THE SEQUENCE OF SPACERS BETWEEN THE CONSENSUS SEQUENCES MODULATES THESTRENGTH OF PROKARYOTIC PROMOTERS

We constructed a library of synthetic promoters for Lactococcus lactis in which the known consensus sequences were kept constant while the s equences of the separating spacers were randomized, The library consis ts of 38 promoters which differ in strength from 0.3 up to more than 2 ,000 relative units, the latter among the strongest promoters known fo r this organism, The ranking of the promoter activities was somewhat d ifferent when assayed in Escherichia coli, but the promoters are effic ient for modulating gene expression in this bacterium as well. DNA seq uencing revealed that the weal;er promoters (which had activities belo w 5 relative units) all had changes either in the consensus sequences or in the length of thf spacer between the -35 and -10 sequences, The promoters in which those features were conserved had activities from 5 to 2,050 U, which shows that by randomizing the spacers, at least a 4 00-fold change in activity can be obtained. Interestingly, the entire range of promoter activities is covered in small steps of activity inc rease, which makes these promoters very suitable for quantitative phys iological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories.