Abstract
To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in ...sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in
Escherichia coli
. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain
ihfA
L−
-
aidB
+
-
ryfA
M−
-
gadA
H−
, producing 30.0 g L
−1
FFAs in fed-batch fermentation, the maximum titer in
E. coli
reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.
Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a ...condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (L-glutamate, L-aspartate, L-leucine and L-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear.
In this study, we aimed to improve surfactin production in B. subtilis by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. Among the 20 recombinant strains, 16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain (the surfactin production of BS168NU-Sd with only dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the yrpC, racE or murC genes were inhibited individually produced 0.54, 0.41, or 0.42 g/L surfactin, respectively. All three genes are related to the metabolism of L-glutamate, whose acylation is the first step in the surfactin condensation reaction. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of yrpC and racE produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of bkdAA and bkdAB, which are related to the metabolism of L-leucine and L-valine, not only improved surfactin production but also increased the proportion of the C
isoform.
This study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals.
•A salt-eluted polysaccharide (SPS-2) was isolated and purified from hawthorn.•Monosaccharide composition and FT-IR analysis indicated that SPS-2 may be pectin.•The antiglycation activity of SPS-2 ...was significantly higher than WPS in vitro.•MM-POS had the highest antiglycation activity among SPS-2 degradation products.•Ultrasound increased neutral sugar content and decreased antiglycation activity of MM-POS.
The crude polysaccharide was extracted from hawthorn (Crataegus pinnatifida Bunge. Var. major) and a salt-eluted polysaccharide (SPS-2) was fractionated by DEAE cellulose-52 chromatography. Monosaccharide composition and Fourier-transform infrared (FT-IR) analysis indicated that the SPS-2 contained 72.3% galacturonic acid and may be pectin. The results of antiglycation activity in vitro showed that the medium molecular weight pectin oligosaccharide (MM-POS) had the highest antiglycation activity among SPS-2 degradation products. Moreover, the MM-POS obtained by enzymatic degradation had stronger antiglycation activity than that by ultrasonic assisted enzymatic degradation. Composition analysis of MM-POS obtained from ultrasound-assisted enzymatic degradation showed that polygalacturonans content decreased from 85.1% to 61.9% but the arabinan and rhamnogalacturonans increased from 10.6% to 22.7% and from 3.5% to 13.2%, respectively, compared to the MM-POS obtained from enzymatic degradation. This study will provide a theoretical basis for the application of POSs in the food field, especially the exploitation of antiglycation agents.
•Pectin was degraded by enzymatic and ultrasound-assisted enzymatic method.•Antiglycation and prebiotic activities of pectin oligosaccharide were compared.•Antiglycation activity was positively ...correlated with oligogalacturonide content.•Prebiotic activity was positively correlated with galactose and arabinose content.•Degree of esterification was less important for antiglycation and prebiotic activity.
Pectin oligosaccharides (POSs) have prebiotic and antiglycation activities in vitro, but the specific structure-activity relationship is unclear. In this study, POSs were obtained by enzymatic and ultrasound-assisted enzymatic degradation of pectin polysaccharide (PPS), respectively. Based on the chemical characterization, the antiglycation in vitro and prebiotic activities of POSs were compared and the structure-activity relationship was studied. The results showed that the antiglycation activity of POSs in vitro was proportional to the galacturonic acid content and GalA:Rha molar ratios except for the low molecular weight POSs (LM-POSs), and inversely proportional to its branching degree, such as Ara:Rha and Gal:Rha molar ratios. In addition, it was also found that the prebiotic activity of POSs was positively correlated with Ara:Rha and Gal:Rha molar ratios in molecule composition and the neutral sugar content, especially galactose and arabinose. The degree of esterification (DE) was less important for both antiglycation and prebiotic activity of POSs. These results provided an important theoretical basis for POSs application in food.
