Adventures in Directed Evolution
Adventure I. The Origins of Molecular Breeding
In 1994, Willem “Pim” Stemmer (1957–2013) invented molecular breeding for directed evolution, the key feature of which is recombination of amino acid diversity. The 1998 Nature paper of Crameri et al established the power of using natural diversity to achieve functional improvements of proteins.The value of this approach is highlighted in a classic 2004 Science paper (PDF) by Linda Castle et al showing that the activity of an enzyme that inactivates glyphosate can be increased nearly 10,000-fold.
Adventure II. A Nobel Prize for Directed Evolution
In 2018, Frances Arnold, Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry, won the Nobel Prize in Chemistry “for the directed evolution of enzymes.” Although Manfred Eigen (himself a Nobel Laureate) proposed a theoretical framework for this process in 1984, the first successful implementation was the 1993 paper by Chen and Arnold who used error-prone PCR to create libraries of protein-coding DNA sequences. In this seminal paper, Frances Arnold “had mastered the whole work flow for directed evolution of enzymes” (from Scientific Background on the Nobel Prize in Chemistry 2018).
Much has been done and much has changed since these early studies but the principle remains the same: genetic diversity when suitably screened can lead to remarkable improvements in protein function.
Adventure III. SuperFolder Protein Variants
Directed evolution is widely used to optimize enzyme activities. However, the method is applicable to a much wider range of protein functions. A recent article by Laurent et al from Michael Traxlmayr’s group in Vienna studied the human CD19 protein, a B-cell-specific protein. The isolated extracellular domain (ECD) of this protein is subject to aggregation and misfolding. Using a library of random mutations and surface display in yeast, they identified stabilized CD19-ECD variants that could be well expressed and efficiently purified. The concept of SuperFolder variants can be of use for many applications.
Adventure IV. Continuous Evolution of Proteins in vivo
Prof. Chang Liu and his colleagues at UC Irvine have developed a very intriguing and powerful directed evolution approach using an orthogonal error-prone DNA polymerase in yeast. In the OrthoRep system, genes continuously and rapidly evolve through serial passaging. Scalable continuous cultures can be automated, allowing for precise regulation of growth conditions. Results from the OrthoRep system also provide insight into some of the characteristics of protein evolution.
Adventure V. COVID and Directed Evolution
In laboratory-based directed evolution experiment, a “library” of protein variants is submitted to selective pressure. Given sufficient diversity, variants are likely to exist that can overcome the selection.
Now consider what is happening in areas of the United States that are highly vaccinated but are also “hot” spots of the delta variant of SARS-CoV-2. This would appear to be a real-world directed evolution experiment.
The delta virus can mutate to create variants, some of which might avoid vaccine-induced immunity in the population and cause disease. Such variants might aggravate an already serious situation.
Science Magazine has an extensive discussion of the evolution of SARS-CoV-2.