Biotechnology and Bioengineering最新文献

A series of Escherichia coli streamlined strains was developed by removing the expression of genes encoding extracellular structures and unessential enzymes. The streamlined strains exhibited improved metabolic performance, including lower overflow metabolism and ATP maintenance coefficient, as well as a higher growth rate, compared to their parental strain. The intracellular levels of ATP were monitored using a genetic sensor, showing the improved resource stewardship of the streamlined cells. The streamlined strains were tested as cell factories to produce plasmid DNA (pDNA) in batch cultures, exhibiting a 23% increase in the specific pDNA production rate, compared to the parental strain. Recombinant protein expression was evaluated in microbioreactors in batch and fed-batch modes. In batch mode, recombinant protein yield from biomass was up to 82% higher in the streamlined strains than in the parental strain. Furthermore, in fed-batch mode, the recombinant protein yield was 79% greater in the streamlined cells compared to the parental strain. Our results show the benefits of reducing cellular complexity on the biomanufacturing of pDNA and recombinant proteins in culture schemes typical of industrial settings.

Amid growing concerns about climate change, environmental pollution, and fossil resource depletion, bio-based chemicals and materials have garnered significant attention. Succinic acid (SA), a four-carbon dicarboxylic acid, is a key platform chemical for producing industrially important compounds such as poly(butylene succinate) (PBS), a biodegradable polymer. In this paper, we demonstrate the enhanced production of SA using metabolically engineered Mannheimia succiniciproducens PALK (pMS3-gltA) strain, which achieves high SA yields through enhanced biomass production and optimized metabolic pathways. High-purity SA, purified from the fermentation broth, was used in an environmentally friendly two-step polymerization process under mild conditions. In the first step, diethyl succinate was synthesized from SA and ethanol using Amberlyst as a catalyst. This was followed by polymerization with 1,4-butanediol, catalyzed by Candida antarctica lipase B. The resulting PBS exhibited comparable thermal stability and molecular weight distribution to its petrochemical counterpart, with enhanced biodegradability. By integrating metabolic engineering with sustainable polymer synthesis, this study underscores the potential for an eco-friendly approach to developing bio-based polymers, offering a promising solution to the environmental challenges posed by conventional plastics.

There is growing interest in newly isolated lactic acid bacteria from natural sources and traditional food manufacturing and transformation. Streptococcus thermophilus is able to improve the safety of dairy products, showing antioxidant and antimicrobial properties and also to produce bioactive molecules, such as exopolysaccharides (EPSs), usable in foods applications. An integrated bioprocess development was performed using S.thermophilus D4, for the production of viable biomass and EPSs using a media optimized through design of experiments. Plackett–Burman screening method was used twice to optimize biomass and exopolysaccharide production. Different fermentation strategies (batch and fed-batch) were performed to increase yield and productivity. Fed-batch processes increased biomass production approximately 2.5- and 7.5-fold than batch processes with optimized and M17 media, respectively. Furthermore, a significant increase of 1.7- and 6-fold EPS biosynthesis was observed. Finally downstream processes based on membranes were conducted to purify bioactive molecules (EPSs) used on Caco-2 cells showing a significant effect in accelerating wound healing. Finally, purification and recovery of EPSs tested on Caco-2 cells. The results obtained confirm the potential of newly defined medium to replace M17, which has animal-derived nitrogen sources, for probiotic biomass and EPSs production with many applications in different fields.

This paper proposes the controlled release of adeno-associated virus (AAV) by polymer mesh structures of alginate hydrogel microbeads (gel-beads). The polymer mesh structure of alginate hydrogel can be controlled by adjusting the number of cross-linking points depending on the molecular structure (M/G ratio) of the alginate hydrogel. Scanning electron microscopic analysis on the mesh structures of alginate hydrogels with M/G ratios of 0.96 (MG-0.96) and 0.42 (MG-0.42) indicates that the mesh size distribution of MG-0.42 hydrogel was smaller than that of MG-0.96 hydrogel. Using these characteristics, controlled release of AAVs (AAV-1 or AAV-5) from M/G-ratio-tuned gel-beads (MG-0.96 or MG-0.42), Janus-shaped gel-beads consisting of hemispheres of the MG-0.96 and MG-0.42, and MG-0.64 gel-beads consisting of a mixture of MG-0.96 and MG-0.42 were examined with ELISA for AAV capsids and the GFP fluorescent signals from the transfected cells. The precise control of AAV release by the mesh structure of the alginate hydrogels could be an effective method for creating AAV micro-carriers for gene therapy.

Advancing immunosensing technologies hinges on the development of next-generation surface functionalization methods, as the precise anchoring of antibodies on transducer interface is essential for achieving high sensitivity and selectivity. Among the diverse methodologies explored, bioengineered materials have shown significant potential to improve antibody orientation, stability, and functional performance. In this study, we present a chimeric protein created by fusing the adhesive Class I hydrophobin Vmh2 from Pleurotus ostreatus, with the Fc-binding region of protein A from Staphylococcus aureus (SpA). This fusion protein spontaneously adheres on gold nanoparticles (AuNPs) without requiring chemical modification, forming a robust bio-interactive layer for antibody attachment. The platform's adaptability and effectiveness were assessed using an immunoglobulin specific to a fungal laccase to establish the performance of the system, and antibodies against two clinically significant targets- mesothelin, a tumor-associated glycoprotein, and the SARS-CoV-2 spike protein- to showcase the diagnostic potential of the system. A two-step method based on the induced aggregation of the AuNps not bound to the analyte allows underscoring the platform's promise in biosensing applications. Overall, this approach represents a sustainable, versatile, and low-cost route for fabricating biologically active surfaces, with wide-ranging relevance in medical diagnostics, environmental analysis, and biotechnological innovation.

Amylases are key industrial enzymes, and thermostable variants are particularly valuable for robust bioprocessing. This study investigates amylase production via solid-state fermentation (SSF) using non-sterile potato peel as substrate, comparing the performance of the autochthonous microbial population with that of the inoculated fungus Thermomyces lanuginosus. Non-inoculated batch reactors reached maximum productivities of 3920 U g⁻¹DM day⁻¹, more than double of the inoculated ones (1823 U g⁻¹DM day⁻¹), highlighting the potential of native thermophiles. Sequential batch reactor (SBR) strategies were applied to promote microbial selection and monitor T. lanuginosus persistence over seven cycles. Although the inoculum initially increased overall microbial activity (sOUR), T. lanuginosus was not detected, and no sustained improvement in amylase productivity was observed. Three thermophilic amylase-producing strains were isolated: Bacillus coagulans, Pseudoxanthomonas taiwanensis, and Pseudomonas thermotolerans, the last two being newly reported amylase producers. These findings demonstrate that efficient, scalable amylase production can be achieved through non-sterile SSF relying on native microbial communities, supporting circular bioeconomy strategies and potentially reducing the need for external inoculation. Further work is needed to confirm the generalizability of these results and to better understand the interactions between inoculated and native strains.