title: Functional analysis and randomization of the nisin-inducible promoter for tuning gene expression in Lactococcus lactis.
abstract: Three putative promoter regions were identified preceding the nisZ gene in Lactococcus lactis HSM-22. To investigate their function in the control of nisZ biosynthesis, green fluorescence protein (GFP) was adopted as probe to determine activities of the three promoters. The results showed that PnisZ-0 containing two sets of the -35 and -10 regions exhibited the same maximum activity as promoter PnisZ-2 containing the putative promoter region near the start codon. However, the GFP expression level directed by PnisZ-0 was twofold higher than that found with PnisZ-2 under low-dose nisin, indicating that promoter PnisZ-1 distant from the start codon could be important in response to the inducer nisin. Then, Pnis-2 was randomized to develop functional promoters through the degenerate oligonucleotide approach in L. lactis. 35 inducible promoters and 14 constitutive promoters were obtained, covering 3-5 logs of expression levels in small increments of activity. Sequence analysis revealed that base changes in both consensus sequence and spacing sequence resulted in remarkable decrease of promoter activity, while the sequence outside -35 and -10 regions would influence the promoter function radically. The functional promoters were evaluated for the efficiency and stability to control β-galactosidase (Gal) expression in L. lactis. High correlation was obtained between the Gal activity and promoter strength, suggesting that promoters developed here have the potential for fine tuning gene expression in L. lactis.
authors: State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nanlu, Jinan 250100, People's Republic of China.
authors: Tingting
authors: T
authors: Guo
authors: Shumin
authors: S
authors: Hu
authors: Jian
authors: J
authors: Kong
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D009561:Nisin / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D009561:Nisin; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase