Trends in biotechnology最新文献

Through integration with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems, personal glucose meters (PGMs) have been repurposed to detect non-glucose targets. PGMs enable the intuitive readout of electrochemical signals, while CRISPR/Cas offers rapid detection and signal amplification capability. Their integration can realize point-of-care (POC) diagnostics. Herein, we critically discuss their advances, pitfalls, and future perspectives.

β-Alanine is an important platform chemical whose biosynthesis efficiency is limited by pathway bottlenecks and enzyme constraints. In this study, to overcome genetic interactions for β-alanine production, we systematically engineered Escherichia coli MG1655 through modular pathway optimization and combinatorial regulation. To overcome the limitations of L-aspartate-α-decarboxylase from Bacillus subtilis (PanD_bsu), we developed an in vivo evolution platform combining base-editing systems with biosensor guidance, thus generating PanD_bsu variants with enhanced activity. This system facilitated high-throughput screening and real-time monitoring of β-alanine production and accelerated mutant selection. Furthermore, site saturation and iterative mutations identified a beneficial PanD_bsu^T4E mutant, which enhanced specific β-alanine production in engineered strain MA31 by 62.45%. Structural and functional analysis revealed that PanD_bsu^T4E stabilized its quaternary structure via a Glu-Lys salt bridge. This work describes a scalable strategy for addressing pathway bottlenecks and highlighted the potential of integrating protein engineering with biosensor-guided evolution to optimize microbial cell factories.

Unicellular algae are appealing for nutritional and biotechnological utility, but show wide variation across strains and can be challenging to produce. Species in the thermo-acidophilic genus Galdieria use diverse organic carbon sources for fermentative growth, which can include waste-stream feedstocks, while having complete amino acid compositions for human nutrition. Here, we investigated the metabolic dynamics of Galdieria to catalog organic carbon conversion to biomass. Tested strains had enhanced growth upon 3% CO₂ supplementation, triggering efficient glucose uptake to reach ~5 ± 0.3 g dry biomass l^-1. Stable-isotope analysis revealed that organic carbon uptake dominated CO₂ fixation in darkness under mixotrophy, with CO₂ an apparent metabolic trigger. Galdieria yellowstonensis 5587.1 was able to consume up to 8.3 g carbon l^-1 day^-1 from industrial confectionery waste, with C-phycocyanin (C-PC) reaching 3.8% of dry biomass and remaining thermostable at 72°C. Thus, this framework can be used to optimize Galdieria-based bioprocesses for the conversion of inexpensive waste into high-value biomass, and identifies CO₂ as a trigger of organic carbon assimilation, even in heterotrophic conditions.

The important food microorganism Lactococcus lactis is limited by its fermentative metabolism and low energy yield. This limitation can be partially overcome by activating aerobic respiration which, in a food context, can be difficult to achieve as heme and oxygen must be supplied. Supplying oxygen is particularly challenging due to oxygen mass transfer limitations in food matrices. To address these challenges, we propose to use electro-fermentation to grow L. lactis anaerobically. We propose a technical concept that could potentially increase the biomass productivity by >60% and decrease cooling cost by >90%, and we discuss how this new approach can reshape and enhance the sustainability of the food industry.

The field of 3D extrusion bioprinting has rapidly evolved, presenting significant advancements in tissue engineering applications. Traditionally, the development of printable hydrogel formulations has relied more on empirical approaches rather than rational design principles. However, to better mimic the dynamic and spatiotemporal nature of the extracellular matrix (ECM), novel bioinks with tunable properties and synthetic flexibility are being developed. Simultaneously, variations of extrusion bioprinting methods have been developed to improve the fabrication of sophisticated tissue patterns to emulate their in vivo counterparts. This review will provide a focused overview of recent advancements in bioink design and extrusion bioprinting technologies. By addressing the latest developments in these strategies, this review aims to offer insights into the future direction of extrusion bioprinting.

Bridge amplification of nucleic acids exploits the use of surface-tethered forward and reverse primers to allow amplification to occur on a 2D surface, in a spatially controlled manner, facilitating multiplexed reactions for the detection of nucleic acid targets with a variety of applications from personalized medicine to advanced forensics.

Therapeutic monoclonal antibodies (mAbs) exhibit significant interindividual pharmacokinetic (PK) variability, necessitating therapeutic drug monitoring (TDM). However, current monitoring methods require complex and time-consuming laboratory analysis, making rapid dose adjustments challenging. This review discusses emerging technologies for on-site mAb monitoring, with a focus on recent advances in recognition elements and detection strategies. Novel recognition elements, such as anti-idiotype molecules and meditopes, enable specific detection, whereas direct detection methods that eliminate washing steps facilitate rapid bedside testing. We examine the prospects for continuous mAb monitoring based on current technological advances and regulatory considerations. Although challenges regarding sensor regeneration and stability remain, the technologies discussed hold promise to enable real-time therapeutic monitoring at the bedside.

Prime editing, first introduced in 2019, is a precise and efficient genome-editing technique with applications across various organisms and cell lines, including human pluripotent stem cells (hPSCs). Despite its revolutionary potential, prime editing of hPSCs often shows low efficiency, hindered by current delivery methods and DNA repair mechanisms. In this review, we explore prime editing of hPSCs, emphasizing the optimization necessary for creating ex vivo and in vitro disease models, which are critical for developing personalized therapeutics. We discuss key prime-editing methods for hPSCs, optimization strategies, tools available for prime editing, and the rigorous quality control required before and after genome engineering for downstream applications.

Cryobioprinting enables simultaneous fabrication and cryopreservation of tissue analogs, surpassing the limitations of print-to-use biofabrication approaches. However, cryopreservation of living constructs remains challenging, requiring optimal cryopreservation conditions tailored to specific cell types and hydrogel bioinks. To address this, we explored the formulation of a decellularized extracellular matrix (dECM)-based hydrogel bioink containing cryoprotective agents (CPAs) to generate shelf-ready tumor-stroma pancreatic cancer models. Combinatorial screening of CPAs led to the discovery of a novel melezitose-glycerol-dECM formulation that exhibited superior cryoprotective properties in both tumor and stroma compartments. Exometabolomics analysis revealed that cryopreserved constructs exhibited similar metabolic activity to nonfrozen counterparts 14 days post thawing. Cryobioprinted tumor-stroma models in dECM-CPA bioinks also exhibited increased cell viability post thawing and suitable features for in vitro drug screening. Thus, our optimized cryoprotective strategy opens new opportunities to potentially explore any type of tissue decellularized bioinks for cryobioprinting off-the-shelf living constructs for widespread drug screening and beyond.

Liquid biopsy for microbiome analysis is an emerging field that aims to study the composition and dynamics of microbial communities by analyzing biological fluids such as blood, urine, or saliva. This noninvasive method offers a useful approach to understanding microbiome dynamics. It provides insights into different pathologies, enables unbiased pathogen detection through noninvasive sampling, and offers rapid turnaround times. Clinical applications of liquid biopsy are emerging for microbiome exploration, particularly in areas such as infectious disease management, cancer diagnostics, and personalized medicine for chronic bowel diseases. This review highlights the role of liquid biopsy in infection and microbiome exploration, potentially revolutionizing diagnostics and tailored medicine by enabling the real-time monitoring of microbial shifts.