Development of a transcriptional regulator-based bioreporter : towards a generic selection method for novel enzymes

Abstract

The biotech industry is continuously seeking for new or improved biocatalysts. The success of these efforts is often hampered by the lack of an efficient screening assay. Thus, to be able to extend the number of enzymes available for industrial applications, high-throughput screening and selection methods are required. In the last few years an impressive range of screening and selection strategies has been developed. In this review, we will mainly focus on in vivo reporter systems in which the activity of a reporter is controlled by the activity of an enzyme of interest. Different mechanisms can be distinguished: (a) binding of the product of the enzymatic reaction to a transcriptional regulator and thereby turning on transcription of the reporter; (b) direct modification of a transcriptional regulator by the enzyme resulting in expression of the reporter; (c) binding of the product to a regulatory riboswitch or ribozyme, resulting in translation of the reporter; and (d) direct modification of the reporter by the enzyme, altering the reporter's activity. The choice for either a selection or a screening strategy depends on the type of reporter, e.g. providing antibiotic resistance (selection) or transmitting a fluorescent signal (screening). Although developing the specificity of each of these reporter-based selection or screening systems towards a certain enzymatic reaction is not yet straightforward, their adjustable modular design appears to be a promise for general applicability in the near future. Reporter-based screening and selection of enzymes