Lac抑制因子
大肠杆菌
操纵子
抑制因子
T7 RNA聚合酶
核糖体结合位点
生物
紫胶操纵子
化学
分子生物学
生物化学
噬菌体
基因表达
基因
信使核糖核酸
翻译(生物学)
作者
Sefli Sri Wahyu Effendi,I-Son Ng
标识
DOI:10.1021/acssynbio.2c00363
摘要
Lac operon is the standard regulator used to control the orthogonality of T7RNA polymerase (T7RNAP) and T7 promoter inEscherichia coli BL21(DE3) strain for protein expression. However,E. coliNissle 1917 (EcN), the unique probiotic strain, has seldom been precisely adapted to the T7 system. Herein, we applied bioinformatics analysis on Lac operon from different strains, and it was observed that a weak promoter for LacI repressor existed in EcN. Furthermore, X-gal assay revealed a strong expression of lacZ in EcN. We demonstrated that Lac operon significantly affected the protein expression in the two T7-derived EcN, in which T7RNAP was integrated at lambda (ET7L) and HK022 (ET7H), respectively. Different combinations of replication origin, chaperonin GroELS, inducer, and medium were explored to fine-tune the best strain with tyrosine ammonia-lyase (TAL) for p-coumaric acid (pCA) production, which is one of the essential bioactive compounds for human health. Finally, the highest pCA conversion of 78.8% was achieved using RRtL (plasmid form) under the optimum condition, and a 51.5% conversion was obtained with L::Rt strain which has integrated T7-RtTAL at HK022 of ET7L in the simulated gut environment. The appropriate reprogramming of T7RNAP expedites EcN as an effective and promising cell factory for live bacterial therapeutics in the future.
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