Search Results (60)

Structure-based strategies are widely used in tuberculosis drug discovery; however, their translational impact remains limited. This review examines how structure-based virtual screening (SBVS) is applied in practice to Mycobacterium tuberculosis targets and explores why docking-derived predictions frequently fail to translate into measurable biological activity. Rather than treating docking scores as quantitative predictors of potency, representative case studies are analyzed to demonstrate that SBVS is most effective when employed as a prioritization framework integrated with appropriate target preparation, physicochemical filtering, and early experimental validation. Across diverse targets, molecular dynamics simulations emerge as a critical discriminator, enabling the identification of binding instability and false-positive hits that persist after static docking. Tuberculosis-specific constraints—including cofactor-dependent catalysis, resistance-associated mutations, membrane-rich environments, and permeability barriers—are discussed as key factors decoupling in silico affinity from whole-cell efficacy. Collectively, these observations support a workflow-oriented view of computational drug discovery in tuberculosis, in which iterative integration of structural modeling and experimental validation is required for meaningful lead identification. Full article

As an isoflavone metabolite with diverse physiological activities, the development of efficient and sustainable manufacturing technologies for (S)-equol holds significant importance. This study focuses on the semi-rational design of daidzein reductase (DZNR), the first key enzyme in the (S)-equol biotransformation pathway. Through multiple sequence alignment and three-dimensional structural analysis, two critical residues, Gly30 and Ala105, were identified in DZNR. A library of single and combinatorial mutants was constructed and screened, yielding the double variant DZNR30S+105S with substantially enhanced catalytic performance. In a whole-cell biocatalytic system, the recombinant E. coli (Escherichia coli) strain harboring this combinatorial mutant achieved a yield of 238.3 mg/L (S)-equol at a substrate concentration of 1 mM daidzein, demonstrating markedly improved catalytic efficiency. Upon increasing the daidzein concentration to 2 mM, the reaction reached equilibrium within 5 h, producing 384.6 mg/L (S)-equol, which highlights the mutant’s excellent potential for high-substrate-concentration applications. This study not only provides novel mechanistic insights into DZNR catalysis but also successfully establishes a DZNR variant with enhanced activity, offering an efficient biocatalytic component for the industrial-scale biomanufacturing of (S)-equol and thereby advancing the development of green biosynthesis technologies for this valuable compound. Full article

The sustainable synthesis of valuable noncanonical amino acids from renewable raw materials holds significant importance. This research developed a viable chemical–biological coupling process, leveraging the synergistic effect of a solid acid catalyst and the whole cell of E. coli PpLTA to selectively synthesize β-(2-furanyl) serine from corncob. Initially, a novel magnetic solid acid catalyst, Fe₃O₄/C-SO₃H, was successfully fabricated and employed to catalyze the degradation of corncob in a toluene–water biphasic system for furfural production. Under the optimal conditions (catalyst loading of 2.0% w/w and reaction at 170 °C for 20 min), the furfural yield could attain 62.3%. After ten cycles of use, the yield of furfural remained at 44.7% and the retention rate of catalytic activity was 71.7%. Furthermore, the biocompatibility verification results demonstrated that the furfural derived from corncob could be completely transformed by E. coli PpLTA at a concentration of 50 mM, and this furfural system did not generate any by-products that hindered the biotransformation process. This chemical–biological coupling approach offers a technical solution for the efficient production of noncanonical amino acids from biomass resources. Full article

