component of OMVs, arouses safety concern. Therefore, genetically modifying bacterial LPS to produce safe and efficient OMVs is a viable approach to enhance the production and application of OMVs. [Methods] We modified Salmonella minnesota Re595 with O antigen and most core antigen deletions by deleting the acyl chain coding gene msbB and inserting the phosphatase coding gene lpxE from Francisella novicida to reduce acyl chains and phosphate groups on lipid A, thus obtaining less toxic LPS. LPS and OMVs were extracted from the starting strain and modified strain, and their pro-inflammatory activities were compared between the two strains. In addition, inactivated foot-and-mouth disease virus vaccines were prepared with OMVs to assess the immune adjuvant activity of OMVs. [Results] The modification of LPS reduced the endotoxin activity and pro-inflammatory responses while significantly increasing the immune adjuvant activity of OMVs. [Conclusion] This study demonstrates that the modification of LPS can attenuate the toxicity and enhance the immune adjuvant activity of bacterial OMVs. These findings provide a theoretical foundation for utilizing OMVs as immune adjuvants in the future.

Abstract:Transfer-message RNA (tmRNA) is a ubiquitous and stable non-coding small RNA in bacteria, with characteristic regions similar with both tRNA and mRNA. tmRNA is known to mediate the ribosome rescue mechanism called trans-translation and has effects on the pathogenicity and stress responses of pathogenic bacteria. [Objective] To study the function of tmRNA in Aeromonas veronii causing threats to aquaculture and human public safety, so as to reveal the molecular mechanism for the pathogenicity of A. veronii. [Methods] IntaRNA 2.0 was used to predict the downstream targets binding to tmRNA. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted to predict the biological processes and signaling pathways involving the predicted targets. Real-time qPCR was employed to compare the expression levels of the candidate target genes among the wild type, tmRNA knockout strain, and smpB knockout strain of A. veronii, thus identifying the potential targets regulated by tmRNA in the form of sRNA. [Results] One hundred potential specific downstream targets might bind to tmRNA at the 3′-end tRNA-like domain (tRNA-like domain, TLD), H2 domain, and PK3 and PK4 regions, thus participating in the general metabolic pathways of the pathogen. The results of qPCR indicated that the expression of WP_201994931.1 was regulated by tmRNA in an SmpB-independent manner, while the expression of WP_201954220.1, WP_005335875.1, WP_265062582.1, WP_265061484.1, and WP_265061494.1 was regulated by SmpB. [Conclusion] We preliminarily identified that WP_201994931.1 might constitute a downstream target regulated by tmRNA as an sRNA. This study provides basic information for expanding the functions of tmRNA in the form of sRNA and facilitates the further studies regarding the molecular mechanisms of the pathogenicity and environmental adaptation of A. veronii.

Abstract:Beneficial bacteria in the gut affect human health, and it is generally believed that the assemblage of healthy gut flora is achieved through vertical transmission of by breastfeeding in early infancy. There is limited evidence for the difference in the composition of beneficial bacteria across different mother-infant cohorts and the presence of population-specific microbial taxa [Objective] To investigate Lactobacillus spp. and the vertical transmission and genetic differences of the dominant species Lacticaseibacillus paracasei among mother-infant cohorts of different ethnic groups, providing a theoretical basis for developing personalized probiotic regimens. [Methods] Lactobacillus strains were isolated from 39 mother-infant pairs of three ethnic groups without mixed marriage in China and identified by repetitive extragenic palindromic PCR (rep-PCR) and groEL sequences. The genetic differences of 83 strains of L. paracasei, a representative species, were analyzed by multilocus sequence typing (MLST). [Results] The species and abundance of Lactobacillus varied among the mother-infant pairs of different ethnic groups. A total of 945 L actobacillus strains were isolated, belonging to 15 species of 4 genera. L. rhamnosus (20.07%), L. paracasei (16.54%), and L. casei (11.90%) were dominant species in the Han ethnic group, while L. casei (13.55%), L. paracasei (12.69%), and Ligilactobacillus salivarius (11.47%) were dominant bacteria in Uighur ethnic group in Hotan. The dominant species in the Li ethnic group in Hainan were Limosilactobacillus oris (24.55%), L. paracasei (15.85%), and Lactobacillus gasseri (10.87%). The 83 strains of L. paracasei were classified into 11 phylogenetic groups by rep-PCR and 31 sequence types (STs) by MLST, demonstrating ethnic specificity. L. paracasei isolates from the same mother-infant pair had the same STs, and isolates from the mother-infant pairs of the same ethnic group had higher genetic similarity. [Conclusion] L actobacil lus species varied in the mother-infant pairs of different ethnic groups, and L. paracase i strains from the same origin displayed higher genetic similarity, which supported vertical transmission at strain level and ethnic specificity.

