Search Results (92)

The growing threat of infectious diseases requires novel therapeutics with different mechanisms of action. Antimicrobial peptides (AMPs), which are crucial for innate immunity, are a promising research area. The medicinal leech (Hirudo medicinalis) is a potential source of bioactive AMPs that are vital while interacting with microorganisms. This study aims to investigate the antimicrobial properties of peptides found in the H. medicinalis genome using a novel high-throughput screening method based on the expression of recombinant AMP genes in Escherichia coli. This approach enables the direct detection of AMP activity within cells, skipping the synthesis and purification steps, while allowing the simultaneous analysis of multiple peptides. The application of this method to the first identified candidate AMPs from H. medicinalis resulted in the discovery of three novel peptides: LBrHM1, NrlHM1 and NrlHM2. These peptides, which belong to the lumbricin and macin families, exhibit significant activity against E. coli. Two fragments of the new LBrHM1 homologue were synthesised and studied: a unique N-terminal fragment (residues 1–23) and a fragment (residues 27–55) coinciding with the active site of lumbricin I. Both fragments exhibited antimicrobial activity in a liquid medium against Bacillus subtilis. Notably, the N-terminal fragment lacks homologues among previously described AMPs. Full article

CRISPR technology, which is derived from the bacterial adaptive immune system, has transformed traditional genetic engineering techniques, made strain engineering significantly easier, and become a very versatile genome editing system that allows for precise, programmable modifications to a wide range of microbial genomes. The economies of fermentation-based manufacturing are changing because of its quick acceptance in both academic and industry labs. CRISPR processes have been used to modify industrially significant bacteria, including the lactic acid producers, Clostridium spp., Escherichia coli, and Corynebacterium glutamicum, in order to increase the yields of bioethanol, butanol, succinic acid, acetone, and polyhydroxyalkanoate precursors. CRISPR-mediated promoter engineering and single-step multiplex editing have improved inhibitor tolerance, raised ethanol titers, and allowed for the de novo synthesis of terpenoids, flavonoids, and recombinant vaccines in yeasts, especially Saccharomyces cerevisiae and emerging non-conventional species. While enzyme and biopharmaceutical manufacturing use CRISPR for quick strain optimization and glyco-engineering, food and beverage fermentations benefit from starter-culture customization for aroma, texture, and probiotic functionality. Off-target effects, cytotoxicity linked to Cas9, inefficient delivery in specific microorganisms, and regulatory ambiguities in commercial fermentation settings are some of the main challenges. This review provides an industry-specific summary of CRISPR–Cas9 applications in microbial fermentation and highlights technical developments, persisting challenges, and industrial advancements. Full article

A novel bivalent oral vaccine candidate against H5N1 and H9N2 avian influenza virus (AIV) was developed using Lactobacillus surface display technology without genetic modification. The hemagglutinin subunit 1 (HA1) antigens from both subtypes were fused to the surface layer-binding domain of Lactobacillus crispatus K313, expressed in Escherichia coli, and purified. Wild-type Lactobacillus johnsonii H31, isolated from chicken intestine, served as a delivery vehicle by adsorbing and stably displaying the HA1 proteins on its surface. This approach eliminates the need for bacterial engineering while utilizing lactobacilli’s natural capacity to protect surface-displayed antigens, as evidenced by HA1’s protease resistance. Mouse immunization studies demonstrated induction of strong systemic IgG and mucosal IgA responses against both H5N1 and H9N2 HA1. The system offers several advantages, including safety through non-GMO probiotics, potential for multivalent vaccine expansion, and intrinsic antigen protection by lactobacilli. These findings suggest this platform could enable development of cost-effective, multivalent AIV vaccines. Full article

