Selection and screening strategies in directed evolution to improve protein stability

Abstract

Protein stability is not only fundamental for experimental, industrial, and therapeutic applications, but is also the baseline for evolving novel protein functions. For decades, stability engineering armed with directed evolution has continued its rapid development and inevitably poses challenges. Generally, in directed evolution, establishing a reliable link between a genotype and any interpretable phenotype is more challenging than diversifying genetic libraries. Consequently, we set forth in a small picture to emphasize the screening or selection techniques in protein stability-directed evolution to secure the link. For a more systematic review, two main branches of these techniques, namely cellular or cell-free display and stability biosensors, are expounded with informative examples.

An enzyme-based biosensor for monitoring and engineering protein stability in vivo

“…Since any fluorescence readout from CysG A depends on its enzymatic activity-which is closely related to the correct folding of CysG A -we envisioned that CysG A could be converted into a stability biosensor, specifically by coupling its capacity for proper folding to the stability of a given POI. To avoid defects with head-to-tail construction reported from previous biosensors (24), such as generating intact and active CysG A upon proteolytic cleavage of unstable POIs, we decided to create a tripartite, sandwich fusion biosensor comprising split CysG A on either side of the POI (Fig. 1A).…”

“…Our design is conceptually similar to other tripartite fusion systems (49). Tripartite fusion design is advantageous compared to the "head-to-tail" design [e.g., the original GFP sensor (50)] in that it can avoid the false positive caused by proteolytic cleavage of a poorly folded POI and alternative translation because of frameshifting or the presence of internal cryptic ribosome-binding sites within the POI gene (24). Many of the available tripartite stability biosensors link cell viability to protein folding by engineering an antibiotic decomposing enzyme such as β-lactamase (51), aminoglycoside 3′-phosphotransferase (17), aminoglycoside 3″-adenylyltransferase (17), or nourseothricin acetyltransferase (17) or using an enzyme essential to cell growth under selective conditions such as orotate phosphoribosyl transferase (52) or DsbA (26).…”

Production of Jet Biofuels by Catalytic Hydroprocessing of Esters and Fatty Acids: A Review

“…In this sense, through metagenomic methodologies, it is possible to access all biodiversity; even enzymes from non-cultivable and/or no longer existing microorganisms may be screened, facilitating the search for the best available enzyme for a given process [122][123][124]. Furthermore, direct evolution allows the mimicry of natural evolution, but in a shorter time period, aiming to improve specific features of the enzymes [124][125][126]. For instance, mutagenesis may alter the enzyme activity by modifying the active site or even ex novo active sites may be formed for creating enzymes with double active sites [127,128].…”

Evolutionary coupling-inspired engineering of alcohol dehydrogenase reveals the influence of distant sites on its catalytic efficiency for stereospecific synthesis of chiral alcohols

“…Currently, directed evolution is a kind of protein engineering that allows proteins to evolve in the desired direction by simulating natural selection. The required protein properties are obtained through the commonly used methods error-prone PCR and DNA shuffling coupled with a large number of screenings [15] , [16] , [17] , [18] . However, the associated workload is quite heavy due to random mutagenesis.…”