Abstract:[Objective] To obtain a yeast strain efficiently producing the acidic protease PrA for applications in food processing, feed additives, and other related industries. [Methods] We constructed a recombinant strain of Pichia pastoris expressing PrA by fermentation in shake flasks and measured the enzymatic properties of the expressed PrA. Several strategies, such as signal peptide modification, gene dosage optimization, and co-expression with molecular chaperones, were employed to enhance the production of PrA. Additionally, high-density fermentation was employed to further improve the expression level. [Results] The expressed enzyme PrA showcased the specific activity of 3 974.00 U/mg, with the optimum performance at pH 3.0 and 45 ℃. The production of PrA by the parental strain was 738.03 U/mL. The modification of the MF4I signal peptide increased the production of PrA to 1 206.52 U/mL. Moreover, an increase in the copy number of prA further increased the PrA production to 2 406.47 U/mL. Additionally, co-expression with single or combined molecular chaperones increased the PrA production to 4 091.27 U/mL. After undergoing high-density fermentation, the enzyme activity reached 43 088.00 U/mL within 168 h, representing a 58.4-fold increase compared with the initial production. [Conclusion] High-level expression of PrA was achieved in P. pastoris, which laid a foundation for the future industrial applications. The results provide valuable insights into the research and development of PrA for applications in food processing and feed additives.

Abstract:[Objective] Keratinases, a class of serine proteases capable of degrading keratin, have important application potential and research value in the utilization of keratin resources. The efficient industrial production of keratinase is helpful to promoting its application in leather, textiles, feed, chemical fertilizers, daily chemicals, and medicine. In this study, we optimized the fermentation conditions of Bacillus subtilis WB600-pMA5-KerBv, a recombinant keratinase-producing strain constructed in our laboratory, to improve the enzyme production. Furthermore, we explored the potential application of keratinase in the degradation of fibrin. [Methods] First, the composition of the fermentation medium was determined by single factor experiments. Then, response surface methodology was employed to optimize the medium formula for producing keratinase, and the factors significantly affecting the growth and enzyme production of bacteria and the optimum concentrations were determined. Subsequently, the DoseResp model was adopted to predict the optimal growth point of the strain and thus guide the expansion of enzyme production in a 5 L fermenter. Finally, the blood clot and fibrinogen degradation experiments were carried out to evaluate the degradation performance of the keratinase. [Results] The formula of the fermentation medium for producing keratinase by the recombinant strain was optimized as follows (g/L): glucose 25.0, yeast powder 25.0, soybean meal 15.0, dipotassium phosphate 14.04, potassium dihydrogen phosphate 2.58, and magnesium chloride 0.3. The optimal growth point of the strain was predicted based on the DoseResp model to guide the expansion of production in a 5 L fermenter. Under the optimized conditions, the OD₆₀₀ (bacterial biomass) increased from 2.45 in a shake flask to 77.80, and the enzyme activity increased by about 4.76 times from 4 471 U/mL in a shake flask to 21 301.67 U/mL. In addition, the keratinase showcased remarkable degradation ability on fibrinogen and blood clots. [Conclusion] The systematic fermentation optimization and model-based prediction of enzyme production in fermenters improved the production of keratinase in Bacillus subtilis. The findings provided a research basis for the application of keratinase in thrombolysis.

