Biosynthesis of nonnutritive monosaccharide d-allulose by metabolically engineered Escherichia coli from nutritive disaccharide sucrose.

Sucrose is a commonly utilized nutritive sweetener in food and beverages due to its abundance in nature and low production costs. However, excessive intake of sucrose increases the risk of metabolic disorders, including diabetes and obesity. Therefore, there is a growing demand for the development of nonnutritive sweeteners with almost no calories. d-Allulose is an ultra-low-calorie, rare six-carbon monosaccharide with high sweetness, making it an ideal alternative to sucrose. In this study, we developed a cell factory for d-allulose production from sucrose using Escherichia coli JM109 (DE3) as a chassis host. The genes cscA, cscB, cscK, alsE, and a6PP were co-expressed for the construction of the synthesis pathway. Then, the introduction of ptsG-F and knockout of ptsG, fruA, ptsI, and ptsH to reprogram sugar transport pathways resulted in an improvement in substrate utilization. Next, the carbon fluxes of the Embden-Meyerhof-Parnas and the pentose phosphate pathways were regulated by the inactivation of pfkA and zwf, achieving an increase in d-allulose titer and yield of 154.2% and 161.1%, respectively. Finally, scaled-up fermentation was performed in a 5 L fermenter. The titer of d-allulose reached 11.15 g/L, with a yield of 0.208 g/g on sucrose.

Humic acid-mediated mechanism for efficient biodissolution of used lithium batteries.

Resource recovery of valuable metals from spent lithium batteries is an inevitable trend for sustainable development. In this study, external regulation was used to enhance the tolerance and stability of strains in the leaching of spent lithium batteries to radically improve the bioleaching efficiency. The leaching of Li, Ni, Co and Mn increased to 100 %, 85.06 %, 74.25 % and 69.44 % respectively after targeted cultivation with HA as compared to the undomesticated strain. In the process of microbial leaching of spent lithium batteries, the metabolites in the Ⅰ, Ⅳ, and Ⅴ regions of the metabolism of the undomesticated bacterial colony had a positive correlation to the dissolution of spent lithium batteries. The metabolites of Ⅰ, Ⅱ, and Ⅴ regions were directly affected by the HA domesticated flora on the dissolution of spent lithium batteries. The excess metabolism of protein substances can significantly promote the reduction of Ni, Co, Mn leaching, and at the same time in the role of a large number of humic substances complexed the toxic metal ions in the system, to ensure the activity of the bacterial colony. It can be seen that the bacteria were domesticated by humic acid, which promoted the bacteria's own metabolism, and the super-metabolised EPS promoted the solubilisation of spent lithium batteries.

Enhanced Biosynthesis of d-Allulose from a d-Xylose-Methanol Mixture and Its Self-Inductive Detoxification by Using Antisense RNAs in Escherichia coli.

d-Allulose, a C-3 epimer of d-fructose, has great market potential in food, healthcare, and medicine due to its excellent biochemical and physiological properties. Microbial fermentation for d-allulose production is being developed, which contributes to cost savings and environmental protection. A novel metabolic pathway for the biosynthesis of d-allulose from a d-xylose-methanol mixture has shown potential for industrial application. In this study, an artificial antisense RNA (asRNA) was introduced into engineered Escherichia coli to diminish the flow of pentose phosphate (PP) pathway, while the UDP-glucose-4-epimerase (GalE) was knocked out to prevent the synthesis of byproducts. As a result, the d-allulose yield on d-xylose was increased by 35.1%. Then, we designed a d-xylose-sensitive translation control system to regulate the expression of the formaldehyde detoxification operon (FrmRAB), achieving self-inductive detoxification by cells. Finally, fed-batch fermentation was carried out to improve the productivity of the cell factory. The d-allulose titer reached 98.6 mM, with a yield of 0.615 mM/mM on d-xylose and a productivity of 0.969 mM/h.

Transporter mining and metabolic engineering of Escherichia coli for high-level D-allulose production from D-fructose by thermo-swing fermentation.

D-Allulose is an ultra-low-calorie sweetener with broad market prospects in the fields of food, beverage, health care, and medicine. The fermentative synthesis of D-allulose is still under development and considered as an ideal route to replace enzymatic approaches for large-scale production of D-allulose in the future. Generally, D-allulose is synthesized from D-fructose through Izumoring epimerization. This biological reaction is reversible, and a high temperature is beneficial to the conversion of D-fructose. Mild cell growth conditions seriously limit the efficiency of producing D-allulose through fermentation. FryABC permease was identified to be responsible for the transport of D-allulose in Escherichia coli by comparative transcriptomic analysis. A cell factory was then developed by expression of ptsG-F, dpe, and deletion of fryA, fruA, manXYZ, mak, and galE. The results show that the newly engineered E. coli was able to produce 32.33 ± 1.33 g L-1 of D-allulose through a unique thermo-swing fermentation process, with a yield of 0.94 ± 0.01 g g-1 on D-fructose.

