Search Results (138)

The mevalonate pathway is crucial for synthesizing isopentenyl pyrophosphate (IPP), the universal precursor of terpenoids, with 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) serving as the rate-determining enzyme that catalyzes the reduction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, requiring NAD(P)H as an electron donor. Improving the cofactor promiscuity of HMGR can facilitate substrate utilization and terpenoid production by overcoming cofactor specificity limitations. In this study, we heterologously expressed rpHMGR from Ruegeria pomeroyi in Escherichia coli BL21(DE3) for the first time and established that it predominantly utilizes NADH. To broaden its cofactor usage, we employed Molecular Operating Environment (MOE)-assisted design to engineer the cofactor binding site, creating a dual-cofactor-utilizing mutant, D154K (the substitution of aspartic acid with lysine at residue 154). This mutant exhibited a significant 53.7-fold increase in activity toward NADPH, without compromising protein stability at physiological temperatures. The D154K mutant displayed an optimal pH of 6, maintaining over 80% of its catalytic activity across the pH range of 6–8, regardless of whether NADH or NADPH was the cofactor. These findings highlight the value of rational design, enhance our understanding of HMGR-cofactor recognition mechanisms, and provide a foundation for future efforts to optimize and engineer HMGR for broader cofactor flexibility. Full article

Pine wilt disease (PWD), a destructive pine forest disease caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, has led to huge economic losses and ecological environment damage. Thaumatin-like proteins (TLPs) are the products of a complex gene family involved in host defense and a wide range of developmental processes in fungi, plants, and animals. In this study, a tlp gene of B. xylophilus (Bxtlp) (GenBank: OQ863020.1) was amplified via PCR and cloned into the expression vector pET-15b to construct the recombinant vector PET-15b-Bxtlp, which was then transformed into Escherichia coli BL-21(DE3). The recombinant protein was successfully purified using Ni-NTA affinity chromatography. The effect of the Bxtlp gene on the vitality and pathogenicity of PWNs was elucidated through RNA interference (RNAi) and overexpression. Bxtlp dsRNA significantly reduced the feeding, motility, spawning, and reproduction abilities of PWN; shortened its lifespan; and increased the female–male ratio. In contrast, the recombinant BxTLP markedly enhanced the reproductive ability of PWN. In addition, Bxtlp dsRNA increased reactive oxygen species (ROS) content in nematodes, while the recombinant BxTLP was confirmed to have antioxidant capacity in vitro. Furthermore, the bioassays on Pinus thunbergii saplings demonstrated that Bxtlp could significantly influence PWN pathogenicity. Overall, we speculate that Bxtlp affects the pathogenicity of PWNs mainly via regulating ROS levels, the motility, and hatching of PWN. Full article

Clostridium novyi is a common pathogen in domestic animals and humans, and alpha-toxin is the main cause of its pathogenesis. Because it is a fastidious organism, obtaining alpha-toxin is expensive. Therefore, we proposed an in silico study to synthesize epitopes in cultures of Escherichia coli BL21 pLysS (DE3). First, we used a stirred-tank bioreactor, developing a dry mass yield (DMY) of 0.77 g/L in batch cultures and 1.03 g/L in fed-batch cultures, without acetic acid production. With scale-up using a system without mechanical agitation, there was a higher DMY (1.20 g/L) with 0.56 mmol/mL of alpha-toxin epitope 1 (DE3/Ep1) and 0.61 mmol/mL of alpha-toxin epitope 2 (DE3/Ep2), with a similar profile for O2 consumption, glucose, and no acetic acid production. The kinetic parameters µ(h⁻¹), YX/S, YP/S, QP, and QX did not differ significantly; however, the kinetic data were superior. Our results suggest that in silico tools allow epitope selection and bioprocess standardization. This system provides cost savings and technological advances for the veterinary vaccine industry. Full article

This study reports the construction, expression, and purification of synthetic SARS-CoV-2 spike (S) and nucleoprotein (N) containing immunodominant epitopes. The pET28aS_epit construct included epitopes 287–317, 402, 507, 524–598, and 601–640, while the pET28aN_epit construct included residues 42–62, 153–172, and 355–401. Commercial sequences of both proteins were used as controls. The four constructs were expressed using the Escherichia coli BL21(DE3) star strain at 37 °C. The results show that the S protein constructs were insoluble, unlike the N protein constructs. Both recombinant proteins induced immune responses in mice and were recognized by antibodies present in sera from COVID-19-positive and/or SARS-CoV-2-vaccinated humans. No significant differences in immune recognition were observed between our constructs and the commercially available proteins. In conclusion, S_epit and N_epit could be promising starting points for the development of new strategies based on immunological reactions for the control of SARS-CoV-2 infections. Full article