2,5-Furandicarboxylic acid (FDCA) is an important bio-based platform compound that can be synthesized through the biotransformation of 5-hydroxymethylfurfural (HMF). However, the limited availability of safe microbial strains is a major constraint in the whole-cell catalysis of HMF to FDCA. In this study, a strain capable of catalyzing the conversion of HMF to FDCA, Bacillus subtilis J8M8, was identified. Under optimized whole-cell catalytic conditions, the wild-type strain produced 33.1 mM FDCA with a yield of 41.4%. To enhance FDCA production, HMF/furfural oxidoreductase (HmfH), PQQ-dependent alcohol dehydrogenase (ADH), and aryl-alcohol oxidase (MaAAO) were co-expressed in B. subtilis J8M8. As a result, FDCA production increased to 72.3 mM, with a yield of 90.4%. Further optimization of the engineered strain improved FDCA production to 83.3 mM and yield to 92.6%, representing a 2.52-fold increase over that of the wild-type strain. This study establishes a foundation for the safe and sustainable production of FDCA from HMF. Full article

Carbon dioxide (CO₂) is a cost-effective, abundant, and renewable carbon source, but its utilization technologies face several issues. The reductive glycine pathway (RGP) is recognized as one of the most efficient one-carbon (C1) assimilation routes in nature, with its core component—the glycine cleavage system (GCS: GcvP, GcvH, GcvT, and GcvL)—playing an essential role in C1 metabolism. To develop efficient CO₂ conversion and utilization pathways, we identified NhFtfL and AmFchA-MtdA with high catalytic efficiency through gene mining and constructed a four-plasmid co-expression system in E. coli BL21(DE3) using Gibson Assembly. This system integrated GcvP-GcvH, GcvT-GcvL, NhFtfL-AmFchA-MtdA, and RsPPK2, thereby reconstituting the complete RGP while enhancing ATP supply. The engineered strain functioned as an efficient whole-cell biocatalyst, achieving a glycine space–time productivity of 0.125 mmol/L/h via one-pot conversion of formate. Furthermore, we expanded the application scope by developing a whole-cell electrocatalysis system that directly synthesized glycine from CO₂ and NH₄Cl, achieving a glycine space–time productivity of 0.135 mmol/L/h. This study demonstrates the potential of the engineered RGP system for upgrading C1 resources and supports the transition toward carbon neutrality. Full article

Raspberry ketone (RK) is the primary aromatic compound in raspberry fruit, which is widely utilized in perfume, cosmetics, and food additive industries. Currently, RK is predominantly produced chemically. RK biosynthesis through enzyme or whole cell has garnered significant attention due to the mild reaction conditions and the process being regarded as ‘natural’. This study proposed a ‘dual-microorganism, two-phase’ stepwise cascade strategy to produce RK from an economical precursor, 4-hydroxybenzaldehyde (4-HBD). An acetone-tolerant deoxyribose-phosphate aldolase DERA_Ec (S238D) mutant was obtained through a site-specific rigidification strategy for converting 4-HBD to 4-hydroxybenzylaceton (4-HBA). Then, an engineered E. coli co-expressing isocitrate dehydrogenase and raspberry ketone synthase RiRZS1 with a citrate-sodium citrate buffer to recycle nicotinamide adenine dinucleotide phosphate (NADPH) was constructed for the conversion of 4-HBA to RK. The final concentration of RK was 50.00 ± 1.92 mmol·L⁻¹ with a yield of 86.96%. This strategy provides a scalable coenzyme self-recycling and two-phase catalysis platform for high-value phenolic compounds. Full article

Patchoulene, the characteristic sesquiterpene of patchouli essential oil, is highly valued in the perfume industry for its distinctive woody note and fixative properties. Beyond its olfactory applications, patchoulene has demonstrated promising biological activities, including anti-inflammatory, antimicrobial, and neuroprotective effects. Current production relies mainly on extraction from Pogostemon cablin plants, which requires long growth cycles (≥8 months), exhibits low yields, and imposes significant environmental constraints. To overcome these limitations, this study aimed to enhance the Whole-cell yield of patchoulene synthase (PcPTS) through structure-informed protein engineering. A semi-rational design approach was employed, combining homology modeling, molecular docking, evolutionary analysis, and molecular dynamics simulations to identify functional residues within the enzyme active site. Ala-scanning mutagenesis highlighted Thr532 as essential for catalytic activity, and coevolutionary analysis indicated synergistic effects between Phe456 and Thr532. Site-directed mutagenesis was conducted to generate single (F456M, T532Y) and double (F456M/T532Y, designated M2) mutants. The double mutant M2 showed a 3.62-fold increase in patchoulene production compared to the wild-type enzyme. In silico analyses suggested that the enhanced performance of M2 originates from improved substrate positioning, reduced structural flexibility, and strengthened molecular interactions, collectively contributing to a lower energy barrier for catalysis. This study provides an effective strategy for the rapid optimization of terpenoid synthases and facilitates the development of microbial cell factories for sustainable and high-yield production of plant-derived terpenoids. Full article

