Reporter‐based screening and selection of 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.

In vivo continuous directed evolution

“…While establishing this linkage between desired biomolecule function and cell survival is relatively straightforward for genes that are natively associated with organismal fitness (such as antibiotic resistance, temperature or solvent tolerance, or improved growth rate), the design of selections for continuous evolution is more challenging for genes and gene products that do not intrinsically affect cellular survival. Fortunately, many creative strategies have been described for coupling a wide variety of molecular activities to cell survival, including protein-fragment complementation of enzymatic activities and organelle-specific localization signals, n -hybrid systems for binding activity and bond-formation catalysis [40,41], derepression-based methods for bond-cleaving activities [42], gene regulation-based selections for activities associated with transcription or translation [43], and cis -acting elements for metabolite sensing [44]. We anticipate that the expansion of such selection methods to address an increasingly broad scope of molecular problems of interest will play a key role in fully realizing the potential and defining the future impact of continuous directed evolution.…”

Engineering anAcinetobacterregulon for biosensing and high-throughput enzyme screening inE. colivia flow cytometry

“…Since a transcription factor can sense the concentration of small molecules in the surrounding or inside the cell, it offers a potent opportunity to select biocatalysts (5–7) under directed evolution or screen large DNA libraries for function (8,9). In a transcription factor-based assay method, a reporter gene is added downstream of the promoter region.…”

A Cell-Based High-Throughput Screen Addressing 3′UTR-Dependent Regulation of the MYCN Gene

“…Reporter cell-based assays offer a versatile and technically simple way to test molecules in high-throughput format [6]. However, until recently this kind of assay has been mainly focused on the identification of promoter-targeting compounds [7]. A similar approach in which a reporter gene is flanked by the 5′UTR and/or 3′UTR of a gene of interest could become a valuable tool for identification of molecules targeting post-transcriptional control mechanisms [8–10].…”