以葡萄醣醛酸酶基因gusA為報告基因探討nisin調控表現系統於乳酸菌Lactobacillus paracasei之調控表現研究

The Nisin-Controlled Expression System Using β-Glucuronidase Gene as a Reporter Gene in Lactobacillus paracasei

葉娟美(Chuan-Mei Yeh) ； 許家榮(Jia-Rong Hsu)

《臺灣農業化學與食品科學》 43卷2期 (2005/04) Pp. 86-96

https://doi.org/10.6578/TJACFS.2005.012

摘要

Nisin調控表現系統(nisin-controlled expression system, NICE system)是一個誘導表現系統，以細胞膜上NisK蛋白質接受環境nisin信號誘導而自我磷酸化，並透過磷酸基團轉移，活化NisR蛋白質，進而活化nisA啓動子表現下游的基因。本研究以葡萄醣醛酸酶基因gusA作爲報告基因，探討乳酸菌Lactobacillus parcasei作爲nisin調控表現系統表現宿主之最佳條件。結果顯示，以雙質體NICE系統，在乳酸菌L. paracasei生長至OD600約爲0.5至0.6之間開始以40μg/mL濃度之nisin在30℃下誘導1.5h，所得之β-葡萄醣醛酸酶活性最高，爲260比活性單位；高於37℃下之最高表現量（175比活性單位）。30℃下，0.01μg/mL之nisin濃度即可誘導nisA啓動子表現。0.01-0.10μg/mL間之誘導表現為劑量反映階段。在半乳糖及NisK、NisR蛋白質存在下，不需誘導即有活性表現。本研究之結果，除建立NICE系統於L. paracasei宿主誘導表現外源蛋白質之條件外，未來可開發生產功能性胜肽或蛋白質之益生菌。

關鍵字

NICE系統 ； Lactobacillus paracasei ； nisin ； 葡萄醣醛酸酶基因 ； 誘導表現

並列摘要

The nisin-controlled expression system (NICE system) is an efficient gene expression systembased on the autoregulation mechanism of nisin in the Lactococcus lactis. In NICE system, the membrane sensor protein NisK senses the inducing signal nisin and autophosphorylates, then transfers phosphorous group to response regulator protein NisR which activates nisA promoter to expresses the downstream gene(s). In this study, the optimal induction parameters of a dual plasmid NICE system were tested by using gusA as a reporter gene in Lactobacillus paracasei host cells. The results indicate that nisA promoter is well controlled in L. paracasei. Optimal induced β-glucuronidase specific activity (260 U/OD600) is obtained by induction the L. paracasei at 0.5 to 0.6 OD600 cell density with 40μg/mL nisin after 1.5 h cultivation at 30℃, which is higher than the optimal β-glucuronidase specific activity (175 U/OD600) obtained at 37℃ cultivation. The induced expression can be achieved at nisin concentration even as low as 0.01μg/mL and the dose-response induction is observed at concentrations of 0.01 to 0.10 μg/mL nisin. Nisin-independent β-glucuronidase activity is observed under the presence of NisR, NisK proteins, and galatose. This study provides optimal parameters for the NICE system in L. paracasei as well as a means to construct genetically modified probiotic bacteria with the ability to produce functional peptides or proteins.

並列關鍵字

NICE system ； Lactobacillus paracasei ； nisin ； gusA ； inducible expression