The increasing global demand for energy and the urgent need to mitigate climate change have driven the search for sustainable alternatives to fossil fuels, with biofuels emerging as a promising solution. However, the low yields and inefficiencies in biofuel production processes necessitate advanced strategies to enhance their commercial viability. Metabolic engineering has become a pivotal tool in optimizing microbial pathways to improve biofuel production, addressing these challenges through innovative genetic and synthetic biology approaches. This review highlights the role of metabolic engineering in enhancing biofuel production by focusing on microbial engineering for lignocellulosic biomass utilization, strategies to overcome inhibitor effects, and pathway optimization for biofuels like n-butanol and iso-butanol. It also explores the production of advanced biofuels from fatty acid and isoprenoid pathways, emphasizing the use of model organisms such as Escherichia coli and Saccharomyces cerevisiae. Key insights include the application of CRISPR/Cas9 and multiplex automated genome engineering for precise genetic modifications, as well as metabolic flux analysis to optimize pathway efficiency. Additionally, the review discusses synthetic biology methodologies to rewire metabolic networks and improve biofuel yields, providing a comprehensive overview of current advancements and their implications for industrial-scale production. Full article

The increasing prevalence of multidrug-resistant (MDR) bacterial infections necessitates the exploration of alternative antimicrobial strategies, with phage therapy emerging as a viable option. However, the effectiveness of naturally occurring phages can be significantly limited by bacterial defense systems that include adsorption blocking, restriction–modification, CRISPR-Cas immunity, abortive infection, and NAD+ depletion defense systems. This review examines these bacterial defenses and their implications for phage therapy, while highlighting the potential of phages’ bioengineering to overcome these barriers. By leveraging synthetic biology, genetically engineered phages can be tailored to evade bacterial immunity through such modifications as receptor-binding protein engineering, anti-CRISPR gene incorporation, methylation pattern alterations, and enzymatic degradation of bacterial protective barriers. “Armed phages”, enhanced with antimicrobial peptides, CRISPR-based genome-editing tools, or immune-modulating factors, offer a novel therapeutic avenue. Clinical trials of bioengineered phages, currently SNIPR001 and LBP-EC01, showcase their potential to safely and effectively combat MDR infections. SNIPR001 has completed a Phase I clinical trial evaluating safety in healthy volunteers, while LBP-EC01 is in Phase II trials assessing its performance in the treatment of Escherichia coli-induced urinary tract infections in patients with a history of drug-resistant infections. As “armed phages” progress toward clinical application, they hold great promise for precision-targeted antimicrobial therapies and represent a critical innovation in addressing the global antibiotic resistance crisis. Full article

Background/Objectives: Heparosan is a component of the capsular polysaccharide in Escherichia coli K5 and Pasteurella multocida Type D. It shares a similar glycan structure with heparin and can be enzymatically modified to produce bioactive heparin. Methods: In this study, the probiotic strain E. coli Nissle 1917 (EcN), which naturally produces heparosan, was genetically engineered to utilize sucrose as a carbon source for growth while achieving high-yield heparosan biosynthesis. Results: By expressing the sucrose hydrolase genes sacA (from Bacillus subtilis) or spI (from Bifidobacterium adolescentis), EcN was enabled to utilize sucrose, achieving heparosan titers of 131 mg/L and 179 mg/L, respectively. Further metabolic engineering was performed to block the glycolytic and pentose phosphate pathways, thereby redirecting sucrose-derived glucose-6-phosphate and fructose-6-phosphate toward heparosan biosynthesis, while glycerol was supplemented as an auxiliary carbon source to support cell growth. Finally, the key biosynthesis genes galU, kfiD, and glmM were overexpressed, resulting in an engineered strain with a heparosan titer of 622 mg/L. Conclusions: This study represents the first successful engineering of EcN to utilize sucrose as the carbon source for growth, while achieving enhanced heparosan production through synergistic carbon source utilization. These findings establish a foundational strategy for employing this strain in the sucrose-based biosynthesis of other glycosaminoglycans. Full article

