The main active compound, 3,4-dihydroxyacetophenone(3,4-DHAP), in the leaves of Ilex pubescens var. glaber, exhibits various pharmacological activities, including vasodilation and heart protection. Currently, natural extraction and chemical synthesis are the primary methods for obtaining 3,4-DHAP, but both approaches have inherent challenges. To address these problems, this study explored the whole-cell bioconversion of 1-(4-hydroxyphenol)-ethanol to 3,4-DHAP using recombinant Escherichia coli, cultivated in a green, cost-effective medium at room temperature and atmospheric pressure. Firstly, this study successfully constructed recombinant E. coli S1, which contained only the HpaBC gene, and recombinant E. coli S3, which contained both the Hped and HpaBC genes. The ability of S1 and S3 to synthesize 3,4-DHAP from their respective substrates was then evaluated through whole-cell bioconversion. Based on these results, the effects of four factors, i.e., substrate concentration, IPTG concentration, induction temperature, and transformation temperature, on the whole-cell bioconversion yield of S3 were investigated using an orthogonal experiment. The results showed that the factors influenced the yield in the following order: transformation temperature > induction temperature > IPTG concentration > substrate concentration. The optimal conditions were found to be a transformation temperature of 35 ℃, IPTG concentration of 0.1 mmol·L~(-1), induction temperature of 25 ℃, and substrate concentration of 10 mmol·L~(-1). Finally, the effect of transformation time on the yield of 3,4-DHAP was further examined under the optimal conditions. The results indicated that as the transformation time increased, the yield of 3,4-DHAP steadily increased. The highest yield of 260 mg·L~(-1) with a productivity of 17% was achieved after 72 hours of transformation. In conclusion, this study successfully achieved the whole-cell bioconversion of 1-(4-hydroxyphenol)-ethanol to 3,4-DHAP using recombinant E. coli for the first time, laying the groundwork for further optimization and development of the biosynthesis of 3,4-DHAP.
Escherichia coli/genetics* ; Acetophenones/chemistry* ; Ethanol/chemistry* ; Drugs, Chinese Herbal/chemistry* ; Biotransformation

Objective The study aims to investigate the immunological functions of the nucleocapsid (N) protein of the novel coronavirus Omicron (BA.1, BA.2) and evaluate the differences among different N proteins of mutant strains in immunogenicity. Methods By aligning sequences, the mutation sites of the Omicron (BA.1, BA.2) N protein relative to prototype strain of the novel coronavirus (Wuhan-Hu-1) were determined. The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination. The three kinds of N protein were expressed in prokaryotic system, purified through Ni-NTA affinity chromatography, and then immunized into mice. The titer and reactivity of the polyclonal antibody, as well as the expression level of IL-1β and IFN-γ in mouse spleen cells, were detected using indirect ELISA and Western blot assay. Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N, BA.1 N, and BA.2 N proteins in E.coli BL21(DE3) at 37 DegreesCelsius for 4 hours. The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1:51 200. All three N proteins can increase the expression of IFN-γ and IL-1β cytokines, but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein. Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies, and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity. This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
Animals ; Mice ; SARS-CoV-2/genetics* ; Coronavirus Nucleocapsid Proteins/immunology* ; Nucleocapsid Proteins/isolation & purification* ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Mice, Inbred BALB C ; Interferon-gamma/metabolism* ; Interleukin-1beta/metabolism* ; Female ; Escherichia coli/metabolism* ; Mutation ; Humans

Gut microbial communities are likely remodeled in tandem with accumulated physiological decline during aging, yet there is limited understanding of gut microbiome variation in advanced age. Here, we performed a metagenomics-based enterotype analysis in a geographically homogeneous cohort of 367 enrolled Chinese individuals between the ages of 60 and 94 years, with the goal of characterizing the gut microbiome of elderly individuals and identifying factors linked to enterotype variations. In addition to two adult-like enterotypes dominated by Bacteroides (ET-Bacteroides) and Prevotella (ET-Prevotella), we identified a novel enterotype dominated by Escherichia (ET-Escherichia), whose prevalence increased in advanced age. Our data demonstrated that age explained more of the variance in the gut microbiome than previously identified factors such as type 2 diabetes mellitus (T2DM) or diet. We characterized the distinct taxonomic and functional profiles of ET-Escherichia, and found the strongest cohesion and highest robustness of the microbial co-occurrence network in this enterotype, as well as the lowest species diversity. In addition, we carried out a series of correlation analyses and co-abundance network analyses, which showed that several factors were likely linked to the overabundance of Escherichia members, including advanced age, vegetable intake, and fruit intake. Overall, our data revealed an enterotype variation characterized by Escherichia enrichment in the elderly population. Considering the different age distribution of each enterotype, these findings provide new insights into the changes that occur in the gut microbiome with age and highlight the importance of microbiome-based stratification of elderly individuals.
Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Bacteroides ; China ; Diabetes Mellitus, Type 2/microbiology* ; Escherichia coli/classification* ; Gastrointestinal Microbiome/genetics* ; Metagenomics ; East Asian People

