The sequence of spacers between the consensus sequences modulates the strength of procaryotic promoters

Abstract

We constructed a library of synthetic promoters for Lactococcus lactis in which the known consensus sequences were kept constant while the sequences of the separating spacers were randomized. The library consists 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 for this organism. The ranking of the promoter activities was somewhat different when assayed in Escherichia coli, but the promoters are efficient for modulating gene expression in this bacterium as well. DNA sequencing revealed that the weaker promoters (which had activities below 5 relative units) all had changes either in the consensus sequences or in the length of the spacer between the 235 and 210 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 400-fold change in activity can be obtained. Interestingly, the entire range of promoter activities is covered in small steps of activity increase, which makes these promoters very suitable for quantitative physiological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories. Metabolic engineering has promising perspectives with respect to improving the properties and performances of microorganisms used as industrial bioreactors, as cell factories, and in food fermentations. The importance of tuning gene expression in this context, i.e., to perform metabolic optimization rather than massive overexpression or gene inactivation, is now far more appreciated. However, the more subtle approach of metabolic optimization is hampered by the lack of proper expression systems for tuning gene expression in many microorganisms. Also, the fundamental understanding of a biological system through metabolic control analysis (5, 10) requires the tuning of enzyme activities in order to calculate the socalled control coefficients. For some organisms, expression systems that allow for changing gene expression for scientific purposes and for a limited set of experimental conditions have been developed. Thus, for Escherichia coli, the lac system, the cI-regulated lambda pR/pL, and many derivatives of these systems have been widely applied, and such systems have also been adapted for use in other organisms (for a recent review, see reference 12). With respect to changing steady-state gene expression, these systems can sometimes be difficult to apply, particularly when it comes to changing gene expression on an industrial scale. Besides, in most food fermentation processes, the addition of chemicals as inducers of gene expression or the changing of other process parameters is not acceptable; in such cases, there are virtually no expression systems available for tuning gene expression and thus for performing accurate metabolic optimization. Lactic acid bacteria are widely used in food fermentation, e.g., cheese and yoghurt production, but besides lactic acid, these bacteria excrete a spectrum of organic compounds. Some of these are desirable with respect to the development of texture and flavors or for bioconservation purposes, and some are undesirable for similar or different reasons. The lactic acid bacteria are therefore obvious candidates for attempts to optimize the pattern of formation of these compounds for specific applications. But the experimental tools for manipulating gene expression are not well developed for these bacteria. An exception is the nisin-inducible system, developed recently by de Ruyter et al. (2). This system appears to be well suited for inducing gene expression in Lactococcus lactis by adding the antibiotic nisin (which is accepted as a food additive). A question that perhaps needs to be addressed in this context is whether the nisin expression system is also suitable for achieving a steady level of gene expression. In addition, for effective metabolic optimization, it is often necessary to optimize the expression of a number of genes, which is not feasible with the systems developed so far. Here we describe a method for tuning steady-state gene expression in L. lactis. We overcome many of the limitations discussed above by using libraries of synthetic promoters which cover a wide range of promoter activities and show that the strength of prokaryotic promoters can be modulated by randomizing the spacer sequences that separates the consensus sequences. The system is food grade and well suited for use in industrial bioreactors and food fermentation processes. In addition, the system should be applicable to a broad range of biological systems. (Potential commercial users should be aware that the approach for obtaining the synthetic promoters, as well as the promoter sequences, were filed for patent worldwide [7a]).