Journal of Biological Engineering最新文献

Background: Tuberculosis, a persistent global health threat, necessitates a comprehensive understanding of the genes and pathways crucial for the survival and virulence of the causative pathogen, Mycobacterium tuberculosis. Working with M. tuberculosis (M.tb) presents significant challenges; therefore, the use of M. smegmatis as a surrogate system for conducting genetic studies of M.tb has proven to be highly valuable. Development of novel genetic tools to probe cellular processes accelerates the progress in the field of drug development and also helps in understanding the basic physiology of the bacterium.

Results: This study reports the successful implementation and evaluation of the CRISPR-Cas12a system for gene repression in Mycobacterium smegmatis, a surrogate for M. tuberculosis. We engineered a Cas12a-based CRISPR interference (CRISPRi) system and assessed its functionality. Targeting 45 genes with a single sgRNA per gene, we achieved efficient gene repression, leading to marked phenotypic changes. Each knockdown strain was evaluated individually for growth phenotypes, and a comparison of the results with the reported essential gene library probed with dCas9 demonstrated congruous results across diverse gene categories. The study shows that CRISPR/Cas12a system can be effectively utilised with a single gene specific target for efficient silencing of the gene and highlights the importance of subsequent growth assays required to evaluate the vulnerability of targeted gene silencing.

Conclusion: Our findings reveal the robustness and versatility of the dCas12a-CRISPRi system in M. smegmatis, providing a valuable tool for functional genomics research. This work showcases the potential of the dCas12a-CRISPRi system in investigating essential genes, enabling a deeper understanding of the biology and potential therapeutic targets in mycobacterium species.

Background: A transgenic strain of Escherichia coli has been engineered to directly assimilate gaseous CO₂ into its biomass through hydrogen-powered anaerobic respiration. This was achieved by expressing key components of the reverse tricarboxylic acid (rTCA) cycle, including genes encoding α-ketoglutarate: ferredoxin oxidoreductase (KOR) and ATP-dependent citrate lyase (ACL) from Chlorobium tepidum. These enzymes were selected for their essential roles in enabling CO₂ fixation and integration into central metabolism.

Results: This study found that KOR alone can support cellular maintenance under chemolithotrophic conditions, while additional expression of ACL enhances CO₂ assimilation. Using isotopic ¹³CO₂ tracing, it was demonstrated that KOR alone facilitates CO₂ assimilation into TCA metabolites. However, co-expression of ACL with KOR redirected carbon fluxes from TCA cycle toward essential metabolic pathways, particularly those involved in protein and nucleotide biosynthesis. Compared to KOR alone, ACL co-expression significantly increased isotopic enrichments in amino acids (e.g., methionine, threonine, glycine) and nucleotides (e.g., deoxythymidine, deoxycytidine). These results suggest that ACL supports the synthesis of nitrogen-containing metabolites when inorganic nitrogen is sufficient, while KOR alone sustains core metabolic functions under chemolithotrophic conditions.

Conclusions: This study demonstrates a novel strategy to engineer E. coli for CO₂ fixation using only one or two heterologous enzymes under chemolithotrophic conditions. These findings reveal the minimal genetic and nutritional requirements for CO₂ assimilation and provide insights into metabolic flux partitioning in engineered strains. This research paves the way for sustainable applications in carbon fixation and biotechnological innovation.

Background: Bioconversion of methanol derived from CO₂ reduction into value-added chemicals provides a unique approach for mitigating global warming and reducing fossil fuels dependence. Production of 3-hydroxypropionic acid (3-HP), a key building block for the development of biobased products such as acrylates and 1,3-propanediol, has been successfully achieved using methanol as the sole carbon and energy source in the methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris). However, challenges remain in meeting commercially relevant concentrations, yields and productivities of 3-HP, prompting further strain optimization. In the present study, we have combined metabolic engineering strategies aiming at increasing metabolic precursors supply and redirecting carbon flux towards 3-HP production.

Results: A combinatorial metabolic engineering strategy targeting precursors supply and 3-HP export was applied to the original 3-HP producing K. phaffii strain harboring the synthetic β-alanine pathway and a mutated NADP-dependent formate dehydrogenase from Pseudomonas sp. 101 (PseFDH(V9)). To do so, several genes encoding enzymes catalyzing reactions immediately upstream of the β-alanine pathway were overexpressed to enhance precursors availability. However, only the overexpression of the pyruvate carboxylase PYC2 gene significantly increased the 3-HP yield on biomass (Y_P/X) in small-scale cultivations. Co-overexpression of PYC2 and the lactate permeases ESBP6 and JEN1 genes led to a 55% improvement in 3-HP titer and product yield in methanol deep-well plate cultures compared to the reference strain, mostly due to Esbp6 activity, proving its effectiveness as a 3-HP transporter. Deletion of the native formate dehydrogenase gene FDH1 did not increase methanol flux entering the assimilatory pathway. Instead, knockout strains showed severe growth defects due to toxic intermediates accumulation. Co-expression of the PseFDH(V9) encoding gene in these strains failed to compensate for the loss of the native FDH. The strain combining PYC2, ESBP6, and JEN1 overexpression was further tested in fed-batch cultures at pH 5, achieving a 3-HP concentration of 27.0 g l^- 1, with a product yield of 0.19 g g^- 1, and a volumetric productivity of 0.56 g l^- 1 h^- 1 for the methanol feeding phase of the cultivations. These results represent a 42% increase in final concentration and over 20% improvement in volumetric productivity compared to the original 3-HP-producing strain. Furthermore, bioreactor-scale cultivations at pH 3.5 revealed increased robustness of the strains overexpressing monocarboxylate transporters.

Conclusions: Our results point out the potential of lactate transporters to efficiently drive 3-HP export in K. phaffii, leading to higher titers, yields, and productivities, even at lower pH conditions.

Rickettsia is an intracellular bacteria transmitted to humans through ticks, lice, fleas, or their feces, causing acute symptoms such as fever, headache, rashes, and muscle aches. Detecting rickettsial diseases is challenging due to limitations in current methods such as negative results, low sensitivity, and high cost. These limitations highlight the need for improved detection methods. Dielectrophoresis (DEP) offers a promising alternative to develop a point-of-care economical, label-free, and sensitive diagnostic tool. By exposing cells to non-uniform electric fields one can measure the electrical properties of the cells which are different and unique based on the cell type. By comparing the dielectric profiles of healthy and infected cells, DEP could be utilized to design a rapid, cost-effective diagnostic tool. Initial steps involve characterizing the electrophysiological properties of Vero cells infected with Rickettsia montanensis to develop this new detection tool. This study found significant differences in electrical parameters between healthy and Rickettsia spp. infected Vero cells, particularly at a medium conductivity of 500 µS/cm. Moreover, we found that the dielectric spectrum showed the greatest differences between healthy and Rickettsia spp. infected Vero cells at medium conductivity of 500 µS/cm, with significantly different dielectrophoretic crossover frequencies (no DEP force region). These findings suggest that dielectrophoretic detection of infected cells could serve as a quick, cost-effective, label-free, and sensitive alternative for developing a point-of-care diagnostic tool for Rickettsial infections.

The need for a sensitive, selective, non-invasive and reversible fluorescent sensor for Zn²⁺ monitoring is addressed in this work. A novel guest-host system is developed, including a Zn²⁺ sensitive fluorescent probe, Zinpyr-1, embedded in a porous optically transparent hybrid film. The entrapped probe molecules are accessible and can interact with the external analyte. The immobilized Zinpyr-1 confirms its specificity and selectivity for Zn²⁺, as shown by sensing tests conducted in buffer solutions that mimic the ionic composition of biological media. The uniqueness of the developed sensor system lies in its reversibility, combined with a fast and selective response, allowing dynamic measurements of zinc concentrations in the 1 µM to 1 mM range within few tens of seconds. Unlike most Zn²⁺ sensors, this system is a film-based sensor, making it an interesting minimally invasive tool for future studies on how live cells cultured on it dynamically regulate the Zn²⁺ concentration under controlled physiological conditions.

The fabrication of scaffolds for bone tissue engineering (BTE) applications often involves the utilization of two distinct categories of biomaterials, namely calcium phosphates and calcium silicates. The selection of these materials is based on their biocompatibility, bioactivity, and mechanical characteristics that closely resemble those of natural bone. The present research examined the utilization of hydroxyapatite (HAP) and tri-calcium silicate (TCS), which are among the most commonly utilized materials in calcium phosphates and calcium silicates, in the context of bone scaffolding applications. A molecular dynamics simulation was conducted to investigate the impact of different concentrations of ceramic nanoparticles, when combined with sodium alginate (SA) hydrogel, on the fabrication of bone scaffolds.The stability and self-assembly were assessed through several parameters, such as the solvent-accessible surface area (SASA), radius of gyration (Rg), radial distribution function (g(r)), root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), hydrogen bonding, van der Waals, electrostatic, and total energies. The findings indicate that the addition of 10 wt% HAP and TCS to the SA hydrogel matrix results in a more compact, stable, and potentially less hydrated structure. Accordingly, the experimental validation of these simulation approved our in silico findings. Experimental rheology and mechanical properties evaluation validate our simulation results, indicating a superior characteristic of TCS10 and HAP10 inks and 3D-printed scaffolds among other composition ratios. This could potentially benefit the in vitro and in vivo performance of the scaffold and its interaction with cells. The aforementioned traits are considered fundamental for the successful execution of the scaffold in the field of BTE. The findings indicate that TCS samples exhibit superior properties when compared to HAP samples, specifically in terms of composition with SA hydrogel.

