L-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand. Although microbial cell factories have been widely used for biosynthesis, there are still challenges such as genetic instability and low efficiency in the biosynthesis of L-citrulline. In this study, an efficient, plasmid-free, non-inducible L-citrulline-producing strain of Escherichia coli BL21(DE3) was engineered by combined strategies. Firstly, a chassis strain capable of synthesizing L-citrulline was constructed by block of L-citrulline degradation and removal of feedback inhibition, with the L-citrulline titer of 0.43 g/L. Secondly, a push-pull-restrain strategy was employed to enhance the L-citrulline biosynthesis, which realized the L-citrulline titer of 6.0 g/L. Thirdly, the NADPH synthesis and L-citrulline transport were strengthened to promote the synthesis efficiency, which achieved the L-citrulline titer of 11.6 g/L. Finally, fed-batch fermentation was performed with the engineered strain in a 3 L fermenter, in which the L-citrulline titer reached 44.9 g/L. This study lays the foundation for the industrial production of L-citrulline and provides insights for the modification of other amino acid metabolic networks.
Citrulline/biosynthesis* ; Escherichia coli/genetics* ; Metabolic Engineering/methods* ; Fermentation ; NADP/biosynthesis*

Phytoestrogens refer to a class of compounds in plants that can bind to and activate estrogen receptors in mammalian organisms,exerting varying degrees of protective and improvement effects on the body.Modern pharmacological studies have found that phytoestrogens have therapeutic effects on the cardiovascular system,nervous system,endocrine system,immune system,and so on.It is worth noting that the binding of phytoestrogens to estrogen receptors in the brain can produce central neuroprotective effects and improve learning and cognitive impairment in Alzheimer's pectients through multiple targeted pathways.This paper describes the research progress and related mechanisms of phytoestrogen-like monomers,Chinese herbal medicine,and compound formulations in Alzheimer's disease.The aim is to provide new drug design ideas and solutions for the effective treatment of senile dementia with traditional Chinese medicine.

Objective This study aims to compare the sedative effects of remimazolam and midazolam during im-pacted tooth extraction to provide a comfortable sedation treatment for patients with dental anxiety.Methods A pro-spective randomized controlled trial was conducted,in which 60 patients undergoing intravenous sedation for mandibular impacted third molar extraction were evenly divided into either the remimazolam or midazolam group.Prior to receiving a nerve blocker,the patients were sedated with remimazolam or midazolam.Various parameters were recorded and ana-lyzed,including onset time,awakening time,recovery time,modified dental anxiety scale(MDAS)scores before and af-ter surgery,patient-doctor satisfaction levels,postoperative side effects within 24 hours,heart rate(HR),and mean arteri-al pressure(MAP)at different time points.Results Compared with the midazolam group,patients in the remimazolam group demonstrated significantly shorter onset,awakening,and recovery times as well as lower postoperative MDAS scores and higher levels of patient-doctor satisfaction.Fewer postoperative side effects were reported in the remimazol-am group,although the differences were not statistically significant.Conclusion The use of remimazolam demon-strates faster onset and recovery,superior efficacy in reducing dental anxiety,and enhanced satisfaction among patients and doctors,thereby presenting distinct advantages for sedation treatment for patients with dental anxiety.

Objective To study the relationship between serum Melatonin,macrophage inflammatory pro-tein-1α(MIP-1α)and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflamma-some and prognosis in children with acute respiratory distress syndrome(ARDS).Methods A total of 140 children with ARDS who were admitted to the hospital from January 2021 to January 2023 were enrolled as the ARDS group,and 100 healthy children who underwent physical examination during the same period were enrolled as the control group.According to the clinical outcome on 28 d after admission,the children with ARDS were divided into a death group(29 cases)and a survival group(111 cases).Enzyme-linked immu-nosorbent assay was used to detect the serum levels of Melatonin,MIP-1α,interleukin(IL)-1β,and IL-18.Re-al-time fluorescent quantitative PCR was used to detect the mRNA levels of NLRP3 and Caspase-1.Pearson correlation was used to analyze the correlation between serum levels of Melatonin and MIP-1α and NLRP3 in-flammasome related indicators(NLRP3 mRNA,Caspase-1 mRNA,IL-1β,IL-18)in children with ARDS.Mul-tivariate Cox regression analysis was used to analyze the factors affecting the prognosis of children with ARDS.According to the mean levels of serum Melatonin and MIP-1α,the children with ARDS were divided in-to a high/low Melatonin group and MIP-1α group.The Kaplan-Meier method was used to draw the survival curves of ARDS children with high/low levels of serum Melatonin and MIP-1α.The receiver operating charac-teristic(ROC)curve was used to analyze the predictive value of serum Melatonin and MIP-1α for death in children with ARDS.Results Compared with the control group,the ARDS group had a significantly decreased serum Melatonin level and significantly increased serum MIP-1α,IL-1β,IL-18 and NLRP3 mRNA and Caspase-1 mRNA levels in peripheral blood mononuclear cells(P＜0.05).Pearson correlation analysis showed that the serum Melatonin level of ARDS children was negatively correlated with NLRP3 mRNA,Caspase-1 mRNA,IL-1β and IL-18(P＜0.05),the level of MIP-1α was positively correlated with NLRP3 mR-NA,Caspase-1 mRNA,IL-1β and IL-18(P＜0.05).Multivariate Cox regression analysis showed that acute physiology and chronic health evaluation Ⅱ score,NLRP3 mRNA,Caspase-1 mRNA,IL-1β,IL-18 and MIP-1αwere independent risk factors affecting the prognosis of children with ARDS.Melatonin was an independent protective factor(P＜0.05).Kaplan-Meier survival curve analysis showed that the 28 d survival rate in high Melatonin group was higher than that in low Melatonin group(P＜0.05),and the 28 d survival rate in high MIP-1α group was lower than that in low MIP-1α group(P＜0.05).ROC curve analysis showed that the area under the curve of Melatonin and MIP-1α combined to predict the death of ARDS children was 0.881(95％CI 0.816-0.930).It was higher than 0.785(95％CI 0.708-0.850)and 0.778(95％CI 0.700-0.844)predic-ted by Melatonin or MIP-1α alone.Conclusion The serum Melatonin level is decreased and MIP-1α level is in-creased in children with ARDS,which is closely related to NLRP3 inflammasome and prognosis.Combined de-tection of serum Melatonin and MIP-1α levels has a high predictive value for the prognosis of children with ARDS.