Mandelic acid (MA) is a valuable α-hydroxy acid with applications in pharmaceuticals, cosmetics, and fine chemicals. While chemical synthesis is well established, concerns over toxicity and sustainability have driven interest in microbial production. Here, we engineered Escherichia coli for de novo MA biosynthesis by integrating enzyme screening, metabolic flux optimization, and pathway regulation. We first screened and identified an efficient hydroxymandelate synthase (HMAS) homolog from Actinosynnema mirum for MA synthesis, and subsequently enhanced the shikimate pathway along with the supply of the precursors erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP). Additionally, CRISPR interference (CRISPRi) was employed to repress competing pathways and redirect flux toward MA production. High-cell-density cultivation (HCDC) in a 5 L bioreactor demonstrated the strain’s industrial potential, achieving an MA titer of 9.58 g/L, the highest reported for microbial production. This study provides a systematic metabolic engineering approach for efficient MA biosynthesis from glucose, offering a foundation for sustainable large-scale production, demonstrating not only genetic-level optimizations, but also effective process scaling through high-cell-density cultivation, highlighting the power of pathway engineering in microbial cell factories. Full article

Crop productivity is severely constrained by abiotic and biotic stresses, necessitating innovative strategies to enhance stress resilience. Glycerol-3-phosphate (G3P) is a central metabolite in carbohydrate and lipid metabolism, playing crucial roles in stress responses. In this study, we engineered a novel glycerol-3-phosphate dehydrogenase (GPDH) gene, designated OEGD, by fusing the N-terminal NAD-binding domain of rice OsGPDH1 with the feedback-resistant C-terminal catalytic domain of Escherichia coli gpsA. Overexpression of OEGD in rice enhanced tolerance to drought, phosphorus deficiency, high temperature, and cadmium (Cd²⁺) stresses, while also improving plant growth and yield under drought stress at the adult stage. Notably, the accumulation of glycerol-3-phosphate (G3P) and activities of antioxidant enzymes (SOD, POD, CAT) were significantly elevated in the transgenic plants following osmotic stimuli, and fatty acid profiles were altered, favoring stress adaptation. Transcriptomic analyses revealed that OEGD modulates cell wall biogenesis, reactive oxygen species (ROS) scavenging, and lipid metabolism pathways, with minimal disruption to core G3P metabolic genes. These findings highlight the potential of OEGD as a valuable genetic resource for improving stress resistance in rice. Full article

This study investigates the potential of sugar beet pulp (SBP), a lignocellulosic by-product of sugar production, as a low-cost substrate for biohydrogen and biomass generation using Escherichia coli under dark fermentation conditions. Two strains—BW25113 wild-type and a genetically engineered septuple mutant—were employed. SBP was pretreated via thermochemical hydrolysis, and the effects of substrate concentration, dilution, and glycerol supplementation were evaluated. Hydrogen production was highly dependent on substrate dilution and nutrient balance. The septuple mutant achieved the highest H₂ yield in 30 g L⁻¹ SBP hydrolysate (0.75% sulfuric acid) at 5× dilution with glycerol, reaching 12.06 mmol H₂ (g sugar)⁻¹ and 0.28 mmol H₂ (g waste)⁻¹, while the wild type under the same conditions yielded 3.78 mmol H₂ (g sugar)⁻¹ and 0.25 mmol H₂ (g waste)⁻¹. In contrast, undiluted hydrolysates favored biomass accumulation over H₂ production, with the highest biomass yield (0.3 g CDW L⁻¹) obtained using the septuple mutant in 30 g L⁻¹ SBP hydrolysate without glycerol. These findings highlight the potential of genetically optimized E. coli and optimized hydrolysate conditions to enhance the valorization of agro-industrial waste, supporting future advances in sustainable hydrogen bioeconomy and integrated waste biorefineries. Full article