Abstract:[Objective] To study the effects of adding different concentrations of garlic juice on the fermentation of pickled Chinese cabbage. [Methods] The physicochemical indicators and OD₆₀₀ during the fermentation process were measured, and the effects of garlic juice concentration on the richness, community structure, and abundance of bacteria during the fermentation were studied with natural fermentation as the control. The 16S rRNA gene high-throughput sequencing was carried out in the late fermentation stage for the pickled Chinese cabbage samples with the addition of garlic juice at different concentrations. Fuzzy mathematics-based evaluation was conducted for the sensory quality of garlic-flavored samples to select the lactic acid bacterial strains with excellent fermentation performance. [Results] Different concentrations of garlic juice had minor effects on the pH and OD₆₀₀ but significantly reduced the peak value of nitrite. Adding 0.2%–0.4% garlic juice was beneficial for the growth of Lactiplantibacillus, and the species richness of the product fermented with the addition of garlic juice was higher than that of the naturally fermented product. The fermentation liquid with the addition of 0.3% garlic juice had the highest species richness, with much higher number of genera than other concentration groups. The product from fermentation with 0.3% garlic juice showcased the best quality, presenting better sensory quality and appearance. The product with high species richness and a large number of genera had good sensory quality. A strain of lactic acid bacteria with excellent fermentation performance was screened out and identified as Lactiplantibacillus pentosus, which produced the most acids during fermentation, had the highest nitrite degradation rate, and exhibited tolerance to garlic. [Conclusion] This study provides a strain for the development of lactic acid bacteria fermentation agents and lays a theoretical foundation for the industrialization of garlic-flavored pickled Chinese cabbage.

Abstract:[Objective] To investigate the fungal diversity and community dynamics in the roots and root zone soil of the invasive species Spathoglottis plicata at different development stages and mine potential fungal resources. [Methods] The developmental period of S. plicata was divided into the protocorm, pre-seedling, middle-seedling, late-seedling, adult, and flowering stages. Fungal community composition and diversity in the roots and root zone soil were analyzed by QIIME 2 and other software. Fungal strains were isolated by the tissue culture method from the protocorms and seedlings of S. plicata, and the phylogenetic relationship of the strains was analyzed. [Results] The dominant fungi in the roots and root zone soil of S. plicata were Tulasnellaceae and Onygenaceae, respectively. The fungal community composition of S. plicata varied across different developmental stages. Tulasnellaceae was the most prevalent from the protocorm to middle-seedling stage, while Nectriaceae and Trichocomaceae became dominant during the late-seedling stage and adult stage. At the flowering stage, Ceratobasidiaceae presented high relative abundance. A total of 101 fungal strains were isolated from the protocorms and seedling roots of S. plicata, belonging to 13 families, in which strains of Tulasnellaceae accounted for 35.65%, being dominant. [Conclusion] The present study elucidated the fungal community composition and diversity dynamics in the roots and root zone soil of S. plicata at different development stages. The findings offer a theoretical foundation for comprehensively understanding of the intricate relationship between orchids and symbiotic fungi and facilitating the selection of optimal habitats for the conservation of rare and endangered orchid species.

