A new and efficient phosphate starvation inducible expression system for Lactococcus lactis.
A new expression system for Lactococcus lactis was developed. The system is based on a phosphate starvation inducible pstF promoter of L. lactis MG1363. Intracellular beta-galactosidase and secreted alpha-amylase were produced using this tightly regulated system. No evidence of regulatory sites in regions of the 5'-end of the pstF coding sequence was found. High expression levels of the beta-galactosidase gene were obtained using the original pstF RBS in a phosphate-depleted medium. The results suggested that with the phosphate starvation inducible system, it is possible to achieve expression levels comparable to the ones obtained with the widely used nisin-controlled gene expression system (NICE). A specific beta-galactosidase activity of 670 microkat g(-1) using a phosphate-depleted medium and an alpha-amylase activity of 3.6 microkat l(-1) in a bioreactor cultivation were produced. The advantages of the current expression system include that no prior removal of phosphate from the medium in bioreactor scale is required, and no additions of inducing agents are needed. Furthermore, the system can be operated in L. lactis without introduction of regulatory genes into the host.
Sirén
Noora
N
Laboratory of Bioprocess Engineering, Department of Biotechnology and Chemical Technology, Helsinki University of Technology, Espoo, Finland. noora.siren@tkk.fi
Salonen
Kalle
K
Leisola
Matti
M
Nyyssölä
Antti
A
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors; D015964:Gene Expression Regulation, Bacterial; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009561:Nisin / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D010710:Phosphates; D009561:Nisin; D000516:alpha-Amylases; D001616:beta-Galactosidase