Development and verification of prognostic nomogram for ampullary carcinoma based on the SEER database.

Background: Ampullary carcinoma (AC) is a rare cancer of the digestive system that occurs in the ampulla at the junction of the bile duct and pancreatic duct. However, there is a lack of predictive models for overall survival (OS) and disease -specific survival (DSS) in AC. This study aimed to develop a prognostic nomogram for patients with AC using data from the Surveillance, Epidemiology, and End Results Program (SEER) database. Methods: Data from 891 patients between 2004 and 2019 were downloaded and extracted from the SEER database. They were randomly divided into the development group (70%) and the verification group (30%), and then univariate and multivariate Cox proportional hazards regression, respectively, was used to explore the possible risk factors of AC. The factors significantly related to OS and DSS were used to establish the nomogram, which was assessed via the concordance index (C-index), and calibration curve. An internal validation was conducted to test the accuracy and effectiveness of the nomogram. Kaplan-Meier calculation was used to predict the further OS and DSS status of these patients. Results: On multivariate Cox proportional hazards regression, the independent prognostic risk factors associated with OS were age, surgery, chemotherapy, regional node positive (RNP),extension range and distant metastasis with a moderate C-index of 0.731 (95% confidence interval (CI): 0.719-0.744) and 0.766 (95% CI: 0.747-0.785) in the development and verification groups, respectively. While, marital status, surgery, chemotherapy, regional node positive (RNP),extension range and distant metastasis were significantly linked to AC patients' DSS, which have a better C-index of 0.756 (95% confidence interval (CI): 0.741-0.770) and 0.781 (95% CI: 0.757-0.805) in the development and verification groups. Both the survival calibration curves of 3- and 5-year OS and DSS brought out a high consistency. Conclusion: Our study yielded a satisfactory nomogram showing the survival of AC patients, which may help clinicians to assess the situation of AC patients and implement further treatment.

Exploring the common mechanism of Yindan Xinnaotong soft capsule in the treatment of stroke and coronary heart disease through HIF1α-MMP9-mediated HIF1α signaling pathway / 药学学报

Coronary heart disease (CHD) and stroke are the most well-known cardiovascular diseases, which share many common pathological basis. Yindan Xinnaotong soft capsule (YDXNT) is a commonly used Chinese patent medicine in the treatment of stroke and CHD. However, its action of mechanism of co-treatment for stroke and CHD is still unclear. The aim of this study was to explore the common mechanism of YDXNT in co-treatment of CHD and stroke using network pharmacology, experimental verification and molecular docking. An integrated literature mining and databases of IPA, ETCM, HERB, Swiss Target Prediction, OMIM and GeneCards were used to screen and predict active ingredients and potential targets of YDXNT in co-treatment of CHD and stroke. The protein-protein interaction network, GO analysis and pathway analysis were analyzed by IPA software. The effect of YDXNT on core targets was verified by immunofluorescence. UPLC-QTOF/MS and molecular docking were used to screen and predict the main active constituents of YDXNT and their interactions with core targets. A total of 151 potential targets are predicted for YDXNT in co-treatment of CHD and stroke. Hypoxia-inducible factor-1α (HIF1α)-matrix metalloproteinase-9 (MMP9)-mediated HIF1α signaling pathway serves as one of the common mechanisms. YDXNT could reduce the increase of mitochondrial fluorescence intensity and the protein expression of HIF1α and MMP9 in HL-1 and HA induced by oxygen and glucose deprivation/reperfusion (OGD/R) in a dose-dependent manner. Baicalin may be the material basis for treating stroke and CHD with YDXNT. In conclusion, the HIF1α signaling pathway is one of the common key mechanisms of YDXNT in the co-treatment of stroke and CHD. The study provides support and basis for the in-depth scientific connotation of the traditional Chinese medicine theory of "same treatment to different diseases".