Abstract:[Objective] The application of Bacillus subtilis in soil can promote plant growth, while few studies have reported the bacteriophages infecting B. subtilis. Therefore, the isolation, biological characterization, and genome sequencing of bacteriophages infecting B. subtilis from soil will contribute to the application of B. subtilis and enrich the biological information of bacteriophages infecting B. subtilis. [Methods] B. subtilis SM13 was used as the host to isolate a bacteriophage strain Bac-S from soil. Biological characterization, whole genome sequencing, gene function annotation, and phylogenetic analysis were performed for this bacteriophage strain. [Results] Transmission electron microscopy showed that Bac-S had a head with the diameter of about 43 nm and a tail too short to be measured. The optimal multiplicity of infection of Bac-S was 0.1. The one-step growth curve showed that Bac-S had an incubation period of about 10 min and a burst size of 30 PFU/cell. Bac-S had a wide host spectrum and can infect hosts of different genera. The sequencing results showed that the genome of Bac-S was 150 019 bp, with the G+C content of 42.6% and 237 open reading frames (ORFs). The BLASTn comparison with the sequences in the NCBI database showed that Bac-S was similar to other bacteriophages infecting B. subtilis. [Conclusion] A bacteriophage strain infecting B. subtilis with a short incubation period, a wide host spectrum, tolerance to high temperature, and intolerance to UV light was isolated from soil. The biological and genetic characterization of this bacteriophage enriches our knowledge about the bacteriophages infecting B. subtilis.

Abstract:[Objective] This study explored the application potential of symbiotic fungi and bacteria of Odontotermes formosanus in the degradation of wheat (Triticum aestivum L.) straw resources, aiming to provide a theoretical basis and supplement strain resources for the industrialization of straw biodegradation. [Methods] The lignocellulose-degrading fungi and bacteria were isolated from O. formosanus and screened by Congo red staining and the agar plate supplemented with carboxymethyl cellulose sodium (CMC-Na), and the enzyme production of the strains screened out was examined. Liquid fermentation at room temperature was carried out to evaluate the degradation effects of different strains and strain combinations on wheat straw. Fourier transform infrared spectroscopy (FTIR), X-ray crystal diffraction (XRD), and scanning electron microscopy (SEM) were employed to analyze the physical and chemical properties of wheat straw before and after degradation. [Results] Five species of fungi and three species of bacteria were isolated from the termite combs, including two species of Penicillium, one species of Purpureocillium, one species of Aspergillus, one species of Eutypella, one species of Bacillus, one species of Escherichia, and one species of Stenotrophomonas. Four efficient degrading strains were screened out and identified as Penicillium citrinum, Aspergillus nomiae, Bacillus subtilis, and Escherichia coli. In the liquid fermentation at room temperature, the combination of the four strains had the strongest comprehensive degradation performance, with the degradation rates of 24.35%, 47.24%, 35.75%, and 32.72% for dry matter, cellulose, hemicellulose, and lignin, respectively, in 12 days. The degradation destroyed the chemical bonds, the intermolecular force, and the composite structure of lignocellulose in wheat straw, and decreased the crystallinity of cellulose from 37.40% to 32.97%. [Conclusion] The combination of P. citrinum, A. nomiae, B. subtilis, and E. coli isolated from the combs of O. formosanus had a good degradation effect on wheat straw, demonstrating the application potential in the industrialization of straw biodegradation.

Abstract:[Objective] To investigate the effects of a metabolite cocktail composed of indole-3-propionic acid (IPA), sodium butyrate (SB), and valeric acid (VA) of gut microbiota on the proliferation of hepatocellular carcinoma cells. [Methods] The human hepatocellular carcinoma HepG2 cells were cultured in vitro and treated with the cocktail at different concentrations (1×, 2×, 3×, 4×, and 5×). The total cholesterol (TC) and triglyceride (TG) levels in the cells were determined by the total cholesterol and triglyceride assay kits. The Cell Counting Kit-8 (CCK-8) and colony formation assays were employed to examine the cell proliferation. Twelve BALB/c athymic nude mice were randomized into a control (Ctrl) group and a treatment (Treat) group and then subjected to subcutaneous injections of HepG2 cells. The tumor size was measured every three days, and the tumor volume and tumor inhibition rate were calculated. When the tumor volume reached 100 mm³, the mice in the Ctrl group were administered with sterile water by gavage daily, while those in the Treat group received the cocktail via gavage until euthanized under anesthesia. After 27 days of treatment, the body weights of mice in both groups were measured, and tumors were excised and weighed, with the tumor weight/body weight ratio calculated. The content of Ki-67 protein in the tumors was determined by immunohistochemical (IHC) staining, and the lipid accumulation within tumor cells was assessed by Oil Red O staining. [Results] The cocktail of IPA, SB, and VA lowered the levels of TC and TG in hepatocellular carcinoma HepG2 cells and exerted an inhibitory effect on the proliferation of HepG2 cells. Both CCK-8 and colony formation assays indicated that the cocktail inhibited the proliferation of HepG2 cells in a dose-dependent manner. The oral administration of the cocktail inhibited the growth of hepatocellular carcinoma cells, as evidenced by smaller and lighter tumors and lower tumor weight/body weight ratios in the Treat group than in the Ctrl group (Ctrl: 723 mm³, 0.47 g, 22.23%; Treat: 526 mm³, 0.32 g, 16.65%). IHC and Oil Red O staining further demonstrated reductions in Ki-67 expression and lipid accumulation in the mice administered with the cocktail via gavage. [Conclusion] The cocktail of IPA, SB, and VA can inhibit the proliferation and suppress the lipid synthesis of hepatocellular carcinoma cells.

