Integrated Biofilm Modification and Transcriptional Analysis for Improving Fengycin Production in Bacillus amyloliquefaciens.

Although fengycin exhibits broad-spectrum antifungal properties, its application is hindered due to its low biosynthesis level and the co-existence of iturin A and surfactin in Bacillus amyloliquefaciens HM618, a probiotic strain. In this study, transcriptome analysis and gene editing were used to explore the potential mechanisms regulating fengycin production in B. amyloliquefaciens. The fengycin level of B. amyloliquefacien HM-3 (∆itu-ΔsrfAA) was 88.41 mg/L after simultaneously inhibiting the biosyntheses of iturin A and surfactin. The knockout of gene eps associated with biofilm formation significantly increased the fengycin level of the strain HM618, whereas the fengycin level decreased 32.05% after knocking out sinI, a regulator of biofilm formation. Transcriptome analysis revealed that the differentially expressed genes, involved in pathways of amino acid and fatty acid syntheses, were significantly down-regulated in the recombinant strains, which is likely associated with a decrease of fengycin production. The knockout of gene comQXPA and subsequent transcriptome analysis revealed that the ComQXPA quorum sensing system played a positive regulatory role in fengycin production. Through targeted genetic modifications and fermentation optimization, the fengycin production of the engineered strain HM-12 (∆itu-ΔsrfAA-ΔyvbJ) in a 5-L fermenter reached 1.172 g/L, a 12.26-fold increase compared to the fengycin level in the strain HM-3 (∆itu-ΔsrfAA) in the Erlenmeyer flask. Taken together, these results reveal the underlying metabolic mechanisms associated with fengycin synthesis and provide a potential strategy for improving fengycin production in B. amyloliquefaciens.

Enhancement of polymyxin B1 production by an artificial microbial consortium of Paenibacillus polymyxa and recombinant Corynebacterium glutamicum producing precursor amino acids.

Polymyxin B, produced by Paenibacillus polymyxa, is used as the last line of defense clinically. In this study, exogenous mixture of precursor amino acids increased the level and proportion of polymyxin B1 in the total of polymyxin B analogs of P. polymyxa CJX518-AC (PPAC) from 0.15 g/L and 61.8 % to 0.33 g/L and 79.9 %, respectively. The co-culture of strain PPAC and recombinant Corynebacterium glutamicum-leu01, which produces high levels of threonine, leucine, and isoleucine, increased polymyxin B1 production to 0.64 g/L. When strains PPAC and C. glu-leu01 simultaneously inoculated into an optimized medium with 20 g/L peptone, polymyxin B1 production was increased to 0.97 g/L. Furthermore, the polymyxin B1 production in the co-culture of strains PPAC and C. glu-leu01 increased to 2.21 g/L after optimized inoculation ratios and fermentation medium with 60 g/L peptone. This study provides a new strategy to improve polymyxin B1 production.

An Engineered Microbial Consortium Provides Precursors for Fengycin Production by Bacillus subtilis.

Fengycin has great potential for applications in biological control because of its biosafety and degradability. In this study, the addition of exogenous precursors increased fengycin production by Bacillus subtilis. Corynebacterium glutamicum was engineered to produce high levels of precursors (Thr, Pro, Val, and Ile) to promote the biosynthesis of fengycin. Furthermore, recombinant C. glutamicum and Yarrowia lipolytica providing amino acid and fatty acid precursors were co-cultured to improve fengycin production by B. subtilis in a three-strain artificial consortium, in which fengycin production was 2100 mg·L-1. In addition, fengycin production by the consortium in a 5 L bioreactor reached 3290 mg·L-1. Fengycin had a significant antifungal effect on Rhizoctonia solani, which illustrates its potential as a food preservative. Taken together, this work provides a new strategy for improving fengycin production by a microbial consortium and metabolic engineering.

Enhancing fengycin production in the co-culture of Bacillus subtilis and Corynebacterium glutamicum by engineering proline transporter.

Fengycin possesses antifungal activity but has limited application due to its low yields. Amino acid precursors play a crucial role in fengycin synthesis. Herein, the overexpression of alanine, isoleucine, and threonine transporter-related genes in Bacillus subtilis increased fengycin production by 34.06%, 46.66%, and 7.83%, respectively. Particularly, fengycin production in B. subtilis reached 871.86 mg/L with the addition of 8.0 g/L exogenous proline after enhancing the expression of the proline transport-related gene opuE. To overcome the metabolic burden caused by excessive enhancement of gene expression for supplying precursors, B. subtilis and Corynebacterium glutamicum which produced proline, were co-cultured, which further improved fengycin production. Fengycin production in the co-culture of B. subtilis and C. glutamicum in shake flasks reached 1554.74 mg/L after optimizing the inoculation time and ratio. The fengycin level in the fed-batch co-culture was 2309.96 mg/L in a 5.0-L bioreactor. These findings provide a new strategy for improving fengycin production.

