Search Results (38)

Avian influenza is an economically significant disease affecting poultry worldwide and is caused by influenza A viruses that can range from low to highly pathogenic strains. These viruses primarily target the respiratory, digestive, and nervous systems of birds, leading to severe outbreaks that threaten poultry production and pose zoonotic risks. The ectodomain of the avian influenza virus (AIV) matrix protein 2 (M2e), known for its high conservation across influenza strains, has emerged as a promising candidate for developing a universal influenza vaccine in a mouse model. However, the efficacy of such expression against poultry AIVs remains limited. The objective of this study was to evaluate the immunogenicity of nodavirus-like particles displaying the M2e proteins. In this study, three synthetic heterologous M2e genes originated from AIV strains H5N1, H9N2 and H5N2 were fused with the nodavirus capsid protein (NVC) of the giant freshwater prawn Macrobrachium rosenbergii (NVC-3xAvM2e) prior to immunogenicity characterisations in chickens. The expression vector pTRcHis-TARNA2 carrying the NVC-3xAvM2e gene cassette was introduced into E. coli TOP-10 cells. The recombinant proteins were purified, inoculated into one-week-old specific pathogen-free chickens subcutaneously and analysed. The recombinant protein NVC-3xAvM2e formed virus-like particles (VLPs) of approximately 25 nm in diameter when observed under a transmission electron microscope. Dynamic light scattering (DLS) analysis revealed that the VLPs have a polydispersity index (PDI) of 0.198. A direct ELISA upon animal experiments showed that M2e-specific antibodies were significantly increased in vaccinated chickens after the booster, with H5N1 M2e peptides having the highest mean absorbance value when compared with those of H9N2 and H5N2. A challenge study using low pathogenic AIV (LPAI) strain A/chicken/Malaysia/UPM994/2018 (H9N2) at 10^6.5 EID₅₀ showed significant viral load in the lung and cloaca, but not in the oropharyngeal of vaccinated animals when compared with the unvaccinated control group. Collectively, this study suggests that nodavirus-like particles displaying three heterologous M2e have the potential to provide protection against LPAI H9N2 in chickens, though the vaccine’s efficacy and cross-protection across different haemagglutinin (HA) subtypes should be further evaluated. Full article

Our previous study demonstrated that an Escherichia coli heterologous secretion expression system, mediated by superfolder green fluorescent protein (sfGFP) mutants, significantly enhances recombinant lipase yield and reduces large-scale production costs. In this study, we identified mScarlet3, a fast-folding fluorescent protein, as another effective mediator of secretion expression in E. coli. A novel lipolytic enzyme, named LipHu6, was identified through sequence alignment. Secretion expression of LipHu6 was achieved by fusing mScarlet3 to either its N- or C-terminus. The specific activity of mScarlet3-LipHu6 reached 669,151.75 U/mmol, slightly surpassing that of LipHu6 alone (646,682.69 U/mmol) and markedly exceeding that of sfGFP_(-15)-LipHu6 (492,432.39 U/mmol). Notably, N-terminal mScarlet3 fusion had no impact on LipHu6 hydrolytic activity toward short-chain p-nitrophenyl fatty acyl esters (C2–C8). In contrast, mScarlet3-LipHu6 exhibited approximately 1.5- and 1.7-fold increases in hydrolytic activity toward p-nitrophenyl palmitate (p-NPP, C16) and p-nitrophenyl stearate (p-NPS, C18), respectively. In conclusion, this study establishes a novel E. coli heterologous secretion expression system mediated by mScarlet3, offering a highly efficient and cost-effective strategy for the large-scale production of lipolytic enzymes. Full article