Pectin oligosaccharides (POSs) can not only be used as prebiotics to promote the growth of beneficial bacteria in the intestine but also can be used as natural food-borne antiglycation agents to ...inhibit the formation of advanced glycation end products (AGEs) in vitro, which is related to their structure, including molecular weight and galacturonic acid content. In this study, haw polysaccharides (HPSs) were isolated and purified, and POSs with high antiglycation activity in vitro were prepared. On this basis, the inhibitory effect of POSs on the formation of AGEs in infant formula milk powder was investigated. The results showed that no obvious inhibitory effect of POSs was found on the formation of AGEs in infant formula milk powder under accelerated storage at 25 °C and 45 °C. But, POSs showed a strong inhibitory effect on the formation of furosine, Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL) and the total AGEs in infant formula milk powder under accelerated storage at 65 °C. In addition, POSs also had a strong inhibitory effect on the formation of lipid oxidation products but did not affect the formation of protein degradation products. Simultaneously, the cytotoxicity experiments using human umbilical vein endothelial cells showed that the infant formula milk powder supplemented with POSs had the lowest cytotoxicity compared to the blank control (BC) and GOS/FOS supplementation milk powder under accelerated storage at 65 °C, which may be related to the inhibitory effect of POSs on the formation of AGEs in infant formula milk powder. Furthermore, the in vitro fermentation experiments showed that the antiglycation process did not affect the prebiotic activity of POSs in infant formula milk powder.
Abstract
Miniaturized spectrometers are of considerable interest for their portability. Most designs to date employ a photodetector array with distinct spectral responses or require elaborated ...integration of micro & nano optic modules, typically with a centimeter-scale footprint. Here, we report a design of a micron-sized near-infrared ultra-miniaturized spectrometer based on two-dimensional van der Waals heterostructure (2D-vdWH). By introducing heavy metal atoms with delocalized electronic orbitals between 2D-vdWHs, we greatly enhance the interlayer coupling and realize electrically tunable infrared photoresponse (1.15 to 1.47 μm). Combining the gate-tunable photoresponse and regression algorithm, we achieve spectral reconstruction and spectral imaging in a device with an active footprint < 10 μm. Considering the ultra-small footprint and simple fabrication process, the 2D-vdWHs with designable bandgap energy and enhanced photoresponse offer an attractive solution for on-chip infrared spectroscopy.
Self‐assembled structures of 2D materials with novel physical and chemical properties, such as the good electrical and optoelectrical performance in nanoscrolls, have attracted a lot of attention. ...However, high photoresponse speed as well as high responsivity cannot be achieved simultaneously in the nanoscrolls. Here, a photodiode consisting of single MoS2 nanoscrolls and a p‐type WSe2 is demonstrated and shows excellent photovoltaic characteristics with a large open‐circuit voltage of 0.18 V and high current intensity. Benefiting from the heterostructure, the dark current is suppressed resulting in an increased ratio of photocurrent to dark current (two orders of magnitude higher than the single MoS2 nanoscroll device). Furthermore, it yields high responsivity of 0.3 A W−1 (corresponding high external quantum efficiency of ≈75%) and fast response time of 5 ms, simultaneously. The response speed is increased by three orders of magnitude over the single MoS2 nanoscroll device. In addition, broadband photoresponse up to near‐infrared could be achieved. This atomically thin WSe2/MoS2 nanoscroll integration not only overcomes the disadvantage of MoS2 nanoscrolls, but also demonstrates a single nanoscroll‐based heterostructure with high performance, promising its potential in the future optoelectronic applications.
A MoS2 nanoscroll‐based heterostructure integrated with atomically thin WSe2 is constructed, which exhibits well‐rounded performance with a high external quantum efficiency of 75%, open‐circuit voltage of 0.18 V and a fast response speed of 5 ms. It not only makes up for the inferiority of single MoS2 nanoscroll photodetectors, but also demonstrates the potential of nanoscrolls in nano‐optoelectronics.