Improved Production of Fengycin in Bacillus subtilis by Integrated Strain Engineering Strategy.

Fengycin is a lipopeptide with broad-spectrum antifungal activity. However, its low yield limits its commercial application. Therefore, we iteratively edited multiple target genes associated with fengycin synthesis by combinatorial metabolic engineering. The ability of Bacillus subtilis 168 to manufacture lipopeptides was restored, and the fengycin titer was 1.81 mg/L. Fengycin production was further increased to 174.63 mg/L after knocking out pathways associated with surfactin and bacillaene synthesis and replacing the native promoter (PppsA) with the Pveg promoter. Subsequently, fengycin levels were elevated to 258.52 mg/L by upregulating the expression of relevant genes involved in the fatty acid pathway. After blocking spore and biofilm formation, fengycin production reached 302.51 mg/L. Finally, fengycin production was increased to approximately 885.37 mg/L after adding threonine in the optimized culture medium, which was 488-fold higher compared with that of the initial strain. Integrated strain engineering provides a strategy to construct a system for improving fengycin production.

Bioinformatics and Experimental Analysis of the Prognostic and Predictive Value of the CHPF Gene on Breast Cancer.

Background: Recent studies in the United States have shown that breast cancer accounts for 30% of all new cancer diagnoses in women and has become the leading cause of cancer deaths in women worldwide. Chondroitin Polymerizing Factor (CHPF), is an enzyme involved in chondroitin sulfate (CS) elongation and a novel key molecule in the poor prognosis of many cancers. However, its role in the development and progression of breast cancer remains unclear. Methods: The transcript expression of CHPF in the Cancer Genome Atlas-Breast Cancer (TCGA-BRCA), Gene Expression Omnibus (GEO) database was analyzed separately using the limma package of R software, and the relationship between CHPF transcriptional expression and CHPF DNA methylation was investigated in TCGA-BRCA. Kaplan-Meier curves were plotted using the Survival package to further assess the prognostic impact of CHPF DNA methylation/expression. The association between CHPF transcript expression/DNA methylation and cancer immune infiltration and immune markers was investigated using the TIMER and TISIDB databases. We also performed gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the clusterProfiler package. Western blotting and RT-PCR were used to verify the protein level and mRNA level of CHPF in breast tissue and cell lines, respectively. Small interfering plasmids and lentiviral plasmids were constructed for transient and stable transfection of breast cancer cell lines MCF-7 and SUM1315, respectively, followed by proliferation-related functional assays, such as CCK8, EDU, clone formation assays; migration and invasion-related functional assays, such as wound healing assay and transwell assays. We also conducted a preliminary study of the mechanism. Results: We observed that CHPF was significantly upregulated in breast cancer tissues and correlated with poor prognosis. CHPF gene transcriptional expression and methylation are associated with immune infiltration immune markers. CHPF promotes proliferation, migration, invasion of the breast cancer cell lines MCF-7 and SUM1315, and is significantly enriched in pathways associated with the ECM-receptor interaction and PI3K-AKT pathway. Conclusion: CHPF transcriptional expression and DNA methylation correlate with immune infiltration and immune markers. Upregulation of CHPF in breast cancer promotes malignant behavior of cancer cells and is associated with poorer survival in breast cancer, possibly through ECM-receptor interactions and the PI3K-AKT pathway.

Carboxymethylation of Qingke ß-glucans and their physicochemical properties and biological activities.

In this study, the response surface methodology was used to optimize the reaction conditions of carboxymethylated modification of Qingke ß-glucans (QG), and effects of different degrees of carboxymethylation (high, medium, and low) on their structural characteristics, in vitro antioxidant activities, and in vitro hypolipidemic activities were studied. The optimal reaction conditions of carboxymethylated Qingke ß-glucans (QG-Cs) with high degree of carboxymethylation (DC = 0.90) were as follows: reaction temperature of 62.1 °C, reaction time of 3.29 h, and concentration of chloroacetic acid of 1.75 mol/L. Results demonstrated that the carboxymethylated modification significantly affected the solubilities, molar ratios of constituent monosaccharides, molecular weights, and apparent viscosities of QG. Indeed, the QG-Cs exhibited much higher antioxidant activities (reducing powers, NO, and DPPH radical scavenging activities), in vitro binding properties (fat, bile acid, and cholesterol binding capacities), and pancreatic lipase inhibition activities than that of QG. Furthermore, results showed that bioactivities of QG-Cs were closely correlated to their carboxymethyl groups. Results suggested that the carboxymethylated modification could be an efficient method for enhancing bioactivities of QG, and QG-Cs had good potential applications in bio-pharmaceutical industry.

Comparison of structural characteristics and bioactivities of polysaccharides from loquat leaves prepared by different drying techniques.