Methanol-Dependent Carbon Fixation for Irreversible Synthesis of d-Allulose from d-Xylose by Engineered Escherichia coli.

d-Allulose is a rare hexose with great application potential, owing to its moderate sweetness, low energy, and unique physiological functions. The current strategies for d-allulose production, whether industrialized or under development, utilize six-carbon sugars such as d-glucose or d-fructose as a substrate and are usually based on the principle of reversible Izumoring epimerization. In this work, we designed a novel route that coupled the pathways of methanol reduction, pentose phosphate (PP), ribulose monophosphate (RuMP), and allulose monophosphate (AuMP) for Escherichia coli to irreversibly synthesize d-allulose from d-xylose and methanol. After improving the expression of AlsE by SUMO fusion and regulating the carbon fluxes by knockout of FrmRAB, RpiA, PfkA, and PfkB, the titer of d-allulose in fed-batch fermentation reached ≈70.7 mM, with a yield of ≈0.471 mM/mM on d-xylose or ≈0.512 mM/mM on methanol.

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation.

D-Allulose is an ultra-low calorie sweetener with broad market prospects. As an alternative to Izumoring, phosphorylation-dephosphorylation is a promising method for D-allulose synthesis due to its high conversion of substrate, which has been preliminarily attempted in enzymatic systems. However, in vitro phosphorylation-dephosphorylation requires polyphosphate as a phosphate donor and cannot completely deplete the substrate, which may limit its application in industry. Here, we designed and constructed a metabolic pathway in Escherichia coli for producing D-allulose from D-fructose via in vivo phosphorylation-dephosphorylation. PtsG-F and Mak were used to replace the fructose phosphotransferase systems (PTS) for uptake and phosphorylation of D-fructose to fructose-6-phosphate, which was then converted to D-allulose by AlsE and A6PP. The D-allulose titer reached 0.35 g/L and the yield was 0.16 g/g. Further block of the carbon flux into the Embden-Meyerhof-Parnas (EMP) pathway and introduction of an ATP regeneration system obviously improved fermentation performance, increasing the titer and yield of D-allulose to 1.23 g/L and 0.68 g/g, respectively. The E. coli cell factory cultured in M9 medium with glycerol as a carbon source achieved a D-allulose titer of ≈1.59 g/L and a yield of ≈0.72 g/g on D-fructose.

Restoration Efficacy of Picea likiangensis var. rubescens Rehder & E. H. Wilson Plantations on the Soil Microbial Community Structure and Function in a Subalpine Area.

The knowledge concerning the relationship between vegetation restoration and soil microorganisms is limited, especially at high altitudes. In order to evaluate the restoration efficacy of the reforestation on the soil microbial community, we have examined vegetation composition, edaphic properties and structure and function of different soil microbial groups in two different aged (25- and 40-year-old) Picea likiangensis var. rubescens Rehder & E. H. Wilson (P. rubescens) plantations and the primeval coniferous forest (PCF) dominated by Abies squamata Masters by plot-level inventories and sampling in western Sichuan Province, China. Our results suggested that only the fungal samples in 25-year-old P. rubescens plantation could be distinguished from those in the PCF in both structure and function. The structure and function of the fungal community recovered relatively slowly compared with bacterial and archaeal communities. In addition to the soil chemical properties and tree species composition, the shrub composition was also a key factor influencing the soil microbial community. The P. rubescens plantations were conducive to restoring the soil microbial community in both structure and function. However, there were uncertainties in the variations of the bacterial and archaeal communities with increasing the P. rubescens plantation age.

Nitrogen Fertilization Modified the Responses of Schima superba Seedlings to Elevated CO2 in Subtropical China.

There are very few studies about the effects of relatively higher CO2 concentration (e.g., 1000 µmol·mol-1) or plus N fertilization on woody plants. In this study, Schima superba seedings were exposed to ambient or eCO2 (550, 750, and 1000 µmol·mol-1) and N fertilization (0 and 10 g·m-2·yr-1, hereafter: low N, high N, respectively) for one growth season to explore the potential responses in a subtropical site with low soil N availability. N fertilization strongly increased leaf mass-based N by 118.38%, 116.68%, 106.78%, and 138.95%, respectively, in different CO2 treatments and decreased starch, with a half reduction in leaf C:N ratio. Leaf N was significantly decreased by eCO2 in both low N and high N treatments, and N fertilization stimulated the decrease of leaf N and mitigated the increase of leaf C:N by eCO2. In low N treatments, photosynthetic rate (Pn) was maximized at 733 µmol·mol-1 CO2 in August and September, while, in high N treatments, Pn was continuously increased with elevation of CO2. N fertilization significantly increased plant biomass especially at highly elevated CO2, although no response of biomass to eCO2 alone. These findings indicated that N fertilization would modify the response of S. superba to eCO2.