A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum.
A synthetic promoter library (SPL) for Lactobacillus plantarum has been developed, which generalizes the approach for obtaining synthetic promoters. The consensus sequence, derived from rRNA promoters extracted from the L. plantarum WCFS1 genome, was kept constant, and the non-consensus sequences were randomized. Construction of the SPL was performed in a vector (pSIP409) previously developed for high-level, inducible gene expression in L. plantarum and Lactobacillus sakei. A wide range of promoter strengths was obtained with the approach, covering 3-4 logs of expression levels in small increments of activity. The SPL was evaluated for the ability to drive beta-glucuronidase (GusA) and aminopeptidase N (PepN) expression. Protein production from the synthetic promoters was constitutive, and the most potent promoters gave high protein production with levels comparable to those of native rRNA promoters, and production of PepN protein corresponding to approximately 10-15 % of the total cellular protein. High correlation was obtained between the activities of promoters when tested in L. sakei and L. plantarum, which indicates the potential of the SPL for other Lactobacillus species. The SPL enables fine-tuning of stable gene expression for various applications in L. plantarum.
Rud
Ida
I
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway.|MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway.
Jensen
Peter Ruhdal
PR
Microbial Physiology and Genetics, BioCentrum, Technical University of Denmark, Kgs Lyngby, Denmark.
Naterstad
Kristine
K
MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway.
Axelsson
Lars
L
MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway.
D000626:Aminopeptidases / Q000032:analysis / Q000235:genetics; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D016384:Consensus Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D015723:Gene Library; D017930:Genes, Reporter; D005813:Genes, Synthetic; D020459:Genes, rRNA; D005822:Genetic Vectors; D005966:Glucuronidase / Q000032:analysis / Q000235:genetics; D048191:Lactiplantibacillus plantarum / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic
D001426:Bacterial Proteins; C075633:pepN protein, Bacteria; D005966:Glucuronidase; D000626:Aminopeptidases