Enzyme and Microbial Technology最新文献

Enzymatic xylitol production is an eco-friendly alternative to the chemical method, which employs extreme reaction conditions. Xylose reductase from Debaryomyces nepalensis NCYC 3413 (DnXR) is a robust and well-characterised enzyme that has not yet been explored for xylitol production. The major challenge is the requirement for high NADPH concentrations, which has been overcome by a cofactor regeneration (CFR) system based on Glucose Dehydrogenase (GDH). This study aims to develop a well characterized cofactor regeneration system for xylitol production with DnXR. The key factors influencing xylitol bioproduction, including temperature, xylose concentration, NADPH level, and XR loading, have been thoroughly investigated. Uniform Design (UD) was further applied to develop a nonlinear regression model for xylitol production, identify significant factors, and optimize the reaction conditions. Validation experiments yielded 10.7 ± 0.7 g/L xylitol with a productivity of 5.35 ± 0.05 g/L.h, closely matching the model's predicted value (5.93 g/L.h). Furthermore, an Artificial Neural Network (ANN), a data-driven modelling technique, was also developed, resulting in a 13.62 g/L xylitol titre and productivity of 4.54 ± 0.01 g/L.h. The results highlight the efficiency of this system for eco-friendly xylitol production, with the highest productivity reported for two-enzyme regeneration systems, along with the detailed investigation of each component and their intricate interactions in the coupled system. Xylose conversion declined in the reaction due to a pH drop toward the end of the reaction, highlighting the need for a pH-stat CFR system with immobilised enzymes for an efficient commercial production of xylitol.

Ergothioneine (ERG), a sulfur-containing amino acid derivative known for its antioxidant activity, has a wide range of applications in healthcare and nutrition. Escherichia coli has been extensively studied as a platform for ERG production due to its rapid growth and well-established genetic tools. However, most engineered strains rely on plasmid-based expression system, which are genetically unstable. Additionally, the requirement for antibiotics to maintain plasmid stability further limits the feasibility of plasmid-based systems for industrial-scale production. Here, we established a plasmid-free E. coli platform for ERG biosynthesis using a multi-copy chromosomal integration CRISPR-associated transposase (MUCICAT) system. We first integrated a three-gene ERG biosynthetic pathway into the E. coli genome at varying copy numbers, resulting in a five-copy strain (P5) that exhibited the highest ERG titer of 222.5 ± 5.0 mg/L. Subsequently, we reinforced the two key catalytic modules-histidine methylation and SAM biosynthesis-through iterative genomic integration of the corresponding genes, yielding a plasmid-free strain P18 that produced 370.0 ± 7.0 mg/L ERG. The engineered strain P18 exhibited excellent genetic stability, as confirmed by serial passaging. When scaled up in a 5-L bioreactor under fed-batch condition, an ERG titer of 10.1 g/L was achieved. This study demonstrates a plasmid-free ERG production strategy based on stable, multi-copy chromosomal integration of the ERG biosynthetic pathway in E. coli, highlighting its potential as an efficient platform for scalable ERG production.

Zearalenone (ZEN) is an estrogenic mycotoxin, posing a serious risk to food and feed safety. In this study, a ZEN-degrading bacterium was isolated from soil and, based on phylogenetic and genomic analyses, was identified as a potential novel Microbacterium sp. and designated RD1. LC-TOF-MS/MS analysis identified a non-estrogenic hydrolysis product, indicating that RD1 degrades ZEN through lactone ring cleavage. A new hydrolase, ZenX, was cloned and heterologously expressed. It also exhibited the highest reported catalytic efficiency toward ZEN, with a specific activity of 28.06 U/mg and optimal reaction conditions of pH 9.0 and 50°C. ZenX showed high thermostability (T₅₀ = 51.9°C), which may be associated with the presence of additional intra-domain salt bridges and terminal hydrogen-bond networks. Molecular docking and sequence alignment suggested that the catalytic triad is likely composed of S112-D137-H287. Moreover, ZenX degraded ZEN in dried distillers grains with solubles, reducing its concentration from 1.1 µg/g to 0.2 µg/g. This is the first report of a ZEN hydrolase and its degradation mechanism in Microbacterium sp. Overall, these findings provide new insights into microbial ZEN degradation and the structure-function relationship of ZEN hydrolases.