Development of therapies that selectively inhibit cancer cells but leave normal cells untouched is an important impediment to progress in cancer therapy. This paper reports the novel strategy employing the R-Tf-D-LP4 peptide, an unconventional cell penetrating peptide selectively targeting liver cancer cells, combined with the potent antitumor cytokine interleukin 15 (IL-15), with selective activity towards liver cancer cells. The fusion protein was constructed as a chimera between the IL-15 and the targeting peptide R-Tf-D-LP4 to improve the targeting specificity of this protein to the desired cells. With the help of modern computer modeling technologies, a fusion protein was designed, and its interactions with the IL-15 receptor complex (IL-15Rα/IL-15Rβ/γc) were evaluated using a molecular docking technique. The structure and physicochemical properties of the chimeric protein were determined using programs such as trROSETTA, I-TASSER, and ProtParam, after which they were further verified using SAVES 6.1 server with an excellent ERRAT quality score of 99.35%. Molecular docking using ClusPro 2.0, along with interaction evaluation using PDBsum program, demonstrated stability of the complex and strong hydrogen bonds and salt bridge interactions. The results were verified using the Desmond module in Schrödinger software to simulate molecular dynamics, which revealed a stable structure and proper functioning of the complex in physiological conditions. The expression analysis forecasted good yields from E. coli fermentation. The construct of the IL15/R-Tf-D-LP4 protein gene was created and inserted into the pET-22b ( +) vector, which was then transformed in BL21 (DE3) cells. The recombinant fusion protein was obtained by affinity chromatography and tested for purity by SDS-PAGE and western blotting. Cytotoxicity activity of the fusion protein was evaluated against the HepG2 cell line with the MTT test. As a result, the cytotoxic effect with an IC50 value of 23 μg/ml was established, compared to the non-chimera protein's IC50 value of 62 μg/ml. The achieved results allow us to speak about the prospects of using IL-15/R-Tf-D-LP4 fusion protein for clinical purposes in the future.

The objective of this study was to obtain Mycobacterium tuberculosis methionyl-tRNA synthetase (MetRS) mutant forms E24A and L293A and investigate the efficiency of M. tuberculosis MetRS inhibitors toward these mutant forms in order to predict binding mechanisms. The recombinant mutant M. tuberculosis MetRS E24A and L293A were obtained using overlap PCR. The recombinant proteins MetRS E24A and L293A were expressed in BL21(DE3) cells. The activity of obtained aminoacyl-tRNA synthetases was studied with aminoacylation assay using BIOMOL® Green Reagent. It was found that the mutations of Glu24 and Leu293 with alanine in the synthetic site of M. tuberculosis MetRS lead to significant decrease of inhibitory activity of investigated compounds. These findings suggest that the amino acid residues Glu24 and Leu293 may contribute to the inhibitory activity of the studied compounds and should therefore be considered during further structural optimization.

Lycopene is a high-value carotenoid with broad applications in the food, pharmaceutical, and cosmetic industries. The oleaginous yeast Rhodotorula toruloides naturally synthesizes mixed carotenoids but fails to accumulate lycopene, primarily due to the bifunctional activity of lycopene cyclase/phytoene synthase Car2, which efficiently converts lycopene into downstream cyclic carotenoids. Here, we engineered R. toruloides for efficient lycopene production using a combinatorial strategy that includes pathway blocking, heterologous enzyme screening, precursor enhancement, and fermentation optimization. CRISPR/Cas9-mediated CAR2 knockout abolished endogenous phytoene synthesizing and lycopene cyclizing activities. Heterologous complementation with exogenous phytoene synthase (CrtB) restored phytoene biosynthesis, and the resultant loss of cyclase activity blocked lycopene cyclization, facilitating robust intracellular lycopene accumulation. Screening three heterologous phytoene synthases identified EgCrtB from Euglena gracilis as the superior enzyme. Co-overexpression of endogenous rate-limiting enzymes involved in the mevalonate pathway further boosted precursor supply. Combined with nitrogen-limited cultivation, which synergistically enhanced lycopene accumulation, the final engineered strain achieved a lycopene content of 0.74 mg/g DCW and a titer of 7.24 mg/L. This work verifies that metabolic pathway modification combined with fermentation regulation can effectively drive lycopene synthesis and accumulation in R. toruloides, offering an early-stage proof-of-concept for further advancing lycopene bioproduction in oleaginous yeast.