Surface display technology has revolutionized whole-cell biocatalysis by enabling efficient enzyme immobilization on microbial cell surfaces. Compared with traditional enzyme immobilization, this technology has the advantages of high enzyme activity, mild process, simple operation and low cost, which thus has been widely studied and applied in various fields. This review explores the principles, optimization strategies, applications in the food industry, and future prospects. We summarize the membrane and anchor protein structures of common host cells (Escherichia coli, Bacillus subtilis, and yeast) and discuss cutting-edge optimization approaches, including host strain genetic engineering, rational design of anchor proteins, innovative linker peptide engineering, and precise regulation of signal peptides and promoters, to maximize surface display efficiency. Additionally, we also explore its diverse applications in food processing and manufacturing, additive synthesis, food safety, and other food-related industries (such as animal feed and PET packaging degradation), demonstrating their potential to address key challenges in the food industry. This work bridges fundamental research and industrial applications, offering valuable insights for advancing agricultural and food chemistry. Full article

Background/Objectives: Codon usage bias affects gene expression and translation efficiency across species. The effective number of codons (ENC) and GC content influence codon preference, often displaying unimodal or bimodal distributions. This study investigates the correlation between ENC and GC rankings across species and how their relationship affects codon usage distributions. Methods: I analyzed nuclear-encoded genes from 17 species representing six kingdoms: one bacteria (Escherichia coli), three fungi (Saccharomyces cerevisiae, Neurospora crassa, and Schizosaccharomyces pombe), one archaea (Methanococcus aeolicus), three protists (Rickettsia hoogstraalii, Dictyostelium discoideum, and Plasmodium falciparum),), three plants (Musa acuminata, Oryza sativa, and Arabidopsis thaliana), and six animals (Anopheles gambiae, Apis mellifera, Polistes canadensis, Mus musculus, Homo sapiens, and Takifugu rubripes). Genes in all 17 species were ranked by GC content and ENC, and correlations were assessed. I examined how adding or subtracting these rankings influenced their overall distribution in a new method that I call Two-Rank Order Normalization or TRON. The equation, TRON = SUM(ABS((GC rank₁:GC rank_N) − (ENC rank₁:ENC rank_N))/(N²/3), where (GC rank₁:GC rank_N) is a rank-order series of GC rank, (ENC rank₁:ENC rank_N) is a rank-order series ENC rank, sorted by the rank-order series GC rank. The denominator of TRON, N²/3, is the normalization factor because it is the expected value of the sum of the absolute value of GC rank–ENC rank for all genes if GC rank and ENC rank are not correlated. Results: ENC and GC rankings are positively correlated (i.e., ENC increases as GC increases) in AT-rich species such as honeybees (R² = 0.60, slope = 0.78) and wasps (R² = 0.52, slope = 0.72) and negatively correlated (i.e., ENC decreases as GC increases) in GC-rich species such as humans (R² = 0.38, slope = −0.61) and rice (R² = 0.59, slope = −0.77). Second, the GC rank–ENC rank distributions change from unimodal to bimodal as GC content increases in the 17 species. Third, the GC rank+ENC rank distributions change from bimodal to unimodal as GC content increases in the 17 species. Fourth, the slopes of the correlations (GC versus ENC) in all 17 species are negatively correlated with TRON (R² = 0.98) (see Graphic Abstract). Conclusions: The correlation between ENC rank and GC rank differs among species, shaping codon usage distributions in opposite ways depending on whether a species’ nuclear-encoded genes are AT-rich or GC-rich. Understanding these patterns might provide insights into translation efficiency, epigenetics mediated by CpG DNA methylation, epitranscriptomics of RNA modifications, RNA secondary structures, evolutionary pressures, and potential applications in genetic engineering and biotechnology. Full article