Skin wounds have the potential to rapidly become infected, with bacteria having the ability to quickly penetrate to the skin's deeper layers. Then they enter the lymph nodes and spread throughout the body; therefore, all wounds should be cleaned and have a permanent cover. Modern wound dressings with effective antibacterial and therapeutic properties are required to create a sterile environment for the acceleration of healing. The aim of this work was to prepare zein electrospun nanofibers containing Scrophularia striata extract for wound healing promotion. Electrospun nanofibers made of zein, a natural polymer, have attracted a lot of attention due to their biocompatibility and biodegradability. The prepared nanofibers were characterized by scanning electron microscopy (SEM), energy dispersive X‑ray analysis (EDX), water contact angle test, and Fourier transform infrared spectroscopy (FT-IR). The parameters affected by the electrospinning process were investigated and optimized. The results revealed that the zein nanofibers (25% w/v, zein) containing Scrophularia striata extract (6.7% w/v) had a smooth and bead-free morphology with improved surface hydrophilicity. The measurement of water contact angle confirmed that nanofibers containing extract showed higher wettability (64.9°) compared to fibers without extract (119.8), so the proposed mat adequately moisturizes the wound environment. The antimicrobial studies show that Scrophularia striata extract incorporated nanofibers has the ability to inhibit the growth of both gram-negative and gram-positive bacteria. The biophenols release profile indicated that nanofibrous mat can release more effective substances to promote wound healing. The biocompatibility and biodegradability of nanofibrous scaffold containing Scrophularia striata extract tested in in vivo and in vitro conditions show a significantly higher survival rate of fibroblast cells. In addition, macroscopic and histological observations confirmed that the implanted nanofibers containing the extract did not exhibit any signs of inflammation or redness after a month when inserted beneath the skin of mice surrounded by vessels containing epidermis.

Background: Molecular and synthetic biology tools enable the design of new-to-nature biological systems, including genetically engineered microorganisms, recombinant proteins, and novel metabolic pathways. These tools simplify the development of more efficient, manageable, and tailored solutions for specific applications, biocatalysts, or biosensors that are devoid of undesirable characteristics. The key aspect of preparing these biological systems is the availability of appropriate strategies for designing novel genetic circuits. However, there remains a pressing need to explore independent and controllable systems for the co-expression of multiple genes.

Results: In this study, we present the characterisation of a set of bacterial plasmids dedicated to recombinant expression in broadly used Escherichia coli. The set includes plasmids with four different, most commonly used bacterial expression cassettes - RhaS/RhaBAD, LacI/Trc, AraC/AraBAD, and XylS/Pm, which can be used alone or freely combined in up to three-gene monocistronic expression systems using Golden Standard Molecular Cloning kit assembly. The independent induction of each of the designed cassettes enables the autonomous expression of up to three recombinant proteins from one plasmid. The expression of a triple-enzyme cascade consisting of sucrose synthase, UDP-rhamnose synthase and flavonol-7-O-rhamnosyltransferase, confirmed that the designed system can be applied for the complex biocatalysts production.

Conclusions: Presented herein strategy for the multigene expression is a valuable addition to the current landscape of different co-expression approaches. The thorough characterisation of each expression cassette indicated their strengths and potential limitations, which will be useful for subsequent investigations in the field. The defined cross-talks brought a better understanding of the metabolic mechanisms that may affect the heterologous expression in the bacterial hosts.

A nanozyme-based aptasensor combines the unique properties of nanozymes with the specificity of aptamers for the detection of various biomolecules. Nanozymes are nanomaterials that possess enzyme-like properties, demonstrating substantial potential for enhancing the sensing capabilities of biosensors. In recent years, the incorporation of nanozymes into biosensors has opened new avenues for the detection of tumor biomarkers. The unique attributes of nanozymes and aptamers lead to biosensors characterized by high sensitivity, specificity, reproducibility and accuracy in analytical performance. This article reviews the research progress of nanozyme-based aptasensors in tumor biomarker detection over the past decade. We categorize these sensors based on their sensing modes and target types, and examine the properties and applications of the nanozymes employed in these devices, providing a thorough discussion of the strengths and weaknesses associated with each sensor type. Finally, the review highlights the strengths and challenges associated with nanozyme-based biosensors and envisions future developments and applications in this field. The objective is to provide insights for improving biosensor performance in tumor biomarker detection, thereby contributing to advancements in precision cancer diagnosis and treatment.

The rise of antibiotic resistance has made bacterial infections a persistent global health issue. In particular, extracellular polymeric substances (EPS) secreted by bacteria limit the effectiveness of conventional antibiotics, making biofilm removal challenging. To address this, we created ND@PDA nanoparticles by coating the surface of nanodiamonds (ND) with polydopamine (PDA). These nanoparticles were then integrated into polyvinyl alcohol to fabricate PVA/ND@PDA nanofiber scaffolds, resulting in an innovative platform with enhanced photothermal, antibacterial and antibiofilm properties. Upon exposure to near-infrared (NIR) light, the scaffolds exhibited a significant photothermal activity, oxidative stress and effectively damaging key bacterial components, such as biofilm, bacterial membranes, and proteins. Additionally, the catechol groups in PDA provided strong cell adhesion and high biocompatibility on the nanofiber surface. Our research proposes a platform that not only effectively addresses antibiotic-resistant infections but also contributes to advancements in wound healing therapies by enabling controlled antibacterial action with minimal toxicity.