Abstract:[Objective] A facultative anaerobic bacterium Klebsiella sp. CW-D3T utilizing sulfate as the terminal electron acceptor for anaerobic respiration was used for degradation of target pollutants in the system with phenanthrene (PHE)-Cd²⁺ co-contamination. The response mechanism of the strain to different Cd²⁺ concentrations in the sulfate reduction system and the anaerobic metabolic pathways of the strain for degrading PHE were studied. [Methods] A sulfate reduction system with an initial sulfate concentration of 20 mmol/L was developed to enhance the growth and metabolic activity of functional bacteria and improve the bacterial performance for remediating PHE-Cd²⁺ co-contamination. The changes in extracellular polymer secretion and the vibrational characteristics of characteristic peaks were analyzed to explore the cellular responses to different Cd²⁺ concentrations. Furthermore, qualitative and quantitative analyses of the metabolic products of PHE in the sulfate reduction system were conducted by GC-MS and HPLC. [Results] In the presence of 0.5–50.0 mg/L Cd²⁺, the sulfate reduction system of Klebsiella sp. CW-D3T enhanced the remediation efficiency of target compounds, with the PHE and Cd²⁺ removal rates above 70.00% when the initial Cd²⁺ concentration was below 10 mg/L. As the concentration of Cd²⁺ increased, the secretion of extracellular polysaccharides in extracellular polymeric substances (EPS) was more than that of extracellular proteins, and the intensity of characteristic peaks of polysaccharides and protein functional groups on the surface of bacterial cells was enhanced. The initial activation of PHE in the sulfate reduction system tended to favor carboxylation to produce 2-phenanthroic acid under Cd²⁺ stress. When the initial Cd²⁺ concentrations were 10 mg/L and 50 mg/L, the content of 2-phenanthroic acid peaked at 15.56 μg/L and 10.23 μg/L on day 5, respectively, which decreased by 27.56% and 52.37% compared with that of the control group without the addition of Cd²⁺. Cd²⁺ stress significantly affected the 2-phenanthroic acid content within the cycle and at the end of the cycle. [Conclusion] The biodegradation efficiency of PHE by Klebsiella sp. CW-D3T was significantly improved when sulfate was used as an electron acceptor in the presence of Cd²⁺. The extracellular polysaccharides and proteins played a positive role in enhancing the microbial tolerance to Cd²⁺ stress by regulating the detoxification process.