L-asparaginase (L-ASNase) is a key enzyme in the treatment of leukemia and lymphoma, with high demand in cancer therapies. Advances in recombinant protein production have improved yields and reduced costs, enabling large-scale production. However, optimizing culture conditions remains crucial for maximizing production. This study focused on optimizing the production of double mutant L-ASNase expressed in Escherichia coli BL21 (DE3) by supplementing media with amino acids. Five amino acids were evaluated at a shake flask scale using the design of experiments, with arginine and aspartate showing the most positive effects. Under optimized conditions (14.5 mM arginine, 12.7 mM aspartate, and 0 mM cysteine), the activity model reached 12,513 U L⁻¹, experimentally validated at 10,089 U L⁻¹. The maximum specific cell growth rate was µ_x,max = 0.74 h⁻¹, with substrate–biomass conversion factor Yx/s = 1.16 g/g and cell–product conversion factor Yp/x = 13,891 U/g_cell. Amino acid supplementation resulted in a ten-fold increase in L-ASNase activity. Finally, at the bioreactor scale, adding amino acids and the inducer at the end of the exponential phase increased activity by 135% compared to conventional MD, demonstrating its potential for industrial-scale production. Full article

The first luciferase from Antarctic krill (LAK) was cloned and successfully expressed in Escherichia coli BL21(DE3). LAK exhibits the unique ability to emit bright violet fluorescence at an emission wavelength of 350 nm, which represents the lowest reported bioluminescence wavelength for luciferases. However, its low thermal stability poses a limitation to its broader application. In this study, we employed a rational design approach to introduce three pairs of artificial disulfide bonds into LAK. Circular dichroism (CD) analysis revealed that the introduction of artificial disulfide bonds resulted in a significant increase in the secondary structural content of α-helices and β-sheets compared to the wild-type (WT) enzyme. However, these modifications did not influence the emission spectrum. Among the resultant mutant strains, two exhibited markedly enhanced thermal stability. Notably, Mut2 demonstrated a 6.18-fold increase in half-life at 50 °C. Molecular docking studies indicated that D-fluorescein can form additional hydrogen bonds with surrounding amino acid residues (A323, T347, and K534). The docking energies between the enzyme and substrate for WT and Mut2 were −19.5 kcal/mol and −23.4 kcal/mol, respectively, thereby establishing strong interactions within the catalytic pocket region. These interactions likely contribute to a 2.92-fold improvement in substrate affinity, as evidenced by a reduced Michaelis–Menten constant (Km). Our thermal stability and catalytic activity analyses revealed that the linker region between the N- and C-domains plays a crucial role in the overall stability of the enzyme. Furthermore, the C-terminus of LAK does not participate in substrate-binding and catalysis; its local excessive rigidity was found to restrict the release of the AMP product, thereby negatively impacting catalytic activity. These findings offer new insights into the mutagenesis of luciferases and pave the way for the further optimization of LAK for various biotechnological applications. Full article

UDP-glycosyltransferases (UGTs) contribute to catalyzing the glycosylation of numerous functional natural products and novel derivatives with improved bioactivities. UDP-glucose sterol glucosyltransferase (SGT) is normally involved in the synthesis of sterol glycosides in a variety of organisms. SGT was derived from Salinispora tropica CNB-440 and heterologously expressed in Escherichia coli BL21 (DE3). Novel 12-O-glucosylginsenoside Rh2 was identified using HPLC, high-resolution MS (HR-MS), and NMR analysis. The cell viability assay was performed on 12-O-glucosylginsenoside-treated AGS stomach cancer, HeLa cervical cancer, U87MG glioma, and B16F10 melanoma cell lines. Protein structure modeling, molecular docking, and dynamics simulations were performed using AutoDock 4.2 and GROMACS 2020.1 software. The SGT gene is comprised of 1284 nucleotides and codes for 427 amino acids. The 12-O-glucosylginsenoside Rh2 may be a potential anticancer agent due to its potent viability inhibition of cancer cells. Structural analysis showed critical perspectives into the intermolecular interactions, stability, and binding energetics of the enzyme–ligand complex, with outcomes complementing the experimental data, thereby deepening our understanding of the structural basis of SGT-mediated glycosylation and its functional implications. This report presents a novel ginsenoside, 12-O-glucosylginsenoside Rh2, utilizing reshuffled SGT derived from S. tropica, and provides a promising candidate for anticancer drug research and development. Full article