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*

O-acetyl-L-homoserine (OAH) is a promising platform compound for the production of L-methionine and other valuable compounds, while its low yield and low conversion rate limit the industrial application. To solve these problems, we constructed a strain for high OAH production with the previously constructed L-homoserine producer Escherichia coli HS33 as the chassis by systematic metabolic engineering. Firstly, PEP accumulation, pyruvate utilization, and OAH synthesis pathway (overexpressing aspB, aspA, and thrA^C1034T) were enhanced to obtain an initial strain accumulating 13.37 g/L OAH. Subsequently, the co-factor synthesis genes were integrated to supply reducing power and energy, which increased the yield to 15.79 g/L. The OAH yield of the engineered strain OAH28 was further increased to 17.49 g/L by strengthening the acetic acid reuse pathway, improving the supply of acetyl-CoA, and regulating the expression of MetX from different sources. Finally, in a 5 L fermenter, OAH28 achieved an OAH titer of 47.12 g/L, with a glucose conversion rate of 32% and productivity of 0.59 g/(L·h). The results lay a foundation for increasing the OAH production by metabolic engineering and give insights into the industrial production of OAH.
Metabolic Engineering/methods* ; Escherichia coli/genetics* ; Homoserine/biosynthesis* ; Fermentation

To screen and identify a chitosanase with high stability, we cloned the chitosanase gene from Bacillus atrophaeus with a high protease yield from the barren saline-alkali soil and expressed this gene in Escherichia coli. The expressed chitosanase of B. atrophaeus (BA-CSN) was purified by nickel-affinity column chromatography. The properties including optimal temperature, optimal pH, substrate specificity, and kinetic parameters of BA-CSN were characterized. The results showed that BA-CSN had the molecular weight of 31.13 kDa, the optimal temperature of 55 ℃, the optimal pH 5.5, and good stability at temperatures below 45 ℃ and pH 4.0-9.0. BA-CSN also had good stability within 4 h of pH 3.0 and 10.0, be activated by K⁺, Na⁺, Mn²⁺, Ca²⁺, Mg²⁺, and Co²⁺, (especially by Mn²⁺), and be inhibited by Fe³⁺, Cu²⁺, and Ag⁺. BA-CSN showcased the highest relative activity in the hydrolysis of colloidal chitosan, and it had good hydrolysis ability for colloidal chitin. Under the optimal catalytic conditions, BA-CSN demonstrated the Michaelis constant K_m and maximum reaction rate V_max of 9.94 mg/mL and 26.624 μmoL/(mL·min), respectively, for colloidal chitosan. In short, BA-CSN has strong tolerance to acids and alkali, possessing broad industrial application prospects.
Bacillus/genetics* ; Hydrogen-Ion Concentration ; Escherichia coli/metabolism* ; Glycoside Hydrolases/biosynthesis* ; Substrate Specificity ; Enzyme Stability ; Chitosan/metabolism* ; Temperature ; Kinetics ; Cloning, Molecular ; Bacterial Proteins/biosynthesis* ; Recombinant Proteins/genetics*

