Development of a glycine-inducible expression system for Bacillus subtilis.
Riboswitches are binding domains for metabolites located within the 5' untranslated regions (UTR) of mRNA molecules. With two exceptions, binding of the metabolite prevents expression of the downstream gene(s). In one of these exceptions, binding of the metabolite glycine triggers expression of the downstream genes in Bacillus subtilis involved in degradation of this amino acid if present at high amounts. First, we confirmed by Northern blot the presence of a small transcript corresponding to the 5' UTR in the absence of glycine which is converted into the full-length transcript after addition of the amino acid. Surprisingly, the truncated transcript continued to be synthesized. We could show that neither increased stability is responsible for the continued presence of this truncated transcript nor does it result from processing of the full-length transcript. Next, we fused the promoter and the downstream riboswitch to the lacZ reporter gene and demonstrated glycine-dependent induction. Furthermore, we increased the promoter strength to obtain higher levels of recombinant proteins as shown by using HtpG and alpha-amylase as model proteins. This is the first report that a naturally occurring riboswitch can be used for controllable overproduction of recombinant proteins using the inexpensive inducer glycine.
Phan
Trang Thi Phuong
TT
Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany.
Schumann
Wolfgang
W
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter; D005998:Glycine / Q000494:pharmacology*; D007763:Lac Operon; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D014157:Transcription Factors; D005998:Glycine