School of Biotechnology

Lacto-N-neotetraose (LNnT) is a key human milk oligosaccharide (HMO) with important prebiotic functions, supporting the growth of beneficial gut microbiota and contributing to infant health. Constructing plasmid-free strains via metabolic engineering for LNnT biosynthesis represents a feasible strategy for efficient industrial-scale production. This study integrates various strategies to construct plasmid-free strains capable of efficiently producing LNnT. Building on the previously developed Escherichia coli MG1655 strain for lacto-N-triose II (LNTri II) production, Hplex2B (encoding β1,4-galactosyltransferase) was incorporated, and its copy number was optimized to construct a complete and efficient biosynthetic pathway. By integrating an extra copy of the multidrug efflux pump gene mdfA, the strain tolerance was improved, resulting in a higher yield of LNnT. The integration of expression cassettes for key genes in the glycosyl donor synthesis pathway (including glmS, glmM, glmU, galU, and galE) with varying promoter strengths optimized the supply and balance. The subsequent removal of a key feedback inhibition circuit directed the reaction toward the synthesis of LNnT. The final strain, after successive optimization, produced LNnT at 7.76 g/L in shake flask culture and 34.24 g/L in a 5 L bioreactor, with precursor LNTri II concentrations of 0.58 g/L and 1.61 g/L, respectively.

The exploration of new strategies to monitor Botrytis cinerea populations is critical for disease management. This study achieved efficient and simultaneous separation of all TCA cycle intermediates in mycelia treated with pyraclostrobin (PYR), tebuconazole (TEB), and carbendazim (CAR) using anionic chromatography. Resolutions of nine organic acids and six inorganic anions, including all TCA cycle intermediates, exceeded 1.5. The fungicides inhibited mycelial growth concentration-dependently, but prolonged exposure reduced the level of inhibition. TEB decreased fumarate and malate levels at 48 h, increased citrate, isocitrate, and oxalate levels and reduced α-ketoglutarate levels at 96 h. PYR showed biphasic effects, elevating fumarate, malate, and succinate at both time points but reducing citrate and succinate at 96 h. CAR attenuated TCA cycle flow, with isocitrate and α-ketoglutarate declining continuously. Moreover, TEB promoted oxalate accumulation, PYR had no effect, and CAR suppressed it. These findings enable precise TCA cycle assessment for fungicide adaption in B. cinerea, aiding Botrytis management.

K. pneumoniae has emerged as a major infectious threat due to its remarkable capacity for intracellular survival, virulence, and antibiotic resistance. It evades immune clearance by resisting phagocytosis through capsular polysaccharides and lipopolysaccharides, modulating macrophage polarization to create a permissive intracellular niche, and autophagic degradation. Additionally, it alters cytokine responses and interferes with host cell death pathways like apoptosis and pyroptosis to suppress inflammasome activation and efferocytosis. These immune evasion strategies are particularly concerning in the context of emerging MDR and hypervirulent strains. In response, host directed therapies (HDTs), including epigenetic and immunomodulatory interventions, offer promising non-antibiotic based strategies. Understanding the host pathogen interaction is essential to guide future therapeutic development.