Improving enzyme properties through directed evolution

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations. A enzymes are remarkable catalysts, their use in many practical applications is limited by the availability of large quantities of stable enzyme with appropriate catalytic properties. Directed evolution can be usefully employed to enhance enzyme stability and alter substrate specificity to engineer tailored biocatalysts.1−4 Mutations are introduced at the genetic level yielding a library of variant enzymes that are screened for a desired trait. It is extremely difficult to evolve an enzyme to have a completely new catalytic function; rather an existing and weak promiscuous activity can be exploited and greatly enhanced. Here we report the multistep process by which the substrate specificity of the enzyme dienelactone hydrolase (DLH) has been altered so that it can act upon ester and lipase substrates with long alkyl chains (C4−C12), while turnover of these substrates by the native enzyme is virtually undetectable. DLH is one of the many hydrolytic enzymes that has an α/β hydrolase fold. While these enzymes do not necessarily share any significant sequence similarity, they do share remarkably similar tertiary structures and a preserved arrangement of catalytic residues, suggesting possible evolution from a common ancestor. The catalytic residues include a highly conserved triad, consisting of a nucleophile (serine, cysteine, or aspartic acid), an acid, and a histidine. The most significant variations among these enzymes are in the residues responsible for substrate binding. As well as hydrolases, enzymes exhibiting this characteristic fold can include proteases, esterases, peroxidases, lipases, and dehalogenases. It appears as if nature has taken an effective catalytic core and altered the substrate binding domain to cater to a variety of substrates. We have set out to do the same with DLH from Pseudomonas knackmussii one of the simplest of known α/β hydrolase fold enzymes.8−10 DLH is 25.5 kDa monomeric protein and is the third of four enzymes that constitute the chlorocatechol branch of the β-ketoadipate pathway.11−14 It consists of 236 amino acids that form eight strands of β-sheet in the core of the enzyme with one buried α-helix and six others on the solvent accessible surface. The catalytic triad consists of a cysteine (C123), an aspartic acid (D171), and a histidine (H202). Previous studies have shown that mutation of the nucleophile to a serine (C123S) gives an enzyme with altered isomerase activity toward the physiological substrate. The DLH C123S variant was the starting point for the present study, referred to as native DLH in the remainder of this report, since unlike the native cysteine, serine is not prone to oxidation. It has low but detectable activity toward simple substrates like pnitrophenyl acetate and α-naphthyl acetate but almost nonexistent activity toward p-nitrophenyl butyrate and longer chain p-nitrophenyl esters, too low to detect in crude lysates during library screening. We set out to generate DLH variants that have good activity toward long chain p-nitrophenyl esters and in doing so alter the Received: October 7, 2014 Accepted: November 24, 2014 Articles pubs.acs. org/acschemicalbiology © XXXX American Chemical Society A dx.doi. org/10.1021/cb500809f | ACS Chem. Biol. XXXX, XXX, XXX−XXX activity of the enzyme from a simple hydrolase to an esterase or lipase. These are the most highly utilized biocatalysts where they are commonly found in cleaning products and have applications in industries such as food processing and pharmaceutics. Our intention was to first increase the activity toward p-nitrophenyl acetate with the understanding that this would also give increased activity toward esters with longer chains so that large libraries could then be screened for activity toward p-nitrophenyl butyrate. At various stages in this process, we monitored DLH expression and solubility. These latter experiments were done because we were aware that increased activity in crude lysates could be achieved through improvements in expression or solubility. Additionally, we were aware that active site mutations could compromise the overall stability of the protein and thus hinder attempts to evolve for further increases in activity; thus we also monitored changes in stability. ■ RESULTS AND DISCUSSION Overview. Three sequential directed evolution experiments were carried out, each consisting of multiple rounds of mutagenesis and screening (Figure 1). The method of library screening was different for each evolution, reflecting the aims of the experiment. The first evolution selected for improvements in activity toward p-nitrophenyl acetate, a small ester substrate to which the native enzyme possesses a low level of promiscuous activity. Although only one property was targeted, due to the nature of the screens, improvements in other physical properties would also give rise to improvements in the targeted property, termed coselectable properties. For example, when we select for improved activity, a mutation improving protein expression, even a silent mutation, would manifest as a positive variant since the presence of more enzyme would increase substrate turnover rate. Hence, improvements in expression, solubility, or stability would provide positive results under the screening conditions for improved enzymatic activity (see Methods). Over the course of this evolution, especially during the later rounds, several surface mutations were accumulated. These mutations were assumed to be stabilizing since SDSPAGE analysis (Supporting Figure S1, Supporting Information) showed no significant changes in expression or solubility, although SDS-PAGE gives only a gross estimate of these properties so slight changes may have occurred. It is widely acknowledged that a more stable protein has a greater capacity to evolve.20−22 The additional stability confers the ability to accommodate the often destabilizing mutations that are required for the acquisition of new and improved enzyme functions. Given that stabilizing mutations were acquired during the first set of evolution experiments and the proven importance of protein stability on evolvability, a second set of evolution experiments were conducted to produce a variant with increased thermal stability. This variant was then used as the starting point for the third and final set of evolution experiments to increase the activity and specificity of DLH toward longer chain p-nitrophenyl esters. At this point, the level of activity toward the longer chain esters was adequate for highthroughput library screening. Like the first evolution, the second and third also had coselectable properties that gave rise to improved variants under the primary selection pressure. The stability of mutants during the second evolution was determined based on residual activity after incubation at an elevated temperature; therefore more active variants could also be identified under this screen. The coselectable traits for the third evolution toward pnitrophenyl butyrate were the same as the p-nitrophenyl acetate evolution. As with the first evolution, there appeared to be minimal change to expression or solubility based on SDSPAGE analysis (Supporting Figures S2 and S3, Supporting Information). To summarize, the following study presents a three-dimensional search along three consecutive evolutionary trajectories to evolve DLH for improved activity, substrate specificity, and stability. Directed Evolution toward an Evolutionary Intermediate. Six rounds of directed evolution were carried out to improve the activity of DLH toward p-nitrophenyl acetate (Supporting Table S1, Supporting Information). Random mutagenesis in the form of error-prone PCR was used to generate genetic diversity, and libraries of 5000 variants were screened. Library and screening details for all three evolutions are given in Table 1. Three important active site mutations, Q35H, F38L, and Y145C, were found individually in generation one and combined through shuffling in generation two (Figure 2). The combined mutations in the generation two variant (A-2) gave a 16-fold improvement in catalytic activity compared with the native enzyme but reduced the stability of the enzyme as shown by the 5.0 ± 0.4 °C decrease in melting temperature. Melting temperature was determined from the residual activity of the enzymes following heat treatment; Supporting Figure S4, Supporting Information, shows the residual activity plots, and Figure 3 shows the melting temperatures determined by analysis based upon a Hill type equation (see Methods). Mutations and kinetic cons