Niacin is a compound with a wide range of applications in pharmaceuticals, healthcare, food nutrition, animal breeding, cosmetics, etc. A recombinant Escherichia coli carrying the afnitA nitrilase gene was created to transform 3-cyanopyridine into niacin in this work. After analyzing the viscosity, surface tension, and Kamlet-Taft (K-T) parameters (π*, α, and β values) of certain deep eutectic solvents (DESs), Betaine:Acetic Acid (Betaine:AA) (1:2, mol/mol) was chosen as the bioreaction medium. Using response surface methodology (RSM), systematic biocatalytic optimization was performed. The optimum medium pH, cell loading, temperature, and DES (Betaine:AA) (1:2, mol/mol) dose were determined to be 7.75, 195 g/L, 44.24 °C, and 18.04 wt%. Under the optimized conditions, whole-cell catalysis facilitated the conversion of 3-cyanopyridine to niacin, achieving a high yield of 98.6% within 40 min. These results demonstrated that recombinant E. coli carrying the afnitA nitrilase gene may have practical value as a biocatalyst for the production of niacin, with promising prospects for future applications. Full article

In this study, CGMCC NO:28566, a strain that can efficiently convert Ethyl 4-chloroacetoacetate(COBE) to (R)-4-chloro-3-hydroxybutyrate((R)-CHBE), was screened by soil-sieving bacteria. In order to improve the transformation effect of the strain, the natural low-eutectic solvent (NADES), which can alter the cell permeability, was utilized for assisted catalysis, and a better catalytic effect was achieved. This study was carried out using a co-culture of strains with NADES and secondary addition of NADES on the basis of co-culture, and 10 NADESs were screened at the same time. The co-catalytic effect of 0.5% (w/v) choline chloride: urea (1:2) (ChCl:U (1:2)) was found to be the most significant, with a yield of (R)-CHBE reaching 89.1%, which was 58.2% higher than that of the control group, with a 99% ee value. Furthermore, the catalytic results demonstrated that the co-culture of the strain with NADES during fermentation yielded superior outcomes to the secondary addition of NADES during the reaction buffer. Furthermore, the catalytic effect of ChCl:U (1:2) was demonstrated to be superior to that of its individual components or single-component blends, due to its distinctive valence bonding advantage. The results indicate that the addition of 0.5% (w/v) ChCl:U (1:2) during the co-culture process has the effect of improving cell permeability to a certain extent, thereby increasing the contact between the substrate and the enzyme during the whole-cell catalytic reactions. Full article

An excessively high serum cholesterol (CHOL) level in humans can easily lead to cardiovascular diseases (CVDs), including hypertension and coronary heart disease. In this study, a CHOL-lowering bacterium, Cellulosimicrobium cellulans YS01, was isolated from healthy human intestinal microbiota and identified via average nucleotide identity (ANI) analysis. The cells of YS01 degraded 74.00% of CHOL within 5 d, which decreased from the initial 1.00 g/L to 0.26 g/L. And its extracellular crude enzymes achieved equivalent efficiency within 24 h, which decreased from the initial 0.50 g/L to 0.13 g/L. The results indicated that YS01 indeed has a strong ability in the biodegradation of CHOL. Furthermore, the whole genome analysis of YS01 revealed that cholesterol oxidase and choloylglycine hydrolase encoded by gene choD and gene cbh, respectively, may play key roles in the conversion of CHOL. Cholest-4-ene-3-one was produced from CHOL through the catalysis by cholesterol oxidase, and choloylglycine hydrolase was also involved in another biodegradation pathway of CHOL. The results provide scientific insights into the mechanisms of biodegrading CHOL using C. cellulans YS01 and lay a solid foundation for the development of new CHOL-lowering strategies based on microbial therapy. Full article

