Journal of Biological Engineering最新文献

Background: Vitamin K₂ (VK₂), as a derivative of the menaquinone family, plays an important role in the prevention of osteoporosis and cardiovascular calcification. The realization of the industrialization of VK₂ and the reduction of its production cost have become the focus of attention.

Results: In this work, an E. coli strain with high VK₂ accumulation was constructed through rational metabolic engineering and stepwise improvement based on regulatory metabolic information and CRISPR/Cas9-mediated gene knockout. We first constructed a recombinant E. coli strain BW-T7/MU to produce menaquinol-8 (MKH₂-8, a reduced form of VK₂) by overexpressing menA and ubiE genes, which encoding the rate-limiting enzymes of the menaquinol pathway. After 24 h and 48 h of fermentation, this strain BW-T7/MU reach a titer of 303 mg/L and 232 mg/L. Secondly, we overexpressed different related genes wrbA (oxidative stress mitigation), qorB (reduction of quinones) and menF (conversion of chorismate to isochorismate), respectively. Among these recombinant strains, the strain BW-T7/MUW (overexpressing menA, ubiE and wrbA genes) reached the highest titer of VK₂ after 48 h of fermentation. The optimization of the medium led to an increase in the accumulation of VK₂. Subsequently, the rational metabolic engineering of gene knockout further increased the titer of VK₂. The recombinant strain ΔB/MUW was selected as the dominant strain for further optimization, with a high VK₂ titer of 724 mg/L. A final attempt is to overexpress ispB gene to increased flux of isoprenoid side chain synthesis, resulting in strain ΔB/MUWI with a titer of 859 mg/L in a shake flask and 1360 mg/L in a 5 L fermenter after 48 h cultivation.

Conclusions: The stepwise engineering strategy raised the VK₂ titer from the initial 303 mg/L to 859 mg/L through rational pathway modification and systematic gene expression. Further optimization in batch fermentation increased the VK₂ titer to 1360 mg/L, which highlights the strong engineering impact of our strategy.

Inflammatory bowel disease (IBD), particularly ulcerative colitis (UC), is increasingly recognized for its systemic effects, including neuroinflammation and cognitive deficits mediated through the gut-brain axis. This study investigates the potential mechanisms for treating UC with low-intensity pulsed ultrasound (LIPUS). A murine model of UC was established using 3% dextran sulfate sodium (DSS) in C57BL/6J mice. Disease progression was monitored via the Disease Activity Index (DAI). Histopathological evaluations were conducted using Hematoxylin and Eosin (H&E) staining. To elucidate molecular alterations, hippocampal tissues underwent quantitative proteomic analysis employing high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were identified and analyzed to understand the impact of both abdominal and transcranial LIPUS treatments. Both abdominal and transcranial LIPUS treatments were found to alleviate symptoms of colitis. Proteomic analysis of hippocampal tissues identified five DEPs-REPS1, MYG1, KRT13, SRSF10, and CDC42BPG-whose expression levels were modulated by LIPUS interventions. Notably, REPS1 and MYG1, which were downregulated in UC conditions, showed increased expression following LIPUS treatment. KEGG pathway enrichment analysis revealed that these DEPs are primarily involved in the Ras/MAPK signaling pathways. The modulation of these pathways by LIPUS suggests a mechanism by which it exerts anti-inflammatory effects, potentially restoring metabolic balance and reducing inflammation in both the gut and brain. These findings highlight the role of the gut-brain axis in mediating the beneficial effects of LIPUS and suggest its potential as a non-invasive therapeutic strategy for UC and associated neuroinflammatory conditions.

Background: Polyfunctional thiols are essential contributors to the aromatic profile of varietal white wines like Sauvignon Blanc. These compounds are released during fermentation by Saccharomyces cerevisiae cells from grape-derived precursors, with the carbon-sulfur β-lyase encoded by the IRC7 gene playing a crucial role. However, most oenological yeast strains lack the fully functional IRC7 allele, limiting their thiol-releasing ability.

Results: In this study, we used CRISPR/Cas9-based cisgenesis to replace the native allele with the fully active IRC7^{L, A553} variant into four oenological S. cerevisiae strains, commonly used to produce different white and red wines. Interestingly, all cisgenic strains showed enhanced thiols release, confirming the direct role of IRC7 in their biosynthesis. Fermentation performance, including the production of ethanol and multiple metabolites, remained however unchanged. GC-MS analyses then confirmed that strain-specific profiles of aromatic molecules were also preserved, indicating that genome editing did not affect other relevant oenological traits. Importantly, the cisgenic strains released thiols even when fermenting musts with low precursors content, without the need for additional supplements.

Conclusions: This work demonstrates that targeted IRC7 gene editing via CRISPR/Cas9 is a precise and efficient strategy to enhance thiol production in oenological yeasts, without compromising fermentative behavior or aromatic identity. Importantly, although the strains are formally GMOs, the modification mimics natural allelic variation. Our findings provide a foundation for developing next-generation wine yeasts optimized for high-aroma varietal wines and support the broader application of genome editing in fermentation biotechnology.