Microcystis aeruginosa is an important species causing cyanobacterial blooms, which can be effectively infected and lysed by cyanophages. Several strategies have been developed by M. aeruginosa to resist cyanophage infections, including the CRISPR-Cas systems. However, detailed information on the CRISPR-Cas systems in M. aeruginosa is rare. In the present study, the CRISPR-Cas systems of M. aeruginosa FACHB-524 were analyzed by genome re-sequencing, which showed that there are two type I (Cluster 1, I-B1; Cluster 2, I-D) and three type III-B (Cluster 3/4/5) CRISPR-Cas systems in the cyanobacteria. Further comparison revealed that spacer sequences of two type III-B systems targeted several genes of the cyanophage MaMV (M. aeruginosa myovirus) strains. One of the type III systems (Cluster 4) was then cloned and expressed in Escherichia coli BL21 (DE3). Protein purification and mass spectrometry identification revealed that a Cmr-crRNA effector complex formed in the E. coli. Subsequently, T4 phage (T4) was used to infect the E. coli, expressing the Cmr-crRNA complex with or without accessory proteins. The results showed that the Cmr-crRNA effector complex exhibited anti-phage activity and the accessory protein Csx1 enhanced the immune activity of the complex. Collectively, our results comprehensively demonstrate the CRISPR systems encoded by a strain of M. aeruginosa, and for the first time, one of the CRISPR systems was constructed into E. coli, providing a foundation for further in-depth analysis of cyanobacterial CRISPR systems. Full article

As a scaffolding protein, Raf kinase binding protein (RKIP) is involved in a variety of cellular pathways, including the Raf–MEK–ERK-cascade. It acts as a negative regulator by binding to its partners, making it an attractive target in the development of therapeutic strategies for cancer. Despite its structural stability as a monomer, RKIP may form a dimer, resulting in the switching of binding partners. It is still unclear how RKIP switches between monomeric and dimeric forms. Here, we identified the role of cysteine 133 in RKIP structural dynamics using recombinant human RKIP (rhRKIP) proteins purified from Escherichia coli BL21(DE3) cells. Mutation of alanine or serine instead of cysteine in RKIP proteins did not affect the biochemical characteristics, while dynamic light scattering and liquid chromatography (LC) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) suggested distinct peaks in solution, which were identified via LC–MS/MS analyses, and further clarified the role of cysteine in RKIP dimerization. rhRKIP dimer formation was abrogated by a 32-aa peptide mimicking the region between two RKIP proteins for dimerization. In addition, the 32-aa peptide and its short derivatives were investigated for effects on cancer cell viability. Taken together, our findings suggest that it may be possible to regulate RKIP function by controlling its dynamics with reducing agents, which could aid the targeting of cancer cells. Full article

Poly(A) polymerase (PAP 1) from Escherichia coli is the primary enzyme responsible for synthesizing poly(A) tails on RNA molecules, signaling RNA degradation in bacterial cells. In vitro, PAP 1 is used to prepare libraries for RNAseq and to produce mRNA vaccines. However, E. coli PAP 1’s toxicity and instability in low-salt buffers complicate its expression and purification. Here, we optimized the conditions for the production of recombinant PAP 1. For that, E. coli PAP 1 was expressed in seven E. coli strains with different origins and genetic backgrounds, followed by assessment of the overall protein yield, solubility, and enzymatic activity. Among the tested strains, BL21 (DE3) pLysS achieved the best balance of cell density, total PAP 1 yield, solubility, and specific activity. Rosetta 2 (DE3) and Rosetta Blue (DE3) hosting the pRARE plasmid exhibited the lowest solubility, likely due to excessive translation efficiency. Higher induction temperatures (>18 °C) exacerbated PAP 1’s insolubility. Interestingly, PAP 1 accumulation correlated with an increase in the plasmid copy number encoding the enzyme, indicating its potential utility as a surrogate marker of PAP 1 activity. These findings provide insights into optimizing E. coli PAP 1 production for biotechnological applications. Full article

Thus far, no manufacturing process able to support industrialization has been reported for the recombinant human brain-derived neurotrophic factor (rhBDNF). Here, we described the setup of a new protocol for its production in Escherichia coli (E. coli) and its purification to homogeneity. A synthetic gene, codifying for the neurotrophin precursor, was inserted into an E. coli expression vector and transformed into BL21 (DE3) strain. The recombinant protein accumulates, at high yields, into inclusion bodies. With the developed strategy, more than 50% of the precursor can be refolded. The protein is successively digested by trypsin and the rhBDNF mature form is finally purified by two additional chromatographic steps If the wild-type precursor can be efficiently obtained by the proposed methodology, its pro-peptide remotion, through enzymatic digestion, is however problematic. To circumvent this difficulty, the precursor hinge region, containing the natural furin recognition site, was engineered to be more specifically cleaved by trypsin. Notwithstanding the substitution of three residues in the pro-region carboxyterminal, the precursor correctly refolds and is efficiently cleaved to generate a biologically active mature rhBDNF. This efficient high-yield process fills the current need of a scalable protocol to produce GMP-grade material and unlocks the rhBDNF employment in future clinical investigations. Full article