β-glucuronidase is an important hydrolase, which plays an important role in drug metabolism, clinical diagnostics, and biotransformation. This study focuses on the heterologous expression, isolation, purification, and its enzymatic properties of β-glucuronidase CpGUS from Clostridium perfringens, as well as its application in the whole-cell transformation of unconjugated bilirubin from pig bile. A recombinant E. coli BL21(DE3)/pET-28a-CpGUS was constructed for the heterologous expression of CpGUS, with the majority of the expressed enzyme being soluble. Enzymatic analysis showed that CpGUS displayed optimal activity at pH 5.0 and 45 °C, and it rapidly lost activity at pH < 4.5. Metal ions, such as Mg²⁺ and Fe²⁺, enhanced CpGUS catalysis, while Zn²⁺, K⁺, Fe³⁺, Mn²⁺, Cu²⁺, and Na⁺ inhibited it. Notably, Cu²⁺ and Fe³⁺ can significantly inhibit β-glucuronidase, resulting in the complete loss of its activity. The results of the whole-cell transformation experiment show that when E.coli BL21(DE3)/ pET-28a-CpGUS at an OD₆₀₀ of 10 was incubated at pH 5.0, a temperature of 45 °C, and a rotation speed of 200 rpm for 12 h, the hydrolysis rate of the conjugated bilirubin in pig bile reached 81.1%, the yield of unconjugated bilirubin was 76.8%, and the purity of unconjugated bilirubin was 98.2%. The three-dimensional structure of CpGUS was predicted using AlphaFold2 (AlphaFold v2.0, DeepMind Technologise Limited, London, UK), and p-Nitrophenyl-β-D-Glucuronide (pNPG) and conjugated bilirubin were then docked to the CpGUS protein model using SWISSDOCK. The best docked conformations of the CpGUS–pNPG and CpGUS–conjugated bilirubin complex systems were simulated by independent 500 ns molecular dynamics (MD) runs with the RSFF2C force field, and the binding dynamic and catalytic mechanism of each system were obtained. The results indicated that π-π stacking, hydrogen bonding, and hydrophobic interactions between the key residue Tyr472 and the benzene ring of pNPG molecules are crucial for its catalytic process. Similarly, for the binding and catalysis of conjugated bilirubin by CpGUS, the π-π stacking and hydrogen bonding and hydrophobic interactions between the sidechains of residues Phe368 and Tyr472 and the benzene ring of conjugated bilirubin play a synergistic role during its catalytic process. Their total binding free energy (∆G_bind) values were calculated to be as high as −65.05 ± 12.66 and −86.70 ± 17.18 kJ/mol, respectively. These results suggest that CpGUS possesses high binding and catalytic hydrolysis properties for both pNPG and conjugated bilirubin. Full article