Industrial biotechnology offers a potential ecological solution for PET recycling under relatively mild reaction conditions via enzymatic degradation, particularly using the leaf branch compost cutinase (LCC) quadruple mutant ICCG. To improve the efficient downstream processing of this biocatalyst after heterologous gene expression with a suitable production host, protein crystallization can serve as an effective purification/capture step. Enhancing protein crystallization was achieved in recent studies by introducing electrostatic (and aromatic) interactions in two homologous alcohol dehydrogenases (Lb/LkADH) and an ene reductase (NspER1-L1,5) produced with Escherichia coli. In this study, ICCG, which is difficult to crystallize, was utilized for the application of crystal contact engineering strategies, resulting in ICCG mutant L50Y (ICCGY). Previously focused on the Lys-Glu interaction for the introduction of electrostatic interactions at crystal contacts, the applicability of the engineering strategy was extended here to an Arg-Glu interaction to increase crystallizability, as shown for ICCGY T110E. Furthermore, the rationale of the engineering approach is demonstrated by introducing Lys and Glu at non-crystal contacts or sites without potential interaction partners as negative controls. These resulting mutants crystallized comparably but not superior to the wild-type protein. As demonstrated by this study, crystal contact engineering emerges as a promising approach for rationally enhancing protein crystallization. This advancement could significantly streamline biotechnological downstream processing, offering a more efficient pathway for research and industry. Full article

Ferrochelatase is the terminal enzyme in heme biosynthesis. Bacillus thuringiensis (Bt) 97-27 contains two ferrochelatases, HemH1 and HemH2, but their regulatory mechanisms and functional differences under virous environmental stimuli remain unclear. This study confirmed that the iron uptake regulator protein (Fur) bound to the promoters of hemH1 and hemH2, with Fe²⁺ or Fe³⁺ enhancing this binding. Heterologous expression of HemH1 and HemH2 in Escherichia coli showed that pEH2/BL grew better than pEH1/BL under different 2,2′-Bipyridyl, Fe²⁺, and Fe³⁺ concentrations. Under iron limitation, the heme precursor ALA production decreased significantly in both strains. The heme production of pEH2/BL decreased sharply under iron-limited conditions, while that of pEH1/BL decreased significantly under iron-rich conditions. The H₂O₂ sensitivity experiment revealed that E. coli pEH1/BL was more tolerant to H₂O₂ than pEH2/BL. In Bt, ΔhemH2 was most sensitive to H₂O₂ stress, but complementation of hemH1 or hemH2 partially restored H₂O₂ resistance, with the overexpressed strain pHH2/Bt being most tolerant. β-galactosidase assays indicated that Fur positively regulated hemH1 and negatively regulated hemH2 under normal conditions, but this regulation reversed with 2.5 mM Fe³⁺. qRT-PCR showed upregulation of genes related to heme synthesis, oxidative stress, and ferrous iron transport. This study reveals the functional differentiation of HemH1 and HemH2 under the joint regulation of Fur and environmental factors, highlighting their synergistic roles in heme synthesis, heavy metal detoxification, and oxidative stress resistance to maintain bacterial physiological homeostasis. Full article

Bilirubin (BR) is an important ingredient of a valuable Chinese medicine, Calculus bovis. Over recent decades, increasing evidence has confirmed that BR offers health benefits in cardiovascular health, stroke, diabetes, and metabolic syndrome. However, BR is mainly produced by extraction from pig bile. In this study, we assembled an efficient pathway for BR production by metabolic engineering of Escherichia coli. First, heme oxygenase (HO1) and biliverdin reductase were co-expressed in E. coli. HPLC and LC–MS confirmed the accumulation of BR in the recombinant E. coli cells. To improve BR production, the catalytic abilities of HO1 from different species were investigated. In addition, the outermembrane-bound heme receptor (ChuA) and the enzymes involved in heme biosynthesis were overexpressed among which ChuA, 5-aminolevulinic acid dehydratase (HemB), protoporphyrin oxidase (HemG), and ferrochelatase (HemH) were found to enhance BR accumulation in E. coli. In addition, expression of ferredoxin (Fd) was shown to contribute to efficient conversion of heme to BR in E. coli. To increase supply of NADPH, isocitrate dehydrogenase (IDH), NAD kinase (nadK), NADP-specific glutamate dehydrogenase (gdhA), and glucose-6-phosphate 1-dehydrogenase (ZWF) were overexpressed and were found to enhance BR accumulation when these proteins were expressed with a low-copy plasmid pACYCduet-1. Modular optimization of the committed genes led to a titer of 17.2 mg/L in strain M1BHG. Finally, fed-batch fermentation was performed for the strains M1BHG and M1, resulting in accumulation of 75.5 mg/L and 25.8 mg/L of BR, respectively. This is the first report on biosynthesis of BR through metabolic engineering in a heterologous host. Full article