Active bacterial xanthine oxidase (XOD) has not been functionally expressed in Escherichia coli, limiting protein engineering of this industrial molybdoflavoprotein. Here, the first production of active XOD from Cellulosimicrobium sp. TH20 in E. coli BW25113 is reported. The key factor was identified as the plasmid pBADp15, combining a low-copy P15A origin with the tightly regulated araBAD promoter, with which an enzymatic activity of 0.2 U/mL was obtained-a 6.7-fold improvement over the high-copy vector pBAD18. Activity was completely abolished upon deletion of xodC from the expression cassette, consistent with its established role as an essential maturation chaperone. The purified enzyme exhibited a specific activity of 8.0 U/mg, a Km of 92 μM for xanthine, and optimal activity at pH 7.0 and 50 °C. It is demonstrated that active bacterial XOD can be produced in E. coli when plasmid copy number and promoter stringency are properly balanced, providing a platform for future protein engineering.

Bacillus sp. HMM can efficiently yield cellulase. However, transcriptome data and sequences of important genes such as cellulase gene are indistinct now, which restricts genetic reconstruction of strain HMM. In current paper, GO, COG and KEGG classification of unigenes from transcriptome sequencing of strain HMM was analyzed in detail, and cellulase gene GH (TR1834) encoding glycosylhydrolase was selected. Moreover, transgenic Bacillus sp. HMM overexpressing GH (GHT) with recombinant plasmid PHY-300PLK-GH was constructed. Avicel activity, CMC-Na activity and filter paper activity of GH transgenic HMM (GHT) were 1.4-, 1.2- and 1.08-fold those of control Bacillus sp. HMM with empty plasmid PHY-300PLK. This study enhanced application value of Bacillus for hydrolysis of cellulose.

Porcine pancreatic lipase, a 1, 3-regioselective lipase, is one of the most widely used biocatalyst in academic as well as industrial processes. This lipase showed remarkable catalytic performance, often outclassed other types of lipases in producing valuable products through varieties of reactions. During hydrolysis of vegetable oil to fatty acid, mono and diacylglycerol, porcine pancreas lipase outclassed Candia rugosa and Thermomyces lanuginosus lipase. Porcine pancreas lipase efficiently catalyzed transesterification of vegetable oil to biodiesel as well. This lipase catalyzed esterification of organic acid and alcohol, glycerol and fatty acid, sugar and fatty acid resulting in organic ester, monoacylglycerol and sugar and fatty acid ester, respectively; outperformed Candia rugosa and Rhizopus niveus lipase. Porcine pancreas lipase manifested catalytic promiscuity by conversion of 4-hydroxycoumarin and aromatic aldehyde to Bis-4-hydroxycoumarin through Knoevenagel condensation; of aromatic or heteroaromatic aldehyde and ketone to aldol through aldol condensation. It outweighed lipases from Aspergillus niger, Pesudomonas fluorescens and Candia rugosa during Michael addition between 4-hydroxycoumarin and α, β-unsaturated enones to warfarin. It outperformed Mucor javanicus lipase during cascade reaction involving aldehyde and indole to Bis(indolyl)alkanes; and Candida cylindracea lipase during enantioselective acylation between amino alcohol and acetate ester to amide and amido-ester. This lipase also catalyzed multicomponent synthesis leading to Pyrido[2,3-d]pyrimidine derivatives from aromatic aldehyde, malononitrile and 6-amino-1,3-dimethyl uracil. This lipase has potential application value as it could catalyze these reactions under milder conditions.

