Study of the Nisin-Controlled Expression System in Lactobacillus paracasei by Using gusA as a Reporter Gene

Abstract

Abstract The Nisin-Controlled Expression system (NICE system) is an efficient gene expression system, and it was based on the mechanism of the autoregulation and biosynthesis of nisin in the Lactococcus lactis strains. The NICE system have been developed consisting of three essential elements: (1) nisin or nisin analogs or nisin mutants as inducer molecule; (2) a Gram-positive strain that expresses the nisRK genes to a desired level; and (3) plasmids containing the nisA or nisF promoter fragments, followed by convenient cloning sites to introduce the gene(s) of interest. The Model of NICE system is that the sensor protein NisK senses the presence of nisin as inducing signals, the phosphate group is transferred to response regulator NisR, which acts activate nisA promoter to expresses the gene(s). The main host of NICE system is Lactococcus lactis strains, other Gram-positive strains also can be used as the hosts. The gusA was used as the reporter gene, and the Lactobacillus paracasei was used as the dual plasmid NICE system host in this study. The results indicate that the regulatory plasmid pNZ9530 and the expressive plasmid pNZ8008 can not be stable harbored in Lb. paracase host, the host might have unknow modification mechanism to the plasmids. The study is also confirmed that nisin induction of the nisA promoter is extremely controlled by NisK and NisR proteins. Galactose also can be used as the inducer. When galactose is present, the downstream gene(s) transcripted from nisA promoter can express, but it seems still needs NisK and NisR as regulators. Higher concentrations of nisin under the cell tolerance level increase the induced expression from nisA promoter, but the optimal growth seems unfavorable for nisin induction. The extremely and flexible regulatory control is one of the main advantages of the NICE system.

摘要 Nisin-Controlled Expression System (NICE system)是一個高效的基因表現系統，係利用Lactococcus lactis內nisin的生合成與自調節機制發展而來。NICE system由三個必要元件組成：(1)以nisin、nisin analogs或nisin mutant作為誘導分子。(2)一個可以將nisRK 基因表現至適當水準的革蘭氏陽性宿主菌株。(3)表現載體必須包含啟動子PnisA或PnisF片段及方便利用的cloning sites以表現目標基因。NICE system的誘導表現模式係以NisK蛋白接受環境裡的nisin誘導信號，透過phosphate group轉移的方式把信號傳給NisR蛋白而活化nisA promoter表現下游的基因。由於NICE system主要以Lactococcus lactis為宿主，以其他革蘭氏陽性菌亦可作為NICE system之宿主，本研究利用gusA作為報告基因，探討以乳酸菌Lactobacillus paracasei作為雙質體NICE system宿主之可行性。 由研究結果發現NICE system調控質體pNZ9530與表現質體pNZ8008不能很穩定地存在Lb. paracase宿主細胞之內，且宿主可能對於質體有未知的修飾；構築之high copy number之pHY300RK與表現質體pNZ8008不能相容於Lb. paracasei，這些問題皆有待克服。 研究結果也證實以nisin誘導nisA promoter表現下游基因是受到NisK、NisR嚴謹的調控；在含有galactose的環境下，nisA promoter是可以啟動下游gusA基因表現的，但似乎仍需要NisK、NisR蛋白的調節；在有限的nisin誘導濃度之下，可以用提高誘導劑濃度的方法來增加nisA promoter的強度，但較快的生長速度並不利於nisA promoter的強度。這種嚴謹且具有彈性的調控正是NICE system主要的優點之一。