Journal of biotechnology最新文献

Recent advances in deep learning have fundamentally transformed protein research by creating a synergistic cycle that connects structure prediction, functional annotation, and rational design. This review presents an integrative framework to demonstrate how breakthroughs in one domain catalytically enable progress in the next. First, for a broad range of single-domain, globular proteins-particularly those with sufficient evolutionary information-end-to-end deep learning models exemplified by AlphaFold2 have achieved near-experimental accuracy, effectively addressing a core challenge in structural biology and generating an unprecedented repository of high-confidence predicted structures. This vast structural substrate then serves as the essential foundation for the "Understand" phase, where multimodal models increasingly integrate 3D coordinates with sequence and interaction data to achieve precise, mechanism-aware functional predictions that transcend homology-based inference. These deep functional insights, in turn, provide the critical biochemical constraints for the final "Create" phase. Here, generative AI and inverse folding models enable the de novo design of novel proteins-from enzymes to therapeutics-with tailored structures and functions, guided by desired activity blueprints. This self-reinforcing cycle is further amplified by hybrid experimental-computational workflows, such as cryo-EM integrated with AI, which resolve complex and dynamic assemblies. While challenges in data scarcity, interpretability, and out-of-distribution generalization persist, this unified "predict-understand-create" paradigm establishes deep learning as the cornerstone of a new era in protein science. It not only accelerates discovery in drug development and synthetic biology but also transforms the field from descriptive observation to principled, programmable engineering of biomolecular function.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have emerged as promising and safe therapeutic agents, however, donor heterogeneities, limited replicative life span and changes in the cellular phenotype throughout in vitro cultivation remain major hurdles for scalable EV production. For these reasons, this study aims to investigate the use of hTERT immortalized ('telomerized') MSCs as a potential source for efficient, standardized, reliable MSC-EVs production by comparing parental primary to their telomerized MSC counterparts. We observed that hTERT expression does not affect cell morphology or cellular doubling time, while ensuring unlimited, stable in vitro propagation. In addition, telomerized WJ-MSCs maintained the canonical expression profile of surface markers and the tri-lineage differentiation potential of their primary counterparts. In terms of EV characteristics, the immortalization by hTERT expression did not affect size, number, cargo composition or biological activity regarding anti-inflammatory, anti-fibrotic and wound healing properties in vitro. In summary, the use of hTERT to immortalize MSCs leads to the creation of cell lines that continuously produce MSC-EVs without altering any key functionalities of the cells or resulting EVs. This suggests that telomerization of human cells from single donors is a promising strategy for generating cell factories that can produce EVs in standardized conditions at scale and with standardization.

Integrating microbiome engineering into microalgal biofilms could offer an environmentally sustainable and economically viable strategy to enhance biomass and lipid yields while leveraging the inherent advantages of biofilm-based systems. Building on this hypothesis, our objective was to improve biomass and lipid production in the naturally biofilm-forming marine benthic diatom, Amphora sp. (NCC169) by co-culturing it with two of its native biofilm associated bacteria, Nitratireductor sp. and Sulfitobacter sp., in a vertical porous substrate biofilm photobioreactor (PSBR) operated in batch mode. Axenic, non-axenic, and co-cultures were maintained in triplicate for 15 days in F/2 enriched artificial seawater at 18 ± 1 °C under continuous light (100 μmol photons.m⁻².s⁻¹). Biomass and lipid content were quantified gravimetrically, followed by fatty acid profiling via gas chromatography-mass spectrometry (GC-MS) at three harvesting days (3, 6, and 15). Results showed that the Amphora-Nitratireductor co-culture achieved the highest surface biomass (2.54 ± 0.39 g.m⁻²) and productivity (0.99 ± 0.26 g.m⁻².day⁻¹) on day 3, further peaking on day 6 (7.27 ± 1.31 g.m⁻²; 1.24 ± 0.16 g.m⁻².day⁻¹) and were significantly different from the non-axenic control. The Amphora-Sulfitobacter co-culture recorded a significantly higher lipid yield on day 3 (1.51 ± 0.31 g.m⁻²), surpassing axenic cultures by 3-fold, non-axenic by 6-fold, and Amphora-Nitratireductor by 5-fold, with a lipid content of 67.03 ± 17.80% of Amphora dry biomass weight. The fatty acid profile of Amphora sp. remained largely consistent across culture conditions and harvest days, dominated by palmitic acid (16:0) and palmitoleic acid (16:1), with low polyunsaturated fatty acid levels, highlighting its potential for pharmaceutical, nutraceutical, cosmetic, and bioenergy applications.