Background The role of RNA-binding fox one homolog 2 (RBFOX2) in the progression of multiple tumors is increasingly supported by evidence. However, the unclearness pertaining to the expression of ...RBFOX2, its prognostic potential, and its correlation with the tumor microenvironment (TME) in pan-cancer persists. This study aims to comprehensively investigate the immunological prognostic value of RBFOX2. Methods The Cancer Genome Atlas Gene Expression Omnibus Genotype-Tissue Expression (GTEx), TIMER2.0, Kaplan-Meier (K–M) Plotter, University of Alabama at Birmingham Cancer data analysis Portal (UALCAN), cbioportal, and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) were utilized for a systematic analysis of RBFOX2. This analysis included studying its expression, prognostic value, DNA methylation, enrichment analysis, immune infiltration cells, and immune-related genes. Additionally, qRT-PCR, CCK-8, colony formation, transwell assays, and immunohistochemistry were employed to analyze the expression and biological function of RBFOX2 in liver cancer. Results Variations in RBFOX2 expression have been observed across diverse tumors and have been identified as indicators of unfavorable prognosis. It is closely linked to immune infiltration cells, immune checkpoints, chemokines, and chemokine receptors in the TME. Higher levels of RBFOX2 have been significantly associated with low response and poor prognosis in patients with non-small cell lung cancer (NSCLC) and melanoma who receive immunotherapy. Furthermore, the DNA methylation of RBFOX2 varies across different types of cancer and has shown better prognosis in patients with BLCA, BRCA, CESC, COAD, DLBC, HNSC, LAML, LGG, LUAD, PAAD, SKCM and THYM. Interestingly, RBFOX2 expression was found to be lower in hepatocellular carcinoma (HCC) patients’ tumor tissues compared to their paired adjacent tissues. In vitro studies have shown that knockdown of RBFOX2 significantly promotes the growth and metastasis of liver cancer cells. Conclusion This study investigates the correlation between DNA methylation, prognostic value, and immune cell infiltration with the expression of RBFOX2 in pan-cancer and indicates its potential role to inhibit metastasis of liver cancer.
A novel eco-friendly material (CS-U@PS) for persulfate slow-release to effectively degrade organic pollutants (methyl orange and pyrene) was synthesized using chitosan and urea as the encapsulated ...framework materials via an emulsion cross-linking method for the first time. The obtained CS-U@PS exhibits spherical shapes with a uniform size of approximately 2–3 µm according to the particle-size distribution and SEM image results. The slow-release mechanism was proposed through a kinetics model study and the Ritger–Peppas model fit well (r2 = 0.9699) to indicate that the slow-release process is non-Fickian diffusion. The influences of urea and PS dosages and oxidative conditions on methyl orange degradation were studied, and all the results suggested that urea played an important role in PS slow-release and can also catalyze the activation of PS by iron to further produce radicals and improve the removal efficiency of pollutants. A pyrene removal rate of 90.53% was achieved in aqueous solutions and an above 80% removal rate was obtained in weakly acidic or neutral soil environments by CS-U@PS activated by Fe2+ with citric acid as the chelating agent. Therefore, the fabricated slow-release oxidation materials exhibit application potential for the remediation of organic polluted groundwater and soil.
The degradation of MO can be inferred to possibly follow two main pathways as a result of preferential attack of ROS (mainly OH• and SO4⋅−) on MO molecules. The peak at m/z = 304 is the parent molecule of MO after losing Na+ due to dissolution in aqueous solution. Therefore, from this peak, it was confirmed that MO was not fully degraded. Initially, OH• oxidized the parent MO molecule to form the oxidation product of MO (m/z = 337.2). The other degradation pathway is that the NC bond of the dimethylamino group is broken down, leading to the replacement of the methyl group with protons to form new products corresponding to MO losing two methyl groups (m/z = 277). Then, the peaks found at m/z 139 and 173 proved the symmetric cleavage of the azo bond. The oxidative cleavage of the C and S bonds of the MO dye by free radicals generated the intermediate (m/z = 197.1). The peak at m/z 96.9 is ascribed to the formation of 2-cyclohexen-1-amine, and the peak at m/z 110 can be produced by hydroxylation. Further oxidation of hydroquinone resulted in the destruction (m/z = 171.9) and opening of aromatic rings and the formation of short linear aliphatic carboxylic acids (m/z = 116). Finally, aliphatic carboxylic acids can be directly converted into CO2 and H2O. Display omitted
•CS-U@PS is a novel chitosan-urea encapsulated material for persulfate slow-release.•The slow-release process is non-Fickian diffusion.•Urea was an excellent and vital sustained release component.•A moderate amount of urea can catalyze the Fe2 + activation of PS to further produce free radicals.•A pyrene removal rate of 90.53% and 80% were achieved in aqueous solutions and soil, respectively.
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