Directed Evolution

This book focuses on some of the most significant advances in enzyme engineering that have been achieved through directed evolution and hybrid approaches. On the 25th anniversary of the discovery of directed evolution, this volume is a tribute to the pioneers of this thrilling research field, and at the same time provides a comprehensive overview of current research and the state of the art. Directed molecular evolution has become the most reliable and robust method to tailor enzymes, metabolic pathways or even whole microorganisms with improved traits. By mirroring the Darwinian algorithm of natural selection on a laboratory scale, new biomolecules of invaluable biotechnological interest can now be engineered in a manner that surpasses the boundaries of nature. The volume is divided into two sections, the first of which provides an update on recent successful cases of enzyme ensembles from different areas of the biotechnological spectrum, including tryptophan synthases, unspecific peroxygenases, phytases, therapeutic enzymes, stereoselective enzymes and CO2-fixing enzymes. This section also provides information on the directed evolution of whole cells. The second section of the book summarizes a variety of the most applicable methods for library creation, together with the future trends aimed at bringing together directed evolution and in silico/computational enzyme design and ancestral resurrection.

Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum “you get what you screen for. ” The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective conditions.

Biological systems are very special substrates for engineering—uniquely the products of evolution, they are easily redesigned by similar approaches. A simple algorithm of iterative cycles of diversification and selection, evolution works at all scales, from single molecules to whole ecosystems. In the little more than a decade since the first reported applications of evolutionary design to enzyme engineering, directed evolution has matured to the point where it now represents the centerpiece of industrial biocatalyst development and is being practiced by thousands of academic and industrial scientists in com- nies and universities around the world. The appeal of directed evolution is easy to understand: it is conceptually straightforward, it can be practiced without any special instrumentation and, most important, it frequently yields useful solutions, many of which are totally unanticipated. Directed evolution has r- dered protein engineering readily accessible to a broad audience of scientists and engineers who wish to tailor a myriad of protein properties, including th- mal and solvent stability, enzyme selectivity, specific activity, protease s- ceptibility, allosteric control of protein function, ligand binding, transcriptional activation, and solubility. Furthermore, the range of applications has expanded to the engineering of more complex functions such as those performed by m- tiple proteins acting in concert (in biosynthetic pathways) or as part of mac- molecular complexes and biological networks.

This volume explores the latest techniques used by researchers to study directed evolution (DE) at each stage of the Design-Build-Test-Learn cycle. Chapters in this book cover topics such as designing overlap extension PCR primers for protein mutagenesis; antha-guided automation of Darwin assembly for the construction of bespoke gene libraries; rapid cloning of random mutagenesis libraries using PTO-Quickstep; and DE of glycosyltransferases by a single-cell screening method. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Directed Evolution: Methods and Protocols is a valuable resource for scientists and researchers who are interested in learning more about this field and incorporating these studies into new experimental workflows.

Authored by one of the world's leading organic chemists, this authoritative reference provides an overview of basic strategies in directed evolution and introduces common gene mutagenesis, screening and selection methods. Throughout the text, emphasis is placed on methodology development to maximize efficiency, reliability and speed of the experiments and to provide guidelines for efficient protein engineering. Professor Reetz highlights the application of directed evolution experiments to address limitations in the field of enzyme selectivity, substrate scope, activity and robustness. He critically reviews recent developments and case studies, takes a look at future applications in the field of organic synthesis, and concludes with lessons learned from previous experiments.

A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.

The Arthur M. Sackler Colloquia of the National Academy of Sciences address scientific topics of broad and current interest, cutting across the boundaries of traditional disciplines. Each year, four or five such colloquia are scheduled, typically two days in length and international in scope. Colloquia are organized by a member of the Academy, often with the assistance of an organizing committee, and feature presentations by leading scientists in the field and discussions with a hundred or more researchers with an interest in the topic. Colloquia presentations are recorded and posted on the National Academy of Sciences Sackler colloquia website and published on CD-ROM. These Colloquia are made possible by a generous gift from Mrs. Jill Sackler, in memory of her husband, Arthur M. Sackler.

Based on a comprehensive review of human and societal evolution the book develops an approach to conscious, self-guided evolution. In the course of the evolutionary journey of our species, there have been three seminal events. The first happened some seven million yeas ago, when our humanoid ancestors entered on the evolutionary scene. Their journey toward the second crucial event lasted over six million years when - as the greatest event of our evolutionary history - homo sapiens sapiens, started the revolutionary process of cultural evolution. Today, we have arrived at the threshold of the third major event, `the revolution of conscious evolution,' when it becomes our responsibility to enter into the evolutionary design space and guide the evolutionary journey of our species. The book tells the story of the first six million years of the journey in just enough detail to understand how evolution had worked in times when it was primarily biological, driven by natural selection. With the human revolution some fifty thousand years ago, with the emergence of self-reflective consciousness, the evolutionary process transformed from biological into cultural. From this point on, the book follows the journey with detailed attention, in order to learn how cultural evolution works. The book is organized in three parts. Part One commences with an exposition of a brief history of the evolutionary idea through time with a focus on a review of the science of general evolution and specifically social and societal evolution. Next, the book unfolds the `evolutionary story' of our species from the time when the first humanoids entered the evolutionary scene to our current era. Part Two develops a systems view of evolution, explores the ways and means of how evolution works, characterizes evolutionary consciousness and develops the idea of conscious evolution. Part Three builds upon the knowledge developed in the first two parts and sets forth the key conditions of conscious, self-guided evolution, elaborating the core condition, which is the acquisition of evolutionary competence through evolutionary learning. The focus of this part is on an approach to the design of evolutionary guidance systems that our families, neighborhoods, communities, organizations, social and societal systems can use to design the future they aspire to attain. The work is set aside from other statements in three important ways. It provides: (1) a comprehensive review of how evolution has worked with a focus on socio-cultural evolution, (2) an explanation of evolutionary consciousness and the conditions of engaging in conscious evolution, and (3) most significantly, it develops a detailed approach and a methodology to the design of evolutionary guidance systems.