Developing galectin-1-based therapeutics faces challenges that are specific for this lectin, such as its differing redox states and reversible monomer-dimer equilibrium. The generation of a cysteine-less mutant of galectin-1 (CSGal-1) stabilized its redox behavior, while the development of a covalently linked galectin-1 dimer avoided its concentration-dependent monomer-dimer equilibrium. However, the type and length of the linker can affect the therapeutic potency of the covalent construct, as well as the protein titer, solubility, domain folding or bioactivity. Therefore, the computational design of a linker with desired properties and predictable behavior is highly desired. In this study, we de novo designed three different linkers to covalently connect the two subunits of CSGal-1. The CSGal-1 subunits were genetically fused via the designed linkers and produced as single-chain (sc) proteins for the first time. The data showed that the sc construct with the longest and most acidic linker exhibited the highest expression titer, solubility, and even ability to refold from inclusion bodies, which was in accordance with the in silico prediction. Further, we characterized the best performing scCSGal-1 construct against the noncovalent dimer of CSGal-1. Data from differential scanning fluorimetry and isothermal titration calorimetry demonstrated that scCSGal-1 retained its functionality with a binding affinity and thermodynamic profile similar to that of noncovalently dimerized CSGal-1. These findings underscore the strength of the computational approach in linker design and encourage its extension to other lectins that require a stable dimeric form in their applications.

Polyhydroxyalkanoates (PHAs) have shown much promise as materials in nerve tissue engineering. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] has excellent biocompatibility, biodegradability, and tuneable mechanical properties, which are much more flexible than Poly(3-hydroxybutyrate), P(3HB). In this study, the potential of P(3HB-co-20mol%3HHx), produced by an engineered Cupriavidus necator strain, was investigated and compared with P(3HB), produced by Bacillus subtilis OK2, and polycaprolactone (PCL) for nerve tissue engineering applications. The PHAs were produced by batch/fed-batch fermentation. Solvent cast films were prepared using P(3HB-co-20mol%3HHx), P(3HB) and PCL, and evaluated for morphology, topography and mechanical properties. In addition, their ability to support proliferation, viability and differentiation of NG108-15 was also evaluated. Mechanical properties showed that P(3HB-co-20mol%3HHx) films were more suitable for soft tissue engineering applications, than the homopolymer P(3HB). In addition, P(3HB-co-20mol%3HHx) films demonstrated superior NG108-15 neuronal cell adhesion, proliferation and viability and supported NG108-15 neuronal cell differentiation, outperforming PCL films, the current FDA approved biopolymer used in peripheral nerve repair.

Euglena gracilis, a microalga, can be cultured under photoautotrophic, heterotrophic, or mixotrophic conditions. This species can produce proteins, vitamins, and lipids. Based on previous reports, paramylon, a storage polysaccharide of E. gracilis and a type of β-1,3-glucan, can inhibit the development of skin lesions. Glucose increases the proliferation of E. gracilis and the accumulation of paramylon. However, excess glucose decreases the proliferation of E. gracilis because of high osmotic pressure. In this study, we found that E. gracilis cells cultured with high concentrations of glucose became hypertrophied rather than dehydrated and shrunken. The addition of ethanol (concentration of 0.5%) improved the proliferation of E. gracilis under a high glucose concentration of 400 mM. The addition of ethanol resulted in spindle-shaped cells similar to those observed under a no-glucose condition. These results indicate that ethanol alleviates glucose stress in E. gracilis, revealing the physiological aspects of E. gracilis under heterotrophic conditions.

Gas fermentation is a promising approach to reduce carbon emissions of production processes by using microorganisms to convert industrial gas streams into valuable carbon-based products. Here, isopropanol production by engineered Cupriavidus necator from CO₂-rich gas streams is presented. Four different engineered strains were tested first on synthetic gas mixture, and one was selected, namely Re2133/pEG7d, for further evaluation on two different industrial CO₂-rich gas streams. Up to 1.6 g L^-1 of isopropanol was obtained in autotrophic gas flasks with this strain. Then, raw biogas from landfill containing 45% methane was successfully used as a CO₂ source for isopropanol production with this strain Re2133/pEG7d producing up to 2.35 ± 0.25 g L^-1, and methane enrichment was demonstrated. Finally, flue gases from a municipal incinerator were used as a source of CO₂ and O₂ for the autotrophic cultivation of the strain Re2133/pEG7d, obtaining a final isopropanol concentration of 2.5 ± 0.3 g L^-1. This work highlighted the robustness of C. necator to potential industrial gas stream impurities and its versatility in using diverse gaseous feedstocks, promising future developments at larger scale.