Engineering of a Stable Whole-Cell Biocatalyst Capable of (S)-Styrene Oxide Formation for Continuous Two-Liquid-Phase Applications

Recombinant strains of Pseudomonas putida KT2440 carrying genetic expression cassettes with xylene oxygenase- and styrene monooxygenase-encoding genes on their chromosomes could be induced in shaking-flask experiments to specific activities that rivaled those of multicopy-plasmid-based Escherichia coli recombinants. Such strains maintained the introduced styrene oxidation activity in continuous two-liquid-phase cultures for at least 100 generations, although at a lower level than in the shaking-flask experiments. The data suggest that placement of target genes on the chromosome might be a suitable route for the construction of segregationally stable and highly active whole-cell biocatalysts.

在染色体上搭载带有二甲苯加氧酶（xylene oxygenase）与苯乙烯单加氧酶（styrene monooxygenase）编码基因的基因表达盒（genetic expression cassettes）的恶臭假单胞菌KT2440（Pseudomonas putida KT2440）重组菌株，经摇瓶（shaking-flask）诱导后，其比酶活（specific activities）可媲美基于多拷贝质粒的大肠杆菌（Escherichia coli）重组菌株。此类重组菌株在连续双液相培养体系中可至少维持100代的外源苯乙烯氧化活性，不过活性水平低于摇瓶实验中的结果。本研究数据显示，将目标基因整合至染色体上，或是构建遗传分离稳定且高活性全细胞生物催化剂（whole-cell biocatalysts）的适宜策略。