Abstract:Monocarboxylates such as lactate, pyruvate, and ketone bodies play an important role in the metabolic activities of organisms. As a monocarboxylate transporter, MpMch2 is mainly responsible for the transmembrane transport of monocarboxylates and the maintenance of glucose metabolism balance. [Objective] To analyze the functions of MpMch2 in Monascus purpureus. [Methods] The MpMch2 in M. purpureus Mp-21 was replaced with the hygromycin gene by homologous recombination to construct the deletion strain ∆MpMch2. The colony and cell morphology of Mp-21 and ∆MpMch2 on different media was observed, and the yield of monascus pigment, γ-aminobutyric acid, conidia and ascospores were determined. The expression levels of genes related to conidia and γ-aminobutyric acid were determined by RT-qPCR. [Results] There was no significant difference in the colony morphology between the wild type and ∆MpMch2 on different media. After knockout of MpMch2, the yields of conidia, ascospores, Monascus pigments, and γ-aminobutyric acid decreased, and the expression levels of related genes were down-regulated. [Conclusion] MpMch2 positively regulated the development of conidia and ascospores and the production of Monascus pigments and γ-aminobutyric acid.

Abstract:ThiD encoded by thiD in Thermoanaerobacter tengcongensis is a key enzyme in the biosynthesis of thiamine. The structure and functions of thiD have been elucidated in fungi, yeasts, and plants, while the role of thiD in thermophiles remains unclear. [Objective] This study aims to explore the thermal adaptation mechanism of T. tengcongensis and reveal the role of thiD in the thermal adaptation of T. tengcongensis at different temperatures. [Methods] The thiD-deleted mutant (ΔthiD) of T. tengcongensis was constructed by homologous recombination. The growth trends of the wild type (WT) and ΔthiD at 50 ℃, 60 ℃, 75 ℃, and 80 ℃ were observed and compared. The differentially expressed genes (DEGs) between ΔthiD and WT cultured at 75 ℃ were determined by RNA-seq. The transcript levels of 13 genes and 3 sRNAs in WT and ΔthiD at 50 ℃, 60 ℃, 75 ℃, and 80 ℃ were compared and analyzed by real-time PCR. [Results] ΔthiD was successfully constructed, with the growth rate not significantly different from WT at 50 ℃. However, ΔthiD show cased slower growth than WT at 60 ℃ and 75 ℃ and did not grow at 80 ℃. The transcriptome results revealed 503 DEGs in ΔthiD compared with WT, including 278 DEGs with up regulated expression and 213 DEGs with down regulated expression. The Kyoto encyclopedia of genes and genomes (KEGG) analysis indicated the following pathways associated with thermophilic adaptation, involving thiamine metabolism, pyrimidine metabolism and purine metabolism, peptidoglycan biosynthesis, fatty acid metabolism, amino acid metabolism, two-component system, DNA replication, homologous recombination, mismatch repair, and phosphotransferase system. The transcript levels of 13 genes and 3 sRNAs related to thermal adaptation in WT and ΔthiD changed at specific temperatures. [Conclusion] thiD plays an important role in the thermal adaptation of T. tengcongensis. This study provides experimental data and a theoretical basis for revealing the role of thiD in the thermal adaptation of thermophiles at different temperatures.

Abstract:Isoquercetin is a flavonoid with antioxidant, anti-inflammatory, and immunomodulatory activities. However, the low content in plants poses a challenge to the large-scale production of isoquercetin by the extraction method. [Objective] α-l-rhamnosidase can specifically hydrolyze the terminal l-rhamnose residues of natural glycosides. In this study, we screened the strains capable of efficiently and specifically transforming rutin to produce isoquercetin with rutin as the sole carbon source and applied the α-l-rhamnosidase to the production of isoquercetin, aiming to provide new elements for the large-scale production of isoquercetin. [Methods] The selective culture medium with rutin as the sole carbon source was used to screen and identify the strains that can specifically hydrolyze rutin into isoquercetin. The transcriptome analysis was carried out to obtain highly efficient and specific α-l-rhamnosidase, the domain composition of which was determined by structural simulation. The enzymatic properties and substrate specificity of the α-l-rhamnosidase were studied. Furthermore, the hydrolysis effect of the enzyme heterologously expressed in Pichia pastoris in a 5 L fermenter was determined. [Results] AfRhase had five domains, including one α-domain (domain A) and four β-domains (domains N, E, F, and C). With rutin as the substrate, the recombinant enzyme AfRhase showcased the best performance at 55 ℃ and pH 4.5. AfRhase had a wide range of substrates including rutin, hesperidin, naringin, and epimedin C. In a 5 L fermenter for scaled-up production of isoquercetin, P. pastoris expressing AfRhase generated 61 g isoquercetin by hydrolyzing 120 g crude rutin (purity of 70%), with the molar conversion rate of 95.4% and production efficiency of 2.0 mmol/(L·h). [Conclusion] This study for the first time discovered a highly efficient and specific α-l-rhamnosidase from Aspergillus sp. XT-1 for the production of isoquercetin from rutin and heterologously expressed this enzyme in P. pastoris. The domain composition, enzymatic properties, substrate specificity, and hydrolysis efficiency in a 5 L fermenter of this enzyme were determined. In conclusion, this study broadened the function of a fungus-derived α-l-rhamnosidase for the transformation of rutin and laid a foundation for the industrial production of isoquercetin.

