Directed evolution - improving enzyme properties

Abstract

Although enzymes are the product of millions of years of natural evolution, their properties are frequently far from what is desired by biochemists. Directed evolution has long been used to improve protein properties: stability, expression, solubility, enantioselectivity, activity, and specificity as well as to dissect the relationship between protein structure and function. It mimics natural evolution as proposed by Darwin, in which large number of mutations are introduced randomly over the course of centuries and followed by selection of the fittest. Directed evolution is a useful and indispensable tool to identify mutations and can be accomplished within months in laboratories. During the course of this study, three proteins were subjected to directed evolution to enhance their solubility and catalytic activity. Selection for soluble protein was based on the ability of bacteria expressing DHFR-fused mutants to grow on high concentration of the antibiotic, trimethoprim. E. coli glucose-6-phosphate dehydrogenase, a critical enzyme in biochemical pathways was evolved to enhance its solubility for use in in vitro ethanol production. This experiment interestingly led to the selection of variants with altered oligomerisation state that retained activity. Klebsiella pneumonia metallo-{u03B2}-lactamase, KpMBL was subjected to molecular evolution to predict antibiotic and inhibitor resistance that may arise in the future. However, these experiments were hindered by inefficiency of the selection technique and toxic metabolic load on the bacteria. This led to experiments as to how bacteria growing on agar plates could cope with high levels of protein expression. As a result, mutations that may lead to increased enzyme activity were identified. Serratia protemaculans metallo-{u03B2}-lactamase, SpMBL was evolved for increased solubility via trimethoprim resistance selection when in a fused form or through enhanced ampicillin resistance of the protein. These two strategies led to identification of mutants possessing several mutations that consequently provided insights into the enzymes structure and function and how future resistance could arise. The ability of directed evolution to produce new enzymes as well as elucidate how enzymes function illustrates the versatility and power of directed evolution. Show more