Recombinant mutant holotoxin BoNTs (rBoNTs) are being evaluated as possible vaccines against botulism. Previously, several rBoNTs containing 2–3 amino acid mutations in the light chain (LC) showed significant decreases in toxicity (2.5-million-fold–12.5-million-fold) versus wild-type BoNT/A1, leading to their current exclusion from the Federal Select Agent list. In this study, we added four additional mutations in the receptor-binding domain, translocation domain, and enzymatic cleft to further decrease toxicity, creating 7M rBoNT/A1. Due to poor expression in E. coli, 7M rBoNT/A1 was produced in an endogenous C. botulinum expression system. This protein had higher residual toxicity (LD50: 280 ng/mouse) than previously reported for the catalytically inactive rBoNT/A1 containing only three of the mutations (>10 µg/mouse). To investigate this discrepancy, several additional rBoNT/A1 constructs containing individual sets of amino acid substitutions from 7M rBoNT/A1 and related mutations were also endogenously produced. Similarly to endogenously produced 7M rBoNT/A1, all of the endogenously produced mutants had ~100–1000-fold greater toxicity than what was reported for their original heterologous host counterparts. A combination of mutations in multiple functional domains resulted in a greater but not multiplicative reduction in toxicity. This report demonstrates the impact of production systems on residual toxicity of genetically inactivated rBoNTs. Full article

Microbial alkane degradation pathways provide biological routes for converting these hydrocarbons into higher-value products. We recently reported the functional expression of a methyl-alkylsuccinate synthase (Mas) system in Escherichia coli, allowing for the heterologous anaerobic activation of short-chain alkanes. However, the enzymatic activation of methane via natural or engineered alkylsuccinate synthases has yet to be reported. To address this, we employed high-throughput screening to engineer the itaconate (IA)-responsive regulatory protein ItcR (WT-ItcR) from Yersinia pseudotuberculosis to instead respond to methylsuccinate (MS, the product of methane addition to fumarate), resulting in genetically encoded biosensors for MS. Here, we describe ItcR variants that, when regulating fluorescent protein expression in E. coli, show increased sensitivity, improved overall response, and enhanced specificity toward exogenously added MS relative to the wild-type repressor. Structural modeling and analysis of the ItcR ligand binding pocket provide insights into the altered molecular recognition. In addition to serving as biosensors for screening alkylsuccinate synthases capable of methane activation, MS-responsive ItcR variants also establish a framework for the directed evolution of other molecular reporters, targeting longer-chain alkylsuccinate products or other succinate derivatives. Full article

Spodoptera frugiperda, the fall armyworm (FAW), is a highly invasive polyphagous insect pest that is considered a source of severe economic losses to agricultural production. Currently, the majority of chemical insecticides pose tremendous threats to humans and animals besides insect resistance. Thus, there is an urgent need to develop new pest management strategies with more specificity, efficiency, and sustainability. Chitin-degrading enzymes, including chitinases, are promising agents which may contribute to FAW control. Chitinase-producing microorganisms are reported normally in bacteria and fungi. In the present study, Serratia marcescens was successfully isolated and identified from the larvae of Spodoptera frugiperda. The bacterial strain NRC408 displayed the highest chitinase enzyme activity of 250 units per milligram of protein. Subsequently, the chitinase gene was cloned and heterologously expressed in E. coli BL21 (DE3). Recombinant chitinase B was overproduced to 2.5-fold, driven by the T7 expression system. Recombinant chitinase B was evaluated for its efficacy as an insecticidal bioagent against S. frugiperda larvae, which induced significant alteration in subsequent developmental stages and conspicuous malformations. Additionally, our study highlights that in silico analyses of the anticipated protein encoded by the chitinase gene (ChiB) offered improved predictions for enzyme binding and catalytic activity. The effectiveness of (ChiB) against S. frugiperda was evaluated in laboratory and controlled field conditions. The results indicated significant mortality, disturbed development, different induced malformations, and a reduction in larval populations. Thus, the current study consequently recommends chitinase B for the first time to control FAW. Full article