Abstract:Bacterium-mediated cancer immunotherapy (BCI) presents numerous advantages in cancer treatment, while the immune response mechanism of dexamethasone (DEX) combined with BCI for tumor treatment remains unclear. [Objective] To investigate the therapeutic efficacy and mechanism of dexamethasone in combination with attenuated Salmonella typhimurium St.ΔppGpp-mediated BCI. [Methods] The inhibitory effects of St.ΔppGpp+DEX on cancer were evaluated in a murine model of colorectal cancer. In vivo imaging was utilized to determine the tumor targeting and colonization duration of St.ΔppGpp. Organ toxicity resulted from St.ΔppGpp+DEX treatment was assessed by hematoxylin and eosin (H&E) staining. Macrophage polarization, neutrophil recruitment, and T-cell responses were analyzed by flow cytometry and immunofluorescence assay of sections. The changes in inflammatory cytokines in the tumor microenvironment were examined via qRT-PCR. A mouse model transplanted with human colorectal cancer was employed to confirm the effect of T cell depletion on the therapeutic efficacy of St.ΔppGpp+DEX. [Results] The combined treatment St.ΔppGpp+DEX significantly decreased tumor size and enhanced the survival rate of mice. DEX extended the colonization of St.ΔppGpp in tumor cells. Furthermore, St.ΔppGpp+DEX did not induce damage to vital immune organs, and it facilitated the polarization of macrophages from M2 to M1 phenotype while suppressing neutrophil recruitment. T cell depletion did not influence the efficacy of St.ΔppGpp+DEX. [Conclusion] DEX can enhance the anti-tumor effects of St.ΔppGpp by inhibiting neutrophil recruitment and increasing the proportion of M1 macrophages in the tumor microenvironment.

Abstract:[Objective] To explore the effects of CbrB from the carbon catabolite repression system on the biocontrol performance of Pseudomonas protegens Pf-5. [Methods] The mutant Pf5274 with the markerless deletion of the coding region of cbrB was constructed by a double-crossover recombination event based on pJQ200SK. Moreover, the complementary strain of cbrB and the control strains were constructed by the plasmid complementation method based on pBBR1Am. Lastly, the effects of CbrB on the growth, biofilm formation, motility, antifungal activity, and pyoluteorin synthesis of Pf-5 were analyzed by the measurement of OD₆₀₀, crystal violet staining, agar plate culture, plate confrontation method, and pltL'-'lacZ fusion report strains, respectively. [Results] CbrB greatly slowed the growth of Pf-5 in the natural medium LB, but greatly sped up the growth of Pf-5 in the basic medium M9-glucose. In addition, CbrB significantly promoted the motility but inhibited the biofilm formation, antifungal activity, and pyoluteorin synthesis of Pf-5. [Conclusion] CbrB plays a role in regulating the growth, biofilm formation, motility, antifungal activity, and pyoluteorin synthesis of P. protegens Pf-5, thus regulating the biocontrol performance of this strain. This study provides a theoretical basis for the biocontrol capabilities of strains by genetic engineering and lays a foundation for probing into the biosynthesis of pyoluteorin.

