Engineering in Life Sciences最新文献

Ferulic acid is a high-value chemical synthesized in plants. The ferulic acid biosynthesis is still affected by the insufficient methylation activity of caffeic acid O-methyltransferase (COMT). In this study, we engineered COMT from Arabidopsis thaliana to match caffeic acid, and the mutant COMT^{N129V-H313A-F174L} showed 4.19-fold enhanced catalytic efficiency for degrading caffeic acid. Then, we constructed the de novo synthesis pathway of ferulic acid by introducing tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), 4-hydroxyphenylacetate 3-hydroxylase from Escherichia coli (EcHpaBC), and mutant COMT^{N129V-H313A-F174L}, and further increased tyrosine synthesis. Furthermore, we overexpressed two copies of COMT^{N129V-H313A-F174L} and enhanced the supply of S-adenosyl-L-methionine (SAM) by expressed S-ribosylhomocysteine lyase (luxS) and 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (mtn) to increase the production of ferulic acid. Finally, the production of ferulic acid reached 1260.37 mg/L in the shake-flask fermentation and 4377 mg/L using a 50 L bioreactor by the engineered FA-11. In conclusion, COMT enzyme engineering combined with global metabolic engineering effectively improved the production of ferulic acid and successfully obtained a fairly high level of ferulic acid production.

Trypanosoma brucei phospholipase A₂ (TbPLA₂) is a validated drug target but the difficulty in expressing its soluble recombinant protein has limited its exploitation for drug and vaccine development for African and American trypanosomiases. We utilized recombinant deoxyribonucleic acid (DNA) technology approaches to express soluble TbPLA₂ in Escherichia coli and Pichia pastoris and biochemically characterize the purified enzyme. Full-length TbPLA₂ was insoluble and deposited as inclusion bodies when expressed in E. coli. However, soluble and active forms were obtained when both the full-length and truncated TbPLA₂ were expressed in fusion with N-terminal FLAG tag and C-terminal eGFP in P. pastoris, and the truncated protein in fusion with N-terminal FLAG tag and C-terminal mClover in E. coli. Truncated TbPLA₂ lacking the signal peptide and transmembrane domain was finally expressed in Rosetta 2 cells and purified to homogeneity. Its migration on sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed its size to be 39 kDa. Kinetic studies revealed that the enzyme has a specific activity of 107.14 µmol/min/mg, a V_max of 25.1 µmol/min, and a K_M of 1.58 mM. This is the first report on the successful expression of soluble and active recombinant TbPLA₂, which will facilitate the discovery of its specific inhibitors for the development of therapeutics for trypanosomiasis.

Deep eutectic solvents (DESs) hold the potential to serve as a sustainable and environmentally friendly substitute for supercritical fluids, ionic liquids, and organic solvents. Moreover, DESs have been demonstrated to assist in stabilizing the structure of enzyme. The enzymatic synthesis of epoxy vegetable oil in a DES-system was developed in this study, and the influence of DESs viscosity on the epoxidation system was investigated for the first time. The results demonstrated that the epoxy value reached 8.97, and the double bond conversion rate was 82.48%. The viscosity of the reaction system decreased from 209.32 to 91.35 (mPa·s). The application of DES in epoxidation was confirmed through structural characterization, indicating that eutectic solvents could serve as substitutes for toxic and volatile organic solvents in synthesizing high-epoxide vegetable oils using an enzymatic method, thus facilitating the production of environmentally friendly plasticizers.

Penicillium sp. (IBWF 040-09) produces a protease inhibitor that can potentially be used against the main protease of human African trypanosomiasis. Since the target substance is formed intracellularly (under nutrient limitation), the fungal pellet is preferred compared to the free mycelia in bioreactor cultivation. The optimization of the production of protease inhibitor became the main focus of this study. The effects of the concentrations of spores, calcium chloride, and Pluronic F68 were investigated with regard to fungal growth, pellet morphology, and the production of protease inhibitor. The combination of adjusting the spore concentration and adding Pluronic F68 and calcium chloride increased the probability of achieving the desired morphology. This ensured better reproducibility of the production of the target substance by Penicillium sp. (IBWF 040-09) with the bioreactor system used. In addition, the protease inhibitor was tested in a resazurin assay and showed no noticeable cytotoxic effects on peripheral blood mononuclear cells isolated from whole blood cells.

The epidermal growth factor (EGF) receptor is commonly targeted in cancer therapy because it is overexpressed in many malignant cells. However, a general problem is to couple the targeting moieties and the drug molecules in a way that results in a homogeneous product. Here, we overcome this issue by engineering a variant of EGF with a single conjugation site for coupling virtually any payload. The recombinant EGF variant K-EGF^RR was expressed in E. coli Rosetta with a 4–6 mg/L yield. To confirm the accessibility of the introduced functional group, the ligand was equipped with a sulfo-cyanine dye with a loading of 0.65 dye per ligand, which enables tracking in vitro. The kinetics and affinity of ligand–receptor interaction were evaluated by enzyme-linked immunosorbent assay and surface plasmon resonance. The affinity of K-EGF^RR was slightly higher when compared to the wild-type EGF (K_D: 5.9 vs. 7.3 nM). Moreover, the ligand–receptor interaction and uptake in a cellular context were evaluated by flow cytometry and quantitative high-content imaging. Importantly, by attaching heterobifunctional polyethylene glycol linkers, we allowed orthogonal click-conjugation of the ligand to any payload of choice, making K-EGF^RR an ideal candidate for targeted drug delivery.