To delve into the structure–function relationship of transmembrane proteins (TMPs), robust protocols are needed to produce them in a pure, stable, and functional state. Among all hosts that express heterologous TMPs, E. coli has the lowest cost and fastest turnover. However, many of the TMPs expressed in E. coli are misfolded. Several strategies have been developed to either direct the foreign TMPs to E. coli’s membrane or retain them in a cytosolic soluble form to overcome this deficiency. Here, we summarize protein engineering methods to produce chimera constructs of the desired TMPs fused to either a signal peptide or precursor maltose binding protein (pMBP) to direct the entire construct to the periplasm, therefore depositing the fused TMP in the plasma membrane. We further describe strategies to produce TMPs in soluble form by utilizing N-terminally fused MBP without a signal peptide. Depending on its N- or C-terminus location, a fusion to apolipoprotein AI can either direct the TMP to the membrane or shield the hydrophobic regions of the TMP, maintaining the soluble form. Strategies to produce G-protein-coupled receptors, TMPs of Mycobacterium tuberculosis, HIV-1 Vpu, and other TMPs are discussed. This knowledge could increase the scope of TMPs’ expression in E. coli. Full article

In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker’s yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species’ several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry. Full article

Phycocyanobilin (PCB) is the bioactive chromophore attached to Phycocyanin (PC) that is of special interest for nutraceutical and therapeutic applications. However, the production of PCB from the heterologous host Escherichia coli is still very low. To facilitate subsequent application of PCB, improving its production in microbial hosts is still a challenge to be solved. In this paper, a strategy involving fusion-expression of apo-proteins with signal peptides was adopted to improve PCB production in E. coli. First, we reconstructed the PCB biosynthesis pathway in E. coli and then optimized its culture media. Subsequently, one PC α (CpcA) subunit and one PC β (CpcB) subunit, which can capture free PCB, were introduced and increased the yield of PCB. Finally, CpcA was fused with seven signal peptides to generate recombinant proteins, among which, the signal peptide N20 fused with CpcA protein drastically improved PCB production in E. coli, providing a maximum flask output of 8.47 ± 0.18 mg/L. The results of this study demonstrate that PCB distribution and transporting manners in E. coli could affect the heterologous production efficiency. By fusing apo-proteins with signal peptides, the secretion of phycocyanin was refined and the production of PCB was successfully enhanced by 3.7-fold, compared with the starting strain (1.80 ± 0.12 mg/L). This work provided an alternative method for improving the production of PCB and other phycobilins. Full article

A novel fungal immunomodulatory protein (FIP), identified as FIP-hma, was discovered in the genome of an edible mushroom Hypsizygus marmoreus. Bioinformatics analysis suggested FIP-hma contained the cerato-platanin (CP) conserved domain and was categorized into Cerato-type FIP. In phylogenetic analysis, FIP-hma was clustered into a new branch of the FIP family, displaying large system divergence from most of the other FIPs. The higher gene expression of FIP-hma was observed during the vegetative growth stages than that during the reproductive growth stages. In addition, the cDNA sequence of FIP-hma was cloned and successfully expressed in Escherichia coli (E. coli) BL21(DE3). The recombinant protein of FIP-hma (rFIP-hma) was neatly purified and isolated by Ni-NTA and SUMO-Protease. The iNOS, IL-6, IL-1β, and TNF-α levels of RAW 264.7 macrophages were upregulated by rFIP-hma, indicating its activation of an immune response by regulating central cytokines. No cytotoxic effects were observed in an MTT test. The findings of this work discovered a novel immunoregulatory protein from H. marmoreus, provided a systematic bioinformatic profile, suggested an effective approach for its heterologous recombinant production, and reported its potent immunoregulatory activity in macrophages. This study sheds light on the physiological function research of FIPs and their further industrial utilization. Full article