Biosensors have become powerful tools for real-time monitoring of specific small molecules and precise control of gene expression in biological systems. High-throughput sensors for 1, 4-butanediamine biosynthesis can greatly improve the screening efficiency of high-yielding 1, 4-butanediamine strains. However, the strategies for adapting the characteristics of biosensors are still rarely studied, which limits the applicability of 1, 4-butanediamine biosensors. In this paper, we propose the development of a 1, 4-butanediamine biosensor based on the transcriptional regulator PuuR, whose homologous operator puuO is installed in the constitutive promoter P_gapA of Escherichia coli to control the expression of the downstream superfolder green fluorescent protein (sfGFP) as the reporter protein. Finally, the biosensor showed a stable linear relationship between the GFP/OD₆₀₀ value and the concentration of 1, 4-butanediamine when the concentration of 1, 4-butanediamine was 0-50 mmol/L. The promoters with different strengths in the E. coli genome were used to modify the 1, 4-butanediamine biosensor, and the functional properties of the PuuR-based 1, 4-butanediamine biosensor were explored and improved, which laid the groundwork for high-throughput screening of engineered strains highly producing 1, 4-butanediamine.
Biosensing Techniques/methods* ; Escherichia coli/metabolism* ; Promoter Regions, Genetic/genetics* ; Green Fluorescent Proteins/metabolism* ; Transcription Factors/genetics* ; Escherichia coli Proteins/genetics* ; Diamines/metabolism* ; Gene Expression Regulation, Bacterial

S-methyl-L-cysteine sulfoxide (SMCO) is a non-protein sulfur-containing amino acid with a variety of functions. There are few reports on the enzymes catalyzing the biosynthesis of SMCO from S-methyl-L-cysteine (SMC). In this study, the flavin-containing monooxygenase gene derived from Schizosaccharomyces pombe (spfmo) was heterologously expressed in Escherichia coli BL21(DE3) and the enzymatic properties of the expressed protein were analyzed. The optimum catalytic conditions of the recombinant SpFMO were 30 ℃ and pH 8.0, under which the enzyme activity reached 72.77 U/g. An appropriate amount of Mg²⁺ improved the enzyme activity. The enzyme kinetic analysis showed that the K_m and k_cat/K_m of SpFMO on the substrate SMC were 23.89 μmol/L and 61.71 L/(min·mmol), respectively. Under the optimal reaction conditions, the yield of SMCO synthesized from SMC catalyzed by SpFMO was 12.31% within 9 h. This study provides reference for the enzymatic synthesis of SMCO.
Schizosaccharomyces/genetics* ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Cysteine/biosynthesis* ; Mixed Function Oxygenases/metabolism* ; Schizosaccharomyces pombe Proteins/metabolism* ; Oxygenases/metabolism* ; Kinetics

Due to the wide application of silver-containing dressings and silver-coated medical devices in clinical treatment; the extensive use of antibacterial agents and heavy metal agents in feed factories, Escherichia coli has formed the tolerance to silver ions. To systematically understand the known silver ion resistance mechanisms of E. coli, this article reviews the complex regulatory network and various physiological mechanisms of silver ion tolerance in E. coli, including the regulation of outer membrane porins, energy metabolism modulation, the role of efflux systems, motility regulation, and silver ion reduction. E. coli reduces the influx of silver ions by missing or mutating outer membrane porins such as OmpR, OmpC, and OmpF. It adapts to high concentrations of silver ions by altering the expression of ArcA/B and enhances the efflux capacity of silver ions under high-concentration silver stress via the endogenous Cus system and exogenous Sil system. Furthermore, the motility of bacteria is related to silver tolerance. E. coli has the ability to reduce silver ions, thereby alleviating the oxidative stress induced by silver ions. These findings provide a new perspective for understanding the formation and spread of bacterial tolerance and provide directions for the development of next-generation silver-based antimicrobials and therapies.
Escherichia coli/genetics* ; Silver/pharmacology* ; Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology* ; Porins/metabolism*

CD20 is a surface marker protein of B-cell lymphoma, and its extracellular region is the target of specific antibodies and drugs. To obtain a cheap and easily modified specific preparation targeting CD20, we optimized the gene of CD20 extracellular region according to codon degeneracy to facilitate its expression in Escherichia coli. The optimized gene was cloned into pGEX-4T-1 vector, and the recombinant vector was transformed into E. coli BL21(DE3) for expression. The purified protein was identified by SDS-PAGE and Western blotting. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to screen the ssDNA aptamer that specifically binds to the fusion protein, and the affinity of the aptamer to CD20 was detected by flow cytometry. Then, the cytotoxicity test was carried out to examine the inhibitory effect of the aptamer on B lymphoma cells. In this study, we established the prokaryotic expression method of CD20 and obtained the aptamer specifically binding to the extracellular region of CD20, which laid a foundation for the development of therapeutic drugs targeting CD20.
Humans ; Escherichia coli/metabolism* ; SELEX Aptamer Technique/methods* ; Aptamers, Nucleotide/genetics* ; Antigens, CD20/metabolism* ; Ligands