Industrial biocatalysis, a multibillion dollar industry, relies on the selectivity and efficacy of enzymes for efficient chemical transformations. However, enzymes, evolutionary adapted to mild biological conditions, often struggle in industrial processes that require harsh reaction conditions, resulting in reduced stability and activity. Enzyme immobilization, which addresses challenges such as enzyme reuse and stability, has therefore become a vital strategy for improving enzyme use in industrial applications. Traditional immobilization techniques rely on the confinement or display of enzymes within/on organic or inorganic supports, while recent advances in synthetic biology have led to the development of solely biological in vivo immobilization methods that streamline enzyme production and immobilization. These methods offer added benefits in terms of sustainability and cost efficiency. In addition, the development and use of multifunctional materials, such as magnetic (nano)materials for enzyme immobilization, has enabled improved separation and purification processes. The combination of both “worlds,” opens up new avenues in both (industrial) biocatalysis, fundamental science, and biomedicine. Therefore, in this review, we provide an overview of established and recently emerging methods for the generation of magnetic protein immobilizates, placing a special focus on in vivo immobilization solutions.

The industrially attractive biopolymer poly-γ-glutamic acid (γ-PGA) is commonly produced by species of the genus Bacillus by co-feeding different carbon- and nitrogen-sources. Recent studies have highlighted the pivotal role of co-metabolization of a rapidly degradable carbon source such as glycerol together with citrate for γ-PGA production, independently fueling biomass generation as well as tricarboxylic acid (TCA) cycle precursor supply. With this study, we report that the sole presence of citrate in the production medium greatly influences growth behavior, γ-PGA production, and the viscosity of microbial cultures during biopolymer synthesis. Independent of the citrate concentration in the medium, only minor amounts of citrate were imported by B. subtilis 168 in the presence of glycerol due to carbon catabolite repression. However, a high citrate concentration resulted in a 6-fold increase in γ-PGA titer compared to low exogenous citrate levels. Data suggests that citrate was not used as a precursor in γ-PGA synthesis but rather influenced the fate of imported glutamate. The citrate concentration also affected medium viscosity as depletion resulted in a remarkable spike in culture broth viscosity. Additionally, cellular proteome analysis at different levels of citrate availability revealed significant changes in protein abundance involved in motility and fatty acid degradation.

Practical Application: This research provides critical insights into optimizing γ-PGA production in Bacillus subtilis, particularly by using citrate supplementation to control medium viscosity and improve production yields. The study reveals that citrate not only plays a role in controlling viscosity but also influences intracellular glutamate metabolism, a key factor for γ-PGA synthesis. Citrate interacts with divalent cations such as Mg²⁺ and Ca²⁺, reducing electrostatic interactions and thus decreasing viscosity in the medium. Additionally, while citrate uptake is limited due to carbon catabolite repression (CCR), even the minimal presence of citrate impacts growth and production. The findings suggest that citrate may trigger unexplored regulatory mechanisms affecting glutamate utilization. Their understanding opens new avenues for industrial optimization, which focus on enhancing glutamate synthesis pathways and exploring novel citrate-sensing mechanisms. Overall, this research lays the groundwork for improving the efficiency and consistency of γ-PGA production by fine-tuning media components and understanding their metabolic effects.

This study focuses on applying microbial self-healing cement in repairing cracks in cement-based materials and enhancing its resistance to water penetration performance. Traditional cement is susceptible to environmental influences, leading to the formation of microcracks and a reduction in durability. This research used Bacillus pseudofirmus to prepare microcapsules through sodium alginate gelation technology. We mixed microcapsules into the cement. The results indicate that the microbial self-healing cement, with a 1% self-healing agent added, increased its resistance to water penetration ability by 29.2% after 28 days. This improvement rose to 39.3% after 84 days. Additionally, we used the embedded needle method to make mortar blocks through microcracks, mimicking the cracks found in real cement. The self-healing effect of the microcapsules was especially noticeable for cracks under 0.3 mm in diameter, compared to the commonly used commercial crystallization penetration technology. This is attributed to the crystalline bodies formed by the self-healing agent in the microcapsules blocking the cracks and preventing water penetration. This study provides an environmentally friendly solution for the repair of cracks in cement-based materials using microbial self-healing technology and lays the foundation for improving the repair efficiency and durability and exploring stability and reliability in the future.

Practical Application: This study investigated the application of microbial self-healing cement in repairing cracks in cement-based materials and enhancing its resistance to water penetration properties. Cement, a material widely used in infrastructure, has low tensile strength and often forms microcracks. These microcracks reducing the durability of cement and posing risks to the economy and safety. Adding 1% self-healing agent to microbial self-healing cement significantly increases the resistance to water penetration pressure of the mortar blocks. Compared to the standard specimens, the resistance to water penetration ability increased by 29.2% at 28 days and further increased to 39.3% at 84 days. Microbial self-healing cement could effectively restore the resistance to water penetration performance of the mortar blocks after repairing cracks. The repairing results are significantly better than the methods of mixing or applying cement crystalline materials.