Discovering novel Cas12a homologs with higher activity, relaxed PAM requirements, or broad temperature adaptability is essential for improving CRISPR/Cas12 rapid detection systems, particularly for portable and field-deployable applications. In this study, we conducted bioinformatic mining of metagenomic databases based on the characteristic sequence features of CRISPR arrays to identify Cas12a homologs. Two novel Cas12a homologs, GbCas12a and CbCas12a, were identified. Among them, CbCas12a exhibited pronounced trans-cleavage activity and a broad operational temperature range, making it well-suited for rapid nucleic acid detection. By integrating CbCas12a with reverse transcription recombinase polymerase amplification, we established a CRISPR/CbCas12a-based detection platform and successfully applied it to the visual detection of Porcine epidemic diarrhea virus. This system demonstrated high sensitivity and specificity, underscoring its potential for diverse rapid diagnostic applications in medicine, agriculture, and other fields.
2-(Furan-2-yl)-2-oxoacetic acid (2-FOA) is a key precursor for constructing the C7 side chain of cefuroxime. Existing biocatalytic routes depend on a copper-dependent galactose oxidase system requiring horseradish peroxidase (HRP) for activation. To expand biocatalytic alternatives, we characterized FAD-dependent 5-hydroxymethylfurfural oxidase from Methylovorus sp. MP688 (MvHMFO), whose redox cycle eliminates demands for exogenous HRP and metal cofactors. However, MvHMFO showed low activity toward the non-natural substrate 2-furyl hydroxymethyl ketone (2-FHMK). Herein, we adopted a three-stage structure-guided stepwise engineering strategy by sequentially evaluating and combining substitutions from the catalytic core to peripheral loops, achieving cavity expansion and local hydration network reorganization. The optimal variant M4 showed 75.2-fold and 132.3-fold improvements in catalytic efficiency toward 2-FHMK and its aldehyde intermediate, respectively. Applying M4 in an elevated-temperature (40 °C) fed-batch process yielded 6.2 g/L of 2-FOA with a conversion of 88%. This work provides an HRP- and Cu-free HMFO platform for 2-FOA synthesis and an engineering framework for adapting oxidoreductases to non-natural substrates.
Abstract To achieve itaconic acid production using non-food feedstocks, this study constructed an engineered Escherichia coli strain capable of efficiently synthesizing itaconic acid using corn stover hydrolysate as the carbon source. By heterologously expressing cis-aconitate decarboxylase, relieving carbon catabolite repression, eliminating by-product synthesis pathways, deleting isocitrate dehydrogenase, introducing the Weimberg pathway to reconstruct the α-ketoglutarate supply route, and incorporating a feedback-resistant citrate synthase from Corynebacterium glutamicum, the carbon flux was progressively optimized toward the directed synthesis of itaconic acid. Furthermore, this study employed a low-waste alkali pretreatment strategy to obtain corn stover cellulose hydrolysate, thereby reducing waste liquid discharge and minimizing the loss of cellulose and hemicellulose. Using the resulting non-detoxified hydrolysate as the substrate, the engineered strain BI18 achieved an itaconic acid titer of 57.1 g/L in fed-batch fermentation, with a total sugar conversion yield of 0.61 g/g. This production routeintegrating strain engineering, non-food feedstocks, and low-waste pretreatmentprovides a technical reference for itaconic acid biorefining from lignocellulosic biomass.
Abstract Single-cell perturbation data have enabled computational models that predict intervention-associated cellular responses. This narrative review organizes explicit gene-regulatory-network simulations, latent and compositional response models, graph, optimal-transport, and dynamical approaches, foundation-model representations, and emerging spatial or cross-modal settings through a virtual-knockout lens. We distinguish representation-level ablation from causal genetic intervention and separate held-out regimes for genes, cell types or contexts, perturbation combinations, chemical structures, and doses. We compare representative methods, experimental resources, metrics, and benchmark frameworks and operationally define a prospective holographic virtual cell by its modalities, regulatory and physical constraints, intervention-conditioned outputs with calibrated uncertainty, and prospective validation requirements. The holographic virtual cell and Design−Build−Test−Learn roadmap are presented as future-facing criteria rather than as an achieved clinical capability.
Abstract Genetic circuit reliability is often limited by growth-mediated dilution. However, developing an efficient strategy for maintaining reliable circuit function against dilution effects remains a challenge. Here, we introduce programmable condensates to stabilize circuits in Bacillus subtilis. First, we engineered synthetic condensates and employed RIAD/RIDD peptide pairs to selectively sequester the transcription factor (TF). Spatial recruitment of these TF-filled condensates to target promoters facilitated a significant enhancement in gene expression. By tailoring the physicochemical properties and spatial distribution of the condensates, we substantially reinforced the buffering capacity of the circuits at higher temperatures. As a proof of concept, we harnessed this strategy to significantly improve the bioproduction of alkaline protease and 2′-fucosyllactose, achieving a 109.3% increase in enzyme activity and a 108.9% increase in product titer, respectively. Overall, this work offers a valuable toolkit for B. subtilis applications and a programmable framework for robust circuit redesign in other hosts.