D-tagatose is a functional sweetener with glucose-regulating and prebiotic properties, but its bioproduction from D-galactose faces many limitations, particularly the high production costs. In particular, the current biosynthesis of D-tagatose suffers from thermal instability and the substrate selectivity issues of L-arabinose isomerase (L-AI) required to convert D-galactose into D-tagatose. In this study, recombinant Escherichia coli BW25113/pQE-80L-araA_F118M/F279I expressing double mutant L-AI was immobilized to enhance its stability and reusability. The optimal conditions for whole-cell catalysis were 60 °C, pH 6.5, 5 mM Mn²⁺, and 20 h, with a yield of 55.2 g/L of D-tagatose. Immobilization with 3% sodium alginate and 2% CaCl₂ retained 90% of the production efficiency displayed by free cells. Notably, the immobilized cells exhibited enhanced heat resistance (60–70 °C) and operational stability, retaining 76% activity after five cycles. The D-tagatose production was further increased to 129.43 g/L by increasing the substrate concentration to 250 g/L. Compared to free cells, immobilized cells retained 83.6% of the initial yield up to 10 batches. This study presents a cost-effective and sustainable method for the production of D-tagatose using optimized whole-cell catalysis through immobilization, which paves the way to solve industrial challenges such as thermal instability and low substrate efficiency. Full article

D-tagatose is an ideal sucrose substitute with potential applications in food and healthcare. The combined catalysis of polyphosphate kinase (PPK), fructose kinase (FRK), D-tagatose-6-phosphate 3-differential anisomerase (FbaA) and phytase provides a low-cost and convenient pathway for the biosynthesis of D-tagatose from D-fructose; however, there is still a problem of low catalytic efficiency that needs to be solved urgently. Therefore, this study enhanced the biosynthesis of D-tagatose by optimizing the expression levels of PPK, FRK and FbaA in a polycistronic system and knocking out the gene pfka of Escherichia coli. With 30 g/L D-fructose as a substrate, the conversion rate increased to 52%, which was the highest after 24 h. In addition, by constructing a multienzyme self-assembly system with SpyTag and SpyCatcher to improve the whole-cell catalytic ability, the conversion rate was further increased to 75%. Finally, through the fed-batch strategy, the optimal strain Ec-7 produced 68.1 g/L D-tagatose from 100 g/L D-fructose. The multienzyme cascade route reported herein provides an efficient and elegant innovative solution for the generation of D-tagatose. Full article

D-tagatose is a rare, naturally occurring low-calorie hexose, with a sweetness of 92% sucrose but only 1/3 of the calories. It has beneficial functions in lowering blood sugar, controlling obesity, preventing dental caries, and improving intestinal flora. In recent years, biotechnological routes to D-tagatose production from renewable raw materials have been regarded as very promising approaches. In this review, we provide an overview of the properties and applications of D-tagatose, with a focus on the current developments in the production of D-tagatose using enzymatic transformation and whole-cell catalytic synthesis. The biosynthetic pathways and the types and characteristics of the catalytic enzymes involved have been summarized, providing a reference for the design of D-tagatose synthesis pathways. We also expect that rapid developments in the fields of systems biology and synthetic biology will accelerate protein and metabolic engineering for microbial D-tagatose production in the future. Full article

Lipases are enzymes of great application importance in the food industry, in the cosmetic and detergent industries, in pharmacy and medicine, and in organic chemistry. Among lipases of various origins, those from microorganisms are currently the most commonly used. An excellent producer of lipases seems to be the nonconventional Yarrowia lipolytica yeast, but the biosynthesis of valuable metabolites depends on many factors. This study aimed to investigate the biodiversity of extracellular enzymes produced by four strains of Y. lipolytica, and to determine the optimal conditions of catalysis for the enzymes, according to temperature and pH, in a model hydrolysis reaction. Based on the obtained results, the biodiversity and strain dependence in lipase biosynthesis were observed. Using a Central Composite Design, it was found that temperature is the main factor in determining lipase activity. The enzymes produced by four different strains exhibited other substrate specificity, which was investigated using Latin square design methodology. Only two examined yeast strains, KKP 379 and W29, produced extracellular lipases at a high activity level towards medium- and long-chain fatty acid esters. Moreover, extracellular lipase from wild-type strain KKP 379 was further characterized, followed by exploring the activity of whole-cell biocatalyst and lyophilized enzyme solutions, and it was acknowledged that it was a “true” lipase with the highest affinity to p-nitrophenyl oleate. Full article