Integrated in silico structural design & upstream expression optimization of an A7G mutant rh-G-CSF.

Rational Design of Flexible Loops Adjacent to the Active Site Enhances the Catalytic Efficiency of Bacillus β-Mannanase.

Mannan oligosaccharides (MOS) are valuable prebiotics, and enzymatic hydrolysis by β-mannanase is the preferred production route. Bacillus-derived GH26 β-mannanase (gmuG) holds industrial promise but suffers from low catalytic efficiency and weak pH stability. Here, we rationally engineered flexible loops near the active site via molecular dynamics, NMsim (normal mode-based geometric simulation), sequence conservation analysis, and MAESTRO prediction, and obtained the optimal double mutant M1 (E301K-T271V). Relative to the wild type, M1 showed 1.54-fold higher specific activity and nearly 2-fold improved pH stability at pH 8, with a nearly doubled kcat while retaining substrate affinity. Mechanistically, M1 enlarged the active site, shortened the attack distance of the catalytic residue, increased α-helix content, and rigidified neighboring loops. Fermentation optimization in Escherichia coli gave a maximum yield of 924.34 U/mL. This work offers an effective strategy for β-mannanase engineering and a high-performance biocatalyst for industrial MOS production.

Proof-of-concept validation of p2GUS, a modular expression system for enhanced Ni-binding purification of functional recombinant proteins in Escherichia coli

Protein expression and purification remain critical steps in basic research, biotechnology, and industrial applications. Here, p2GUS, a modular Escherichia coli expression system generated through targeted modification of Precursor 1, a parental pBAD-derived vector, is described. The principal feature is a tandem His₁₀–GST–His₁₀ architecture designed to enhance interaction with Ni–NTA matrices while retaining compatibility with post-expression processing strategies. The GST module serves primarily as a spacer between the two polyhistidine tags while also contributing solubility-enhancing properties. Engineered PreScission and Enterokinase cleavage sites, enabling controlled post-expression processing and generation of alternative protein forms derived from the same multitagged recombinant protein. As a proof of concept, p2GUS was evaluated using a truncated mitochondrial transcription factor A [mTFAM(p.1_41del)]. Recombinant protein production, purification, protease-mediated processing, and DNA-binding activity were assessed using multitagged mTFAM(p.1_41del) as a model substrate. Purification outcomes were compared with those obtained using the parental construct Precursor 1, encoding a single-His 10 –mTFAM(p.1_41del). Under the experimental conditions examined, the p2GUS-derived construct, purified using a simple single-microcentrifuge tube Ni–NTA batch procedure, showed improved recovery and yielded a processed His 10 –EK–mTFAM(p.1_41del) of substantially higher purity than the corresponding His 10 –mTFAM(p.1_41del) produced by Precursor 1. The purified recombinant protein retained DNA-binding activity, supporting preservation of its functional properties following expression, purification and storage. These results support proof-of-concept validation of p2GUS as a modular system for recombinant protein production, purification, and controlled post-expression processing. Although validated here using a DNA-binding protein, broader applicability remains to be established through evaluation with additional recombinant proteins. • Proof-of-concept validation of the modular p2GUS expression system • Tandem His₁₀–GST–His₁₀ architecture improves Ni-NTA retention and purification • Multitagged p2GUS-derived mTFAM retained DNA-binding activity

Systematic modular engineering of genome-integrated Escherichia coli MG1655 for high-level 2'-fucosyllactose production.

2'-Fucosyllactose (2'-FL), the most abundant human milk oligosaccharide (HMO), has attracted considerable interest for its prebiotic and immunomodulatory functions, with broad applications in infant nutrition. In this study, we report the development of a high-yield, genome-integrated 2'-FL-producing strain based on Escherichia coli MG1655 through systematic modular optimization. Starting from a single-copy BKHT strain (MGC06), we first optimized the copy number of the α-1,2-fucosyltransferase (α-1,2-FT) gene BKHT. Subsequently, the GDP-L-fucose supply was enhanced through coordinated genomic integration of the gene clusters cpsG-cpsB and gmd-fcl, while the multidrug efflux transporter gene mdfA was integrated to improve product export and strain robustness. BKHT copy number was then re-evaluated in the optimized background, with four copies yielding the highest production. The final engineered strain, harboring all genetic modifications stably integrated into the chromosome, produced 17.18 g/L 2'-FL in shake-flask culture. In fed-batch fermentation using a 5-L bioreactor, this strain achieved a titer of 154.12 g/L after 60 h, with a productivity of 2.57 g/L/h. Notably, throughout the entire fermentation process, no antibiotics or inducers were supplemented, underscoring the genetic stability and regulatory compliance of this plasmid-free system. To our knowledge, this represents the highest 2'-FL titer reported to date, positioning our engineered strain as a promising candidate for commercial 2'-FL production.

An expression framework for production of recombinant methionine aminopeptidase in Escherichia coli: a case study of upstream process.

Incomplete removal of the initiator methionine is a frequent bottleneck in Escherichia coli-based production of recombinant proteins, causing heterogeneity and increased immunogenicity of biopharmaceuticals. Methionine aminopeptidase (MAP) is the key enzyme responsible for this post-translational modification, yet its endogenous activity is rapidly saturated under high-level expression conditions. Here, we report a case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli. The map gene from E. coli BL21(DE3) was cloned into the pF644 vector to generate pF1492. During fed-batch cultivation under the tested conditions, specific productivity reached 127.03 ± 8.66 mg·g-1 and volumetric productivity of total cell-associated MAP reached 2.71 ± 0.18 g·L-1 by the final hour of induction. MAP accumulated predominantly as insoluble inclusion bodies, which is a common outcome for recombinant protein expression in E. coli at high rates. This study reports upstream production and inclusion body formation only; functional recovery and enzymatic activity were not assessed. Acetate remained moderate (35-60 mM) and biomass was stable, indicating balanced metabolism. The present study evaluates the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions, without a side-by-side comparison with alternative expression systems.

Directed Mutagenesis of Catalytic Residues in L-Asparaginase II from Salmonella paratyphi: Structural, Functional and Stability Analysis Using In-Vitro, Docking and Simulation Studies.

Site-directed mutagenesis of α-L-rhamnosidase boosts its deglycosylation activity for the production of bioactive flavonoid glycosides.

α-L-Rhamnosidases are valuable biocatalysts for the synthesis of bioactive flavonoids, however, their industrial application is often restricted by limited catalytic efficiency and suboptimal thermal stability. This study reports the rational engineering of an α-L-rhamnosidase (DthRha) to address these limitations and enhance its performance for flavonoid production. A library of mutants was constructed via substrate-binding pocket modeling and site-directed mutagenesis. Among them, the R783A variant exhibited a 3.56-fold increase in enzymatic activity relative to the wild-type enzyme (WT), along with remarkable thermal stability, retaining over 92% of its initial activity after 2 h incubation at 60-90 °C， whereas the WT rapidly lost activity above 60 °C. Kinetic assays revealed a 1.46-fold increase in kcat/Km over the WT, coupled with enhanced substrate specificity. Molecular docking and MD simulations suggested that the enhanced catalytic performance of R783A may arise from favorable steric conformation and a more accessible catalytic tunnel. The practical applicability of the R783A was demonstrated at a 50 mL laboratory scale, affording prunin and isoquercitrin in >98% yield with space-time yields of 3.58 and 1.92 g/L/h, respectively. These findings highlight the R783A mutant as a robust and thermally stable biocatalyst with great potential for the sustainable production of bioactive flavonoids.