Menaquinone-7 (MK-7), a highly preferred form of vitamin K2, has garnered extensive attention due to its long half-life in the human body and numerous nutritional and pharmacological benefits. To establish a cost-effective microbial platform for MK-7 production, we engineered the eukaryotic model organism Saccharomyces cerevisiae. The complete biosynthesis pathway was modularized into three parts: the endogenous mevalonate (MVA) pathway, the endogenous shikimate (SA) pathway, and a heterologous menaquinone (MK) module. Eleven key genes from the MK module were introduced into the yeast chassis using selection markers for His, Leu, and Met, creating the engineered strain YK-1. We then optimized the fermentation medium and conditions, and the MK-7 titer reached 0.413 mg/L. This represents a 31.1% increase over the non-optimized yield, demonstrating the successful de novo synthesis of MK-7 from simple carbon sources in a eukaryotic cell factory. This work not only expands the repertoire of chassis cells for MK-7 production but also provides a valuable framework for engineering biosynthesis pathways for other isoprenoids.

The conversion of brown macroalgae like Sargassum offers a secure route for biofuel production from an abundant and underutilized biomass resource; however, efficient utilization of this feedstock remains a major challenge. This study explores the feasibility of converting unwashed and minimally processed Sargassum biomass to microbial lipids with the oleaginous yeast Rhodosporidium toruloides. Acid, alkaline, and hydrothermal pretreatments with enzymatic hydrolysis using cellulase, hemicellulase, and alginate lyase enzyme cocktails were evaluated to assess their ability to deconstruct Sargassum natans. The resulting hydrolysates were tested for the ability to support R. toruloides growth and lipid accumulation. We observed that alkaline-pretreated hydrolysates yielded higher cell growth and produced 0.08 mg of lipids per mg of dry cell weight. Lipid characterization revealed that the strain produced lipids with fatty acids predominantly comprising C16:0, C18:1, and C18:2 species, comparable to those found in vegetable oils suitable for biodiesel applications. Overall, this work establishes a basis for coupling macroalgal deconstruction with yeast bioconversion and highlights S. natans as a promising substrate for the production of fuels and chemicals.

For biohydrogen production, immobilized systems offer the potential for improved growth and productivity. In this study, microfiber-flocked surfaces were used as a novel strategy for Rhodobacter capsulatus biofilm-based photofermentative biohydrogen production. The surfaces were developed using electrostatic flocking by attaching polyamide microfibers to glass and poly methyl methacrylate (PMMA) surfaces. Parameters such as fiber length, density, and adhesive type were optimized to develop shorter, denser, and vertically oriented microfiber structures. This optimized architecture increased the available surface area for bacterial attachment and biofilm formation while maintaining sufficient light transmittance through the flocked layer. The combination of enhanced biofilm development and adequate light availability supported photosynthetic activity, resulting in enhanced cell growth and hydrogen production. For anaerobic cultures, inclined (vertical) photobioreactor provided superior attachment compared to horizontal setups, presumably due to better light exposure and nutrient availability. These enhanced surfaces led to increased cumulative hydrogen production over 21 days during repeated-batch operations with Rhodobacter capsulatus in a novel photobioreactor. This resulted in a stable biofilm (Abs590: 0.65 ± 0.04) and a hydrogen productivity of 0.32 ± 0.05 mmol/L.h. Thick biofilms formed on the microfiber flocks, exhibiting interconnected extracellular matrices and filamentous extracellular structures. In contrast, horizontally oriented surfaces and aerobic conditions were less effective, as sub-optimal light and oxygen atmosphere levels appear to hinder biofilm growth and hydrogen output. Additionally, a combined ultrasonication and Tween surfactant treatment enabled efficient cell removal and biomass recovery, suggesting reusability of the engineered surfaces. These results highlight microfiber-flocked surfaces as a potential platform for enhanced microbial growth and biohydrogen production. The proposed strategy contributes to the development of biofilm-based continuous processes.

