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

In this study, high-throughput promoter screening was employed to optimize the heterologous expression of Mesorhizobium loti carbonic anhydrase (MlCA) in order to reduce the costs associated with carbon capture and storage (CCS). To simplify the complexity of traditional vectors, a fusion protein expression system was constructed using superfolder green fluorescent protein (sfGFP) and MlCA. The synthetic promoter library in Escherichia coli was utilized for efficient one-step screening. Based on fluorescence intensity on agar plates, a total of 143 monoclonal colonies were identified, forming a library with varying expression levels. The top four recombinants with the highest fluorescence intensity were selected, among which MlCA driven by the promoter 342042/+ exhibited the highest enzymatic activity, with a specific activity of the 34.6 Wilbur-Anderson units (WAU)/mg. Optimization experiments revealed that MlCA exhibited the best performance when cultured for 4 days under pH 7.0 and 40 ℃ conditions. The Michaelis constant (K_m·hdy) and maximum reaction rate (V_max·hdy) for CO₂ hydration were determined to be 62.46 mmol/L and 0.164 mmol/(s·L), respectively. For esterase hydrolysis, MlCA showed the K_m and V_max of 639.8 mmol/L and 0.035 mmol/(s·L), respectively. MlCA accelerated the CO₂ hydration process, promoting CO₂ mineralized into CaCO₃ within 9 min at low pH and room temperature conditions. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses confirmed that the precipitated product was calcite. This study provides a low-cost and environmentally friendly alternative for future CCS applications.
Carbonic Anhydrases/biosynthesis* ; Promoter Regions, Genetic/genetics* ; Escherichia coli/metabolism* ; Carbon Sequestration ; Carbon Dioxide/metabolism* ; Green Fluorescent Proteins/metabolism*

Carbamate pesticides, a new type of broad-spectrum pesticides for controlling pests, mites, and weeds, are developed to address the shortcomings of organochlorine and organophosphorus pesticides. Their widespread use and slow degradation have led to environmental pollution, causing damage to ecosystems and human health. Managing pesticide residues is a pressing issue in the current environmental protection. This study aims to investigate the expression of SyEst870, a member of the SGNH/GDSL hydrolase family in Sphingobium yanoikuyae, in a prokaryotic system and evaluate the ability of the recombinant protein to degrade carbamate pesticides. The prokaryotic expression vector pET-32a-SyEst870 was constructed and transformed into the Escherichia coli BL21 for heterologous expression. The purified protein was studied in terms of enzyme activity and effects of temperature, pH, and metal ions on the enzyme activity, with p-nitrophenol acetate as the substrate and based on the standard curve of p-nitrophenol. LC-MS (liquid chromatography-mass spectrometry) was employed to examine the degradation effects of SyEst870 on carbaryl, metolcarb, and isoprocarb. GC-MS (gas chromatography-mass spectrometry) was employed to detect the degradation products of SyEst870 for the three pesticides. The soluble protein SyEst870 was successfully obtained through the heterologous expression in Escherichia coli, which yielded an enzyme with the activity of 677.5 U after affinity chromatography. SyEst870 exhibited degradation rates of 82.34%, 84.43%, and 92.87% for carbaryl, metolcarb, and isoprocarb, respectively, at an initial concentration of 100 mg/L within 24 h at 30 ℃ and pH 7.0. The primary degradation products of carbaryl were identified as α-naphthol and methyl isocyanate. Metolcarb was mainly degraded into m-cresol and methyl isocyanate, and isoprocarb was mainly degraded into 2-isopropylphenol and methyl isocyanate. Compared with the half-life of carbamate pesticides in the natural environment, which ranges from a few days to several weeks, the recombinant protein SyEst870 can rapidly eliminate the residues of carbamate pesticides. This study lays a foundation for addressing pesticide residues in the environment and in fruits and vegetables.
Escherichia coli/metabolism* ; Sphingomonadaceae/genetics* ; Recombinant Proteins/metabolism* ; Biodegradation, Environmental ; Esterases/metabolism* ; Pesticides/isolation & purification* ; Carbamates/isolation & purification*

The enterotoxigenic Escherichia coli (ETEC) infection is a major factor restricting the development of animal husbandry. However, the abuse of antibiotics will lead to the antibiotic residues and emergence of antibiotic-resistant bacteria. The existing vaccines face challenges in stimulating intestinal immunity, demonstrating limited prevention effects. Therefore, it is indispensable to develop a new vaccine that is safe and suitable as a feed additive to activate intestinal immunity. This study constructed yeast-cell microcapsules (YCM) carrying the DNA vaccine against ETEC by genetic engineering. Furthermore, animal experiments were carried out to explore the regulatory effects of feeding YCM on the intestinal immune system and intestinal microbiota. Saccharomyces cerevisiae was selected as the oral delivery vehicle (microcapsules) of the DNA vaccine. The codon-optimized nucleic acid sequence of K88, the main antigen of mammal-derived ETEC, was synthesized, and the yeast shuttle vector containing the corresponding DNA vaccine expression cassette was constructed by DNA recombination. The recombinant strain of YCM was prepared by transforming JMY1. Additionally, the characteristics of the YCM strain and its feasibility as an oral vaccine were comprehensively evaluated by the fluorescence reporter assay, gastrointestinal fluid tolerance assay, intestinal epithelial cell adhesion assay, intestinal retention assessment, antiserum detection, and intestinal microbiota detection. The experimental results showed that the DNA vaccine expression cassette was expressed in mammals, and the recombinant strain of YCM could tolerate up to 8 hours of gastrointestinal fluid digestion and had good adhesion to intestinal epithelial cells. The results of mouse feeding experiments indicated that the recombinant strain of YCM could stay in the intestinal tract for at least two weeks, and the DNA vaccine expression cassette carried by YCM entered the intestinal immune system and triggered an immune response to induce the production of specific antibodies. Moreover, feeding YCM recombinant bacteria also improved the abundance of gut microbiota in mice, demonstrating a positive effect in regulating intestinal flora. In summary, we prepared the recombinant strain of YCM carrying the DNA vaccine against ETEC and comprehensively evaluated its characteristics and feasibility as an oral vaccine. Feeding the recombinant YCM could induce specific immune responses and regulate intestinal microbiota. The findings provide a reference for the immunoprevention of ETEC-related animal diseases.
Animals ; Enterotoxigenic Escherichia coli/genetics* ; Saccharomyces cerevisiae/metabolism* ; Vaccines, DNA/genetics* ; Mice ; Escherichia coli Infections/immunology* ; Escherichia coli Vaccines/genetics* ; Capsules ; Mice, Inbred BALB C ; Female