This study investigated the role of the Zymomonas mobilis recA gene in conferring stress resistance when expressed in Escherichia coli. The recA gene was cloned and expressed in E. coli BL21(DE3), producing a 39 kDa polypeptide. The results of comparative analyses demonstrated that the recombinant strain significantly enhanced survival rates under various stress conditions. In oxidative stress tests, the recombinant E. coli pET-22b(+)-recA exhibited superior survival at 3 mM and 5 mM H₂O₂ concentrations. Heat stress experiments at 50 °C and 55 °C revealed increased survival for the recombinant strain. Under ethanol stress, particularly at 20% (v/v), E. coli pET-22b(+)-recA displayed higher viability than controls. UV-C exposure tests further highlighted the protective effect of recA expression, with the recombinant strain maintaining viability after 60 min of exposure, while control strains showed no survival. These results indicate that the Z. mobilis recA gene product enhances resistance to oxidative, heat, ethanol, and UV-C stresses when expressed in E. coli. This study elucidates the broad stress-protective functions of the RecA protein across bacterial species and suggests potential applications in developing stress-tolerant bacterial strains for biotechnological purposes. Full article

A novel multifunctional isoamylase, MIsA from Myxococcus sp. strain V11, was expressed in Escherichia coli BL21(DE3). Sequence alignment revealed that MIsA is a typical isoamylase that belongs to glycoside hydrolase family 13 (GH 13). MIsA can hydrolyze the α-1,6-branches of amylopectin and pullulan, as well as the α-1,4-glucosidic bond in amylose. Additionally, MIsA demonstrates 4-α-D-glucan transferase activity, enabling the transfer of α-1,4-glucan oligosaccharides between molecules, particularly with linear maltooligosaccharides. The K_m, K_cat, and V_max values of the MIsA for amylopectin were 1.22 mM, 40.42 µmol·min^–1·mg^–1, and 4046.31 mM·min^–1. The yields of amylopectin and amylose hydrolyzed into oligosaccharides were 10.16% and 11.70%, respectively. The hydrolysis efficiencies were 55%, 35%, and 30% for amylopectin, soluble starch, and amylose, respectively. In the composite enzyme hydrolysis of amylose, the yield of maltotetraose increased by 1.81-fold and 2.73-fold compared with that of MIsA and MTHase (MCK8499120) alone, respectively. Full article

Simvastatin is a widely used statin medication that is commonly prescribed to lower cholesterol levels and reduce the risk of cardiovascular events. It is marketed under the brand name Zocor and is known for its effectiveness in treating high cholesterol and managing cardiovascular disease. Monacolin J is an important precursor used to synthesize simvastatin and is mainly produced by chemical methods in industry. Here, monacolin J was synthesized through an enzymatic method under optimized reaction conditions. One recombinant Escherichia coli BL21 (DE3) strain containing lovastatin hydrolase (encoded by CDV55_102090) from Aspergillus turcosus was constructed, which effectively transformed 100 g/L of lovastatin to monacolin J within 3.5 h at pH 8.0 and 30 °C, with a conversion rate of >99.8%. Furthermore, the T50¹⁰, the temperature at which the residual activity was half of the initial enzymatic activity after 10 min of heat treatment, was >50 °C, indicating the tremendous potential of this bioprocess for synthesizing monacolin J at an industrial scale. Full article

Phycocyanobilin (PCB) is a small chromophore found in certain phycobiliproteins, such as phycocyanins (PCs) and allophycocyanins (APCs). PCB, along with other phycobilins (PBs) and intermediates such as biliverdin (BV) or phycoerythrobilin (PEB), is attracting increasing biotechnological interest due to its fluorescent and medicinal properties that allow potential applications in biomedicine and the food industry. This study aims to optimize PCB synthesis in Escherichia coli BL21 (DE3) and scale the process to a pre-industrial level. Parameters such as optimal temperature, inducer concentration, initial OD₆₀₀, and stirring speed were analyzed in shake flask cultures to maximize PCB production. The best results were obtained at a temperature of 28 °C, an IPTG concentration of 0.1 mM, an initial OD₆₀₀ of 0.5, and an orbital shaking speed of 260 rpm. Furthermore, the optimized protocol was scaled up into a 2 L bioreactor batch, achieving a maximum PCB concentration of 3.8 mg/L. Analysis of the results revealed that biosynthesis of exogenous PBs in Escherichia coli BL21 (DE3) is highly dependent on the metabolic burden of the host. Several scenarios, such as too rapid growth, high inducer concentration, or mechanical stress, can advance entry into the stationary phase. That progressively halts pigment synthesis, leading, in some cases, to its excretion into the growth media and ultimately triggering rapid degradation by the host. These conclusions provide a promising protocol for scalable PCB production and highlight the main biotechnological challenges to increase the yields of the process. Full article