1
Introduction to Directed Evolution and Rational Design as Protein Engineering Techniques

1.1 Methods and Aims of Directed Enzyme Evolution

For decades organic chemists have viewed enzymes as possible catalysts in their toolbox of synthetic methods with a great deal of skepticism, traditional textbooks hardly mentioning them. Indeed, even modern monographs on organic chemistry provide little information on the importance of these biocatalysts [1]. On the other hand, textbooks on enzymes in organic chemistry and biotechnology have interested mainly specialists [2], not organic chemists. Apart from psychological reasons, the actual limitations of enzymes were known to everyone, which are as follows:

• Insufficient robustness under operating conditions.
• Insufficient activity.
• Narrow substrate scope.
• Insufficient or wrong stereoselectivity.
• Insufficient or wrong regioselectivity.
• Sometimes product inhibition.

In addition, organic chemists were not trained to handle enzymes and therefore preferred to be content with the use and development of their own synthetic homogeneous and heterogeneous catalysts, knowing that enzymes cannot catalyze many or most of the important reaction types that dominate modern synthesis. Today all of these problems can be addressed and generally solved by applying directed evolution, as summarized in a 2016 monograph [3] authored by Manfred T. Reetz, and in more recent reviews [4]. It mimics Darwinian evolution as it occurs in nature, but it does not constitute real natural evolution. The process consists of several steps (Scheme 1.1), beginning with mutagenesis of the gene encoding the enzyme of interest. The library of mutated genes is then inserted into a bacterial or yeast host, such as Escherichia coli or Pichia pastoris, respectively, which is plated out on agar plates. After a growth period, single colonies appear, each originating from a single cell, which now begins to express the respective protein variants. This ensures the required linkage between phenotype and genotype. Multiple copies of transformants as well as wild type (WT) appear, which unfortunately decreases the quality of libraries and increases the screening effort. Colony harvesting must be performed carefully because cross contamination leads to the formation of inseparable mixtures of mutants with concomitant misinterpretations. The colonies are picked by a robotic colony picker (or manually using toothpicks) and placed individually in the wells of 96- or 384-format microtiter plates, which contain nutrient broth. Portions of each well content are then placed in the respective wells of another microtiter plate where the screening (Chapter 2) for a given catalytic property ensues. In some (fortunate) cases, a sufficiently improved variant (hit) is identified in such an initial library. If this does not happen, which proves to be the case most often, then the gene of the best variant is extracted and used as a template in the next cycle of mutagenesis/expression/screening (Scheme 1.1). This exerts "evolutionary pressure," the underlying characteristic of directed evolution.

Scheme 1.1 The basic steps in the directed evolution of enzymes.

If a library in a recursive mode fails to harbor an improved mutant/variant, the Darwinian process ends abruptly at a local minimum on the fitness landscape. Fortunately, researchers have developed ways to escape from such local minima ("dead ends") (Chapter 4).

Directed evolution is thus an alternative to so-called "rational design" in which the researcher utilizes structural, mechanistic, and sequence information, possibly flanked by computational aids, to perform site-directed mutagenesis at a given position in a protein (Section 1.3). Needed for this alternative is the molecular biological technique of site-specific mutagenesis with exchange of an amino acid at a specific position in a protein by one of the other 19 canonical amino acids, as established by Michael Smith in the late 1970s [5a], which led to the Nobel Prize [5b]. The method is based on designed synthetic oligonucleotides and has been used extensively by Fersht [6] and numerous other groups in the study of enzyme mechanisms. Application of the Smith technique in rational enzyme design has been shown to be successful mainly when aiming to increase protein robustness (Section 1.3 and Chapter 6). However, when aiming for enhanced or reversed enantioselectivity, diastereoselectivity, and/or regioselectivity, rational design is much more difficult, in which case directed evolution is generally the preferred strategy [3, 4].

Directed evolution of enzymes is not as straightforward as it may appear to be. The challenge in putting Scheme 1.1 into practice has to do with the vastness of protein sequence space. High structural diversity is easily achieved in random mutagenesis, but the experimenter is quickly confronted by the so-called "numbers problem," which in turn relates to the screening effort (bottleneck). When mutagenizing a given protein, the theoretical number of variants N is described by Eq. (1.1), which is based on the use of all 20 canonical amino acids as building blocks [3, 4]:

where M denotes the total number of amino acid substitutions per enzyme molecule and X is the total number of residues (size of protein in terms of amino acids). For example, when considering an enzyme composed of 300 amino acids, 5700 different mutants are possible if only one amino acid is exchanged randomly, 16 million if two substitutions occur simultaneously, and about 30 billion if three amino acids are substituted simultaneously.

Calculations of this kind pinpoint a dilemma that accompanies directed evolution to this day, namely how to probe the astronomically large protein sequence space efficiently. One strategy is to limit diversity to a point at which screening can be handled within a reasonable time, but excessive reduction of diversity reduction should be avoided because then the frequency of hits in a library begins to diminish dramatically. Finding the optimal compromise constitutes the primary issue of this monograph. A very different strategy is to develop selection systems rather than experimental platforms that require screening (Chapter 2). In a selection system, the host organism thrives and survives because it expresses a variant having the catalytic characteristics that the researcher wants to evolve. A third approach is based on the use of various types of display systems, which are sometimes called "selection systems," although they are more related to screening. These issues are delineated in Chapter 2, which serves as a guide for choosing the appropriate system. Since it is extremely difficult to develop genuine selection systems or display platforms for directed evolution of stereo- and regioselective enzymes, researchers had to devise medium- and high-throughput screening systems (Chapter 2).

1.2 History of Directed Enzyme Evolution

Readers of this monograph may not be interested in historical aspects, but certainly doctoral students and postdocs who want to learn directed evolution are advised to read this section carefully. Much can be learned by seeing in some detail how a given field developed, some contributions being interesting but perhaps lacking essential conclusions for further work, others actually opening a new research field by posting seminal publications. At the end of this section, the most important developments are summarized in a "timeline" scheme.

Scientists have strived for a long time to "reproduce" or mimic natural evolution in the laboratory. In 1965-1967 Spiegelman et al., performed a "Darwinian experiment with a self-duplicating nucleic acid molecule" (RNA) outside a living cell [7]. It was believed that this mimics an early precellular evolutionary event. Later investigations showed that Spiegelman's RNA molecules were not truly self-duplicating, but his contributions marked the beginning of a productive new research area on RNA evolution, fueled seminally by Szostak and Joyce [8]. Directed evolution at RNA level is a very different field of research with totally different goals, focusing on selection of RNA aptamers, selection of catalytic RNA molecules, or evolution of RNA polymerase ribozyme and ribozymes by continuous serial transfer. Directed evolution in this area has been reviewed [8b]. The term "directed evolution" in the area of protein engineering was used as early as 1972 by Francis and Hansche, describing an in vivo system involving an acid phosphatase in Saccharomyces cerevisiae [9]. In a population of 109 cells, spontaneous mutations in a defined environment were continuously screened over 1000 generations for their influence on efficiency and activity of the enzyme at pH?6. A single mutational event (M1) induced a 30% increase in the efficiency of orthophosphate metabolism. The second mutational event (M2 in the region of the structural gene) led to an adaptive shift in the pH optimum and in the enhancement of phosphatase activity by 60%. Finally, the third event (M3) induced cell clumping with no effect on orthophosphate...