By 2050, surging global population will intensify food insecurity, making it imperative to develop innovative strategies that boost plant productivity. Plants are our core food supply. Engineered nanoparticles have emerged as promising growth stimulants; however, carbon nanotubes (CNTs), despite their efficacy, are inedible and pose eco-toxicity concerns that restrict their agrobiological deployment. Furthermore, conventional whole-tissue studies obscure individual cell responses, preventing precise characterization of nanoparticle-cell interactions. Here, we introduce magnesium-aluminium layered double hydroxide (MgAl-LDH) nanoparticles, which is a food-grade material derived from commercial antacid formulations, that outperformed CNTs in accelerating plant cell development. This enhancement is suggested to be associated with the presence of magnesium ions and the positively charged LDH surface architecture, although the detailed molecular mechanisms remain to be fully elucidated.

Antimicrobial resistance (AMR), mainly caused by the mutation of antibiotic resistance genes, poses a great threat to human health. Clinically, TEM-1 β-lactamase encoding blaTEM-1 gene is the most frequently occurred antibiotic resistance gene. Identification, characterization, and monitor of high antibiotic resistance blaTEM-1 mutants are crucial to AMR research and would provide the guidance for the development of next-generation antibiotics. To this end, we report here the adaption of the continuous directed evolution (CDE) along with base editor (BE) gene editing strategy to discover TEM-1 mutants. Firstly, TEM-1 mutants were identified in single-copy bacterial artificial chromosome vector via BE-mediated CDE. Then each mutant genotype was verified in high copy number pUC plasmid. Finally, combinatorial antibiotic resistance was observed when the TEM-1 phenotypes generated in CDE were combined with that of the reported TEM-1 variants. The Escherichia coli strain harboring the vector cloned with 10 TEM-1 mutations showed a minimum inhibitory concentration of 1280 μg/mL to antibiotic cefotaxime, which is the highest data reported thus far. The research highlights the application of gene editing methods to provide insight into the molecular basis of antibiotic resistance.

Proliferative enteropathy is a gastrointestinal disease caused by the intracellular bacterium Lawsonia intracellularis, affecting swine populations worldwide and leading to significant economic losses due to reduced growth performance, increased mortality, and treatment expenses. Current vaccines present limitations related to production processes, stability, and overall cost. This study aimed to design and evaluat chimeric proteins containing immunogenic epitopes of L. intracellularis. A recombinant chimera, designated Li_full, was rationally designed in silico by integrating predicted B-cell and MHC epitopes relevant to swine immunity. To further investigate the immunogenic potential of distinct regions, Li_full was subdivided into three fractions (Li_1, Li_2, and Li_3). All constructs were cloned into an Escherichia coli expression system, allowing efficient production and biochemical characterization of the recombinant proteins. Antigenicity was confirmed by strong reactivity with sera from naturally infected pigs. Immunogenicity was assessed through vaccination in mice, which developed significantly (p < 0.05) elevated antibody titers, demonstrating the ability of the chimeras to elicit robust humoral immune responses. The integration of bioinformatics-driven epitope prediction with recombinant DNA technology represents a promising and innovative strategy for controlling proliferative enteropathy. This study underscores the value of in silico design strategies for the development of targeted veterinary immunogens and provides a foundation for further research aimed at evaluating protective efficacy under field conditions.

Squalene, a naturally occurring triterpenoid, is widely utilized in the food, pharmaceutical, and vaccine adjuvant industries. Conventional production methods, which rely on extraction from shark liver oil or plant sources, are limited by ecological sustainability concerns and low extraction efficiency. Recent progress in synthetic biology and metabolic engineering has enabled the development of sustainable and efficient microbial platforms for squalene biosynthesis. This review provides a comprehensive overview of the biosynthetic pathways of squalene in yeast, summarizes key metabolic engineering strategies, and highlights recent advancements in the field, with an emphasis on multi-organelle synergistic engineering and critical distinction of evidence sources. We focus on approaches such as enhancing the mevalonate pathway, modulating competing metabolic routes, applying subcellular compartmentalization engineering, optimizing cofactor balance, and implementing product efflux strategies. Notably, integrated strategies, particularly organelle engineering in Yarrowia lipolytica and Saccharomyces cerevisiae, have increased squalene titers to high levels (up to 55.8 g/L). These advances provide a useful basis for future metabolic engineering efforts aimed at improving squalene production and facilitating the synthesis of related terpenoids.