In the present study, freeze drying, hot-air drying, vacuum drying, and microwave drying at the microwave powers of 400, 600, and 800 W, respectively, were utilized to dry loquat leaves for evaluating the effects of different drying techniques on the physicochemical structures and bioactivities of polysaccharides extracted from loquat leaves (LLPs). Results demonstrated that the physicochemical structures and bioactivities of LLPs significantly affected by different drying techniques. The degrees of esterification, molar ratios of constituent monosaccharides, contents of uronic acids, apparent viscosities, and molecular weights of LLPs were varied by different drying techniques. Additionally, LLPs, particularly LLP-M4 which extracted from loquat leaves prepared by microwave drying at the power of 400 W, exerted remarkable in vitro binding capacities, strong inhibitory effects on α-amylase and α-glucosidase, and obvious antioxidant activities. Results indicated that the microwave drying could be an efficient drying technique before extraction of bioactive LLPs, and LLPs had great potential applications in the functional food and pharmaceutical industries.

Structural characterization, antioxidant activity, and immunomodulatory activity of non-starch polysaccharides from Chuanminshen violaceum collected from different regions.

Chuanminshen violaceum has been used as an important traditional Chinese medicine and a popular tonic food in China. Polysaccharides are considered the major bioactive components in C. violaceum. In this study, in order well understand the chemical structures and bioactivities of non-starch polysaccharides in C. violaceum (CVPs), the physicochemical structures, antioxidant activities, and immunomodulatory activities of CVPs in C. violaceum collected from different regions of China were investigated and compared. Results showed that the constituent monosaccharides and Fourier transform infrared spectra of CVPs in C. violaceum collected from different regions were similar. However, their molar ratios of constituent monosaccharides, molecular weights, and contents of uronic acids were different. Furthermore, CVPs exerted remarkable antioxidant activities (ABTS and nitric oxide radical scavenging capacities) and immunomodulatory activities (promoted production of nitric oxide, IL-6, and TNF-α from RAW 264.7 macrophages in vitro). Meanwhile, the antioxidant and immunomodulatory activities of CVPs extracted from C. violaceum also varied by cultivated regions. Moreover, results indicated that the antioxidant and immunomodulatory activities of CVPs were closely correlated to their α-1,4-d-galactosiduronic linkages. Results are helpful for better understanding of the structure-bioactivity relationships of CVPs, and beneficial for the improvement of their applications in pharmaceutical and functional food industries.

Quantitative Evaluation of Ultrasound-Assisted Extraction of 1,3-ß-glucans from Dictyophora indusiata Using an Improved Fluorometric Assay.

In the present study, an improved fluorometric assay based on aniline blue dye for the specific, accurate, and rapid quantification of 1,3-ß-glucans in edible mushrooms was developed and fully validated. Furthermore, the improved method was successfully applied for the quantitative evaluation of water soluble 1,3-ß-glucans extracted from Dictyophora indusiata by ultrasound-assisted extraction (UAE) with response surface methodology. Results showed that the improved method exhibited high specificity, accuracy, precision, repeatability, and stability, as well as a wide calibration range of 10-600 µg/mL (R2 > 99.9%). The maximum extraction yields of water soluble 1,3-ß-glucans (1.20%) and total polysaccharides (5.41%) were achieved at the optimized extraction parameters as follows: ultrasound amplitude (56%), ultrasound extraction time (15 min), and ratio of liquid to raw material (22 mL/g). The results suggest that the improved fluorometric assay has great potential to be used as a routine method for the quantitative evaluation of 1,3-ß-glucans in edible mushrooms and that the UAE method is effective for the extraction of 1,3-ß-glucans from edible mushrooms.

[Effects of elevated ozone concentrations on enzyme activities and organic acids content in wheat rhizospheric soil.] / O3æµ“åº¦å‡é«˜å¯¹å°éº¦æ ¹é™ åœŸå£¤é ¶æ´»æ€§å’Œæœ‰æœºé ¸å«é‡çš„å½±å“.

The elevated concentration of tropospheric ozone (O3) is an important global climate change driver, with adverse impacts on soil ecological environment and crop growth. In this study, a pot experiment was carried out in an open top chamber (OTC), to investigate the effects of elevated ozone concentration on soil enzyme activities (catalase, polyphenol oxidase, dehydrogenase and invertase), organic acids contents (oxalic acid, citric acid and malic acid) at different growth stages (tillering, jointing, heading and ripening stages) of wheat, and combined with the rhizospheric soil physicochemical properties and plant root characteristics to analyze the underlying reasons. The results showed that, elevated ozone concentration increased soil catalase, polyphenol oxidase, dehydrogenase and invertase activities at wheat ripening period to different degrees, with the effects on the activities of catalase and polyphenol oxidase being statistically significant. At the heading stage, activities of dehydrogenase and invertase were significantly increased by up to 76.7%. At the ripening stage, elevated ozone concentration significantly increased the content of citric acid and malic acid and redox potential (Eh) in rhizospheric soil, but reduced soil pH, electrical conductivity, total carbon and nitrogen. For root characteristics, elevated ozone concentrations significantly reduced the wheat root biomass, total root length and root surface area but increased the average root diameter.