The comparison between traditional Chinese medicine Jinzhen oral liquid (JZOL) and Western medicine in treating children with acute bronchitis (AB) showed encouraging outcomes. This trial evaluated the efficacy and safety of the JZOL for improving cough and expectoration in children with AB. 480 children were randomly assigned to take JZOL or ambroxol hydrochloride and clenbuterol hydrochloride oral solution for 7 days. The primary outcome was time-to-cough resolution. The median time-to-cough resolution in both groups was 5.0 days and the antitussive onset median time was only 1 day. This randomized controlled trial showed that JZOL was not inferior to cough suppressant and phlegm resolving western medicine in treating cough and sputum and could comprehensively treat respiratory and systemic discomfort symptoms. Combined with clinical trials, the mechanism of JZOL against AB was uncovered by network target analysis, it was found that the pathways in TRP channels like IL-1β/IL1R/TRPV1/TRPA1, NGF/TrkA/TRPV1/TRPA1, and PGE2/EP/PKA/TRPV1/TRPA1 might play important roles. Animal experiments further confirmed that inflammation and the immune regulatory effect of JZOL in the treatment of AB were of vital importance and TRP channels were the key mechanism of action.

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.
Bacillus subtilis/metabolism* ; Vitamin K 2/metabolism* ; Bioreactors/microbiology* ; Membrane Microdomains/metabolism*

Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.
N-Acetylneuraminic Acid/metabolism* ; Bacillus subtilis/metabolism* ; Promoter Regions, Genetic/genetics* ; Binding Sites ; Biosensing Techniques

Genome-scale metabolic network model (GSMM) is an extremely important guiding tool in the targeted modification of industrial microbial strains, which helps researchers to quickly obtain industrial microbes with specific traits and has attracted increasing attention. Here we reviewe the development history of GSMM and summarized the construction method of GSMM. Furthermore, the development and application of GSMM in industrial microorganisms are elaborated by using four typical industrial microorganisms (Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae) as examples. In addition, prospects in the development trend of GSMM are proposed.
Corynebacterium glutamicum/genetics* ; Escherichia coli/genetics* ; Metabolic Engineering ; Metabolic Networks and Pathways/genetics*

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.
Bacillus subtilis/genetics* ; Bacterial Proteins/genetics* ; Gene Editing ; Metabolic Engineering ; Promoter Regions, Genetic

ProBiotic-4 is a probiotic preparation composed of , , , and . This study aims to investigate the effects of ProBiotic-4 on the microbiota-gut-brain axis and cognitive deficits, and to explore the underlying molecular mechanism using senescence-accelerated mouse prone 8 (SAMP8) mice. ProBiotic-4 was orally administered to 9-month-old SAMP8 mice for 12 weeks. We observed that ProBiotic-4 significantly improved the memory deficits, cerebral neuronal and synaptic injuries, glial activation, and microbiota composition in the feces and brains of aged SAMP8 mice. ProBiotic-4 substantially attenuated aging-related disruption of the intestinal barrier and blood-brain barrier, decreased interleukin-6 and tumor necrosis factor- at both mRNA and protein levels, reduced plasma and cerebral lipopolysaccharide (LPS) concentration, toll-like receptor 4 (TLR4) expression, and nuclear factor-B (NF-B) nuclear translocation in the brain. In addition, not only did ProBiotic-4 significantly decreased the levels of -H2AX, 8-hydroxydesoxyguanosine, and retinoic-acid-inducible gene-I (RIG-I), it also abrogated RIG-I multimerization in the brain. These findings suggest that targeting gut microbiota with probiotics may have a therapeutic potential for the deficits of the microbiota-gut-brain axis and cognitive function in aging, and that its mechanism is associated with inhibition of both TLR4-and RIG-I-mediated NF-B signaling pathway and inflammatory responses.