Ningxia University

In Escherichia coli, cofactor imbalance serves as a crucial limiting factor in cytidine biosynthesis, with nicotinamide adenine dinucleotide phosphate (NADPH) insufficiency representing the principal metabolic barrier. To overcome this limitation, an integrated engineering strategy targeting the enhancement of NADPH metabolism was implemented. Via CRISPR-Cas9-mediated multiplex genomic editing and strong constitutive promoter replacement, three NADPH-regenerating modules were concurrently enhanced: the membrane-bound transhydrogenase (pntAB), the oxidative pentose phosphate pathway (zwf-encoded glucose-6-phosphate dehydrogenase), and the decarboxylation shunt (gnd-encoded 6-phosphogluconate dehydrogenase). After 54-hour fermentation in 500 mL shake flasks, the cytidine titer of the engineered strain NXBG-20 reached 7.83 g/L, representing a 9.10-fold increase compared to the start strain. Systematic multi-omics profiling revealed that the metabolic network had undergone substantial alterations. These alterations were characterized by the redirection of glycolytic flux towards nucleotide precursor substances and the enhancement of ribose-5-phosphate biosynthesis. This engineering approach not only establishes a novel microbial platform for cytidine bioproduction but also provides mechanistic insights into cofactor-driven metabolic flux control.

This study systematically investigated the quality changes of buckwheat hele noodles (BHN) during frozen storage at -18 °C for 80 days. Pronounced lipid-protein co-oxidation was observed, characterized by a 71.9% rise in malondialdehyde (MDA) and a 133.2% rise in carbonyl content. Sulfhydryl content showed a continuous decline, while the levels of key unsaturated fatty acids progressively decreased. The activities of lipoxygenase (LOX) and catalase (CAT) gradually diminished, thereby inhibiting enzymatic oxidation but promoting non-enzymatic oxidation. Microscopic analysis revealed a disrupted gluten network with enlarged pores, and the proportion of β-sheet structures in protein secondary structures increased significantly, suggesting enhanced protein aggregation and cross-linking. Flavor analysis indicated that the levels of aldehydes and ketones peaked at 40 days, with 2-nonenal and 1-octen-3-ol identified as the key off-flavor compounds. Correlation analysis confirmed that protein and lipid oxidation were the primary driver of quality deterioration in BHN during frozen storage.

Linalool is a key aromatic terpene that imparts the characteristic Muscat flavor in grape berries. However, the upstream transcriptional mechanisms regulating its biosynthesis remain largely unknown. Here, integrated GC-MS and transcriptomic analyses across berry development in Muscat and non-Muscat cultivars identified VvTPS14 as a terpene synthase gene whose expression strongly correlated with the linalool accumulation pattern. Functional characterization confirmed that VvTPS14-2 encodes a chloroplast-localized enzyme that catalyzes linalool biosynthesis from GPP in vitro. Transient and stable overexpression of VvTPS14-2 in Nicotiana benthamiana leaves, grape berries, and Vitis amurensis callus consistently enhanced linalool production, establishing VvTPS14 as a key linalool synthase. Upstream regulatory mechanisms of VvTPS14 were elucidated through yeast one-hybrid screening, which identified VvbZIP3 and VvMADS4 as direct binders of the VvTPS14 promoter. Dual-luciferase and overexpression assays demonstrated that VvbZIP3-4 (a splice variant) activates, whereas VvMADS4 represses, the expression of VvTPS14. This regulatory pattern was conserved across heterologous and homologous systems. These findings reveal a complete transcriptional regulatory module controlling linalool biosynthesis and provide potential targets for molecular breeding of flavor traits in grapes.