Due to the barriers imposed by the restriction–modification (RM) system, Nisin-producing industrial strains of Lactococcus lactis often encounter low transformation efficiency, which seriously hinders the widespread application of genetic engineering in non-model L. lactis. Herein, we present a novel pre-modification strategy (PMS) coupled with optimized plasmid delivery systems designed to systematically evade RM barriers and substantially improve Nisin biosynthesis in L. lactis. Through the use of engineered Escherichia coli strains with methylation profiles specifically optimized for L. lactis C20, we have effectively evaded RM barriers, thereby facilitating the efficient introduction of large Nisin biosynthetic gene clusters into L. lactis. The PMS tools, which significantly improve the transformation efficiency (~10³ transformants per microgram of DNA), have been further improved in combination with a Rolling Circle Amplification, resulting in a higher enhancement in transformation efficiency (~10⁴ transformants per microgram of DNA). Using this strategy, large Nisin biosynthetic gene clusters and the expression regulation of all genes within the cluster were introduced and analyzed in L. lactis, leading to a highest Nisin titer of 11,052.9 IU/mL through a fed-batch fermentation in a 5 L bioreactor. This is the first systematic report on the expression regulation and application of a complete Nisin biosynthesis gene cluster in L. lactis. Taken together, our studies provide a versatile and efficient strategy for systematic evasion and enhancement of RM barriers and Nisin biosynthesis, thereby paving the way for genetic modification and metabolic engineering in L. lactis. Full article

Purpose: This study aimed to utilize genetically engineered Bacillus licheniformis for the production of ergothioneine (EGT). Given the value of EGT and the application of Bacillus licheniformis in enzyme preparation production, we cloned the key enzymes (EanA and EanB) from Chlorbium limicola. Through gene alignment, new ergothioneine synthase genes (EanAN and EanBN) were identified and then expressed in Bacillus licheniformis to construct strains. Additionally, we investigated the factors influencing the yield of EGT and made a comparison with Escherichia coli. Methods: The relevant genes were cloned and transferred into Bacillus licheniformis. Fermentation experiments were conducted under different conditions for yield analysis, and the stability of this bacterium was also evaluated simultaneously. Results: The constructed strains were capable of producing EGT. Specifically, the yield of the EanANBN strain reached (643.8 ± 135) mg/L, and its stability was suitable for continuous production. Conclusions: Genetically engineered Bacillus licheniformis demonstrates potential in the industrial-scale production of EGT. Compared with Escherichia coli, it has advantages, thus opening up new possibilities for the application and market supply of EGT. Full article

Reverse genetics is a useful tool for studying viruses and developing vaccines for coronaviruses. However, constructing and manipulating the coronavirus genome in Escherichia coli can be time-consuming and challenging due to its large size and instability. Homologous recombination, a genetic manipulation mechanism found in organisms, is essential for DNA repair, gene recombination, and genetic engineering. In yeast, particularly Saccharomyces cerevisiae, homologous recombination technology is commonly used for constructing gene expression plasmids and genome editing. In this study, we successfully split and spliced a 30 kb viral genome fragment using yeast homologous recombination. By optimizing the program parameters, such as homologous arm lengths and fragment-to-vector ratios, we achieved a splicing efficiency of up to 97.9%. The optimal parameters selected were a 60 bp homologous sequence size and a vector fragment ratio of 1:2:2:2:2:2 for yeast homologous recombination of large DNA fragments. Full article

Multiple antibiotic resistance regulators (MarRs) control the transcription of genes in the mar operon of Escherichia coli in the presence of salicylic acid (SA). The interaction with SA induces conformational changes in the MarR released from the promoter of the mar operon, turning on transcription. We constructed an SA-specific E. coli cell-based biosensor by fusing the promoter of the mar operon (P_marO) and the gene that encodes an enhanced green fluorescent protein (egfp). Because SA and aspirin are structurally similar, a biosensor for monitoring aspirin can be obtained by genetically engineering MarR to be aspirin (ASP)-responsive. To shift the selectivity of MarR toward ASP, we changed the residues around the ligand-binding sites by site-directed mutagenesis. We examined the effects of genetic engineering on MarR by introducing MarRs with P_marO-egfp into E. coli. Among the tested mutants, MarR T72A improved the ASP responses by approximately 3 times compared to the wild-type MarR, while still showing an SA response. Although the MarR T72A biosensor exhibited mutual interference between SA and ASP, it accurately determined the ASP concentration in spiked water and medicine samples with over 90% accuracy. While the ASP biosensors still require improvement, our results provide valuable insights for developing E. coli cell-based biosensors for ASP and transcription factor-based biosensors in general. Full article