Abstract:[Objective] Monensin is a polyether antibiotic produced by Streptomyces cinnamonensis. To enhance the production of monensin by microbial fermentation, we employed metabolic engineering to strengthen the synthesis pathway of the key precursor methylmalonyl-CoA in S. cinnamonensis 2110. [Methods] Firstly, crotonyl-CoA reductase (CCR) was overexpressed to strengthen the acetoacetyl-CoA pathway. Subsequently, methylmalonyl-CoA mutase (MCM) was overexpressed to improve the succinyl-CoA pathway. Finally, an engineered strain with tandem overexpression of CCR and MCM was constructed and evaluated for the fermentation performance. [Results] The overexpression of CCR increased the strain biomass and monensin titer by 10.4% and 19.0%, respectively, after 10 days of shake-flask fermentation. The overexpression of MCM increased the monensin titer by 9.9%, whereas it did not increase the strain biomass after 10 days of shake-flask fermentation. The tandem overexpression of CCR and MCM increased the biomass and monensin titer by 9.4% and 26.8%, respectively, after 10 days of shake-flask fermentation. In a 5 L bioreactor, the engineered strain 2110-CCR-MCM reached the highest biomass of 54.6 g/L and monensin titer of 11.3 kU/mL, which increased by 12.7% and 36.2%, respectively, compared with those of the starting strain 2110. [Conclusion] CCR and MCM mediated the key metabolic pathway of monensin biosynthesis in S. cinnamonensis, and the overexpression of CCR and MCM was highly favorable for monensin synthesis. This study provides technical reference for the engineering of strains with high yields of other polyketides.

Abstract:[Objective] Over 7 million people die from bacterial infections every year due to the inappropriate or untimely use of antibiotics. Antimicrobial susceptibility test (AST) is a routine method guiding antibiotic therapy in clinical practice, while the existing methods have limitations as they require at least 16–20 h to get the results. We aimed to develop a novel rapid AST method based on bacterial morphology analysis, which can improve the diagnosis efficiency and provide timely treatment for the patients with bacterial infections, especially for urgent cases. [Methods] We developed an in-situ time-lapsed imaging (ISM-TLI) technique for microcolonies by combining bacterial incubation, in vitro antibiotic treatment, microscopic imaging, and image processing algorithm. The combination of the imaging device with the algorithm enabled the tracing of the morphological changes of colonies treated with different concentrations of antibiotics in-situ and the rapid obtainment of AST results. [Results] This technique worked effectively for both Gram-positive and Gram-negative bacteria and could determine the minimum inhibitory concentration (MIC) within 2 h, with the results in agreement with those obtained with the clinical gold standard method. [Conclusion] Our rapid AST technique significantly reduced the turn-around-time for obtaining the AST profile and had great potential for AST of a wide range of other strains.

Abstract:[Objective] To improve the culture medium components of Parvimonas micra, increase the number of live cells, and develop a demonstration method for culturing fastidious bacteria. [Methods] Biochemical analysis was conducted on a strain of P. micra to screen the potential substrates that could promote bacterial growth. A single-factor experiment was carried out for each substrate with three concentrations. The substrate with a significant bacterial enrichment effect was further optimized for concentration, and thus a new culture medium was obtained. [Results] The single-factor experiment results showed that the substrates with significant bacterial enrichment effects included l-serine, l-threonine, and glycyl-l-glutamine. In the medium with the addition of 4.8 g/L l-serine, the live cell count of P. micra reached 3.6×10⁸ cfu/mL, representing a 4.2-fold increase compared with that in the basic medium with tryptone soya broth (TSB) and fetal bovine serum (FBS). Furthermore, the improved culture medium was applied to the culture of another P. micra strain, demonstrating a significant growth-promoting effect. [Conclusion] This study proves that using biochemical identification plates to screen medium supplements is a fast and efficient method, providing a reference for the enlargement culture of fastidious bacteria.