Escherichia coli is the best-known model for the biotechnological production of many biotechnological products, including housekeeping and heterologous primary and secondary metabolites and recombinant proteins, and is an efficient biofactory model to produce biofuels to nanomaterials. Glucose is the primary substrate used as the carbon source for laboratory and industrial cultivation of E. coli for production purposes. Efficient growth and associated production and yield of desired products depend on the efficient sugar transport capabilities, sugar catabolism through the central carbon catabolism, and the efficient carbon flux through specific biosynthetic pathways. The genome of E. coli MG1655 is 4,641,642 bp, corresponding to 4702 genes encoding 4328 proteins. The EcoCyc database describes 532 transport reactions, 480 transporters, and 97 proteins involved in sugar transport. Nevertheless, due to the high number of sugar transporters, E. coli uses preferentially few systems to grow in glucose as the sole carbon source. E. coli nonspecifically transports glucose from the extracellular medium into the periplasmic space through the outer membrane porins. Once in periplasmic space, glucose is transported into the cytoplasm by several systems, including the phosphoenolpyruvate-dependent phosphotransferase system (PTS), the ATP-dependent cassette (ABC) transporters, and the major facilitator (MFS) superfamily proton symporters. In this contribution, we review the structures and mechanisms of the E. coli central glucose transport systems, including the regulatory circuits recruiting the specific use of these transport systems under specific growing conditions. Finally, we describe several successful examples of transport engineering, including introducing heterologous and non-sugar transport systems for producing several valuable metabolites. Full article

Serratiopeptidase is a clinical therapeutic protein for the treatment of human diseases such as arthritis, bronchitis, and thrombosis. Yet production of this protein in a heterologous host (e.g., Escherichia coli) is difficult due to the issue of protein insolubility and the requirement of laborious refolding procedures. Cell-free protein synthesis (CFPS) systems, derived from crude cell extracts, are effective platforms for the expression of recombinant proteins in vitro. Here, we report a new method to produce serratiopeptidase by using an E. coli-based CFPS system. After rational selection of cell extracts and construction of expression vectors, soluble expression of serratiopeptidase was achieved and the enzyme activity could be readily tested in the cell-free reaction mixture. By further optimizing the key parameters, optimum conditions for the enzyme activity assay were obtained, including the pH value at 5, reaction temperature at 45 °C, substrate concentration at 10 mg/mL, and supplementing Ca²⁺ ions at 5 mM. Moreover, the CFPS mixture was freeze-dried and the activity of serratiopeptidase could be regenerated by hydration without losing activity. Overall, the CFPS system enabled soluble expression of serratiopeptidase with catalytic activity, providing a new and promising approach for this enzyme production. Our work extends the utility of the cell-free platform to produce therapeutic proteins with clinical applications. Full article

Cross-reacting material 197 (CRM197) is a non-toxic mutant of the diphtheria toxin and is widely used as a carrier protein in conjugate vaccines. This protein was first obtained from the supernatant of the mutant Corynebacterium diphtheriae strain. This pathogenic bacteria strain is characterized by a slow growth rate and a relatively low target protein yield, resulting in high production costs for CRM197. Many attempts have been made to establish high-yield protocols for the heterologous expression of recombinant CRM197 in different host organisms. In the present work, a novel CRM197-producing Escherichia coli strain was constructed. The target protein was expressed in the cytoplasm of SHuffle T7 E. coli cells without any additional tags and with a single potential mutation—an additional Met [−1]. The fine tuning of the mRNA structure (the disruption of the single hairpin in the start codon area) was sufficient to increase the CRM197 expression level several times, resulting in 150–270 mg/L (1.1–2.0 mg/g wet biomass) yields of pure CRM197 protein. Besides the high yield, the advantages of the obtained expression system include the absence of the necessity of CRM197 refolding or tag removal. Thus, an extensive analysis of the mRNA structure and the removal of the unwanted hairpins in the 5′ area may significantly improve the target protein expression rate. Full article