Directed Evolution in Action
Molecular Breeding, DNA Shuffling
Interest from Industry
Introduction (Back to Top)
Maxygen Inc. (Redwood City, CA) has obtained an exclusive worldwide license from the California Institute of Technology (Pasadena) for the Staggered Extension Process (StEP) directed evolution technology, an iterative, molecular technique that causes fragile, garden-variety proteins to evolve into usable chemical catalysts and drugs. StEP was developed by CalTech professor Frances Arnold, with whom Maxygen has also entered into a research collaboration.
The point of the collaboration and licensing deal is to improve the performance and extend the utility of enzymes and proteins. Potential products include enzymes for the manufacture of fine chemicals and pharmaceuticals, as well as end-use therapeutic proteins and enzymes
Industry's desire to make better proteins is an old story. At the organism level, scientists have tried to induce the evolution of hardy microorganisms and proteins simply by subjecting organisms to harsh chemical conditions or temperature extremes and growing whatever organisms survived. Sometimes it worked, most often it did not. By directed evolution, in combination with Maxygen's other molecular-level protein engineering methods, Maxygen hopes to achieve the same end effect as induced evolution but without the uncertainty of conventional breeding.
Directed Evolution in Action (Back to Top)
The Arnold laboratory at CalTech is one of the leading groups using directed evolution and enzyme optimization for industrial and biotech applications. A recent, and very successful example of directed evolution was carried out by Arnold to produce an industrially viable enzyme that hydrolyzes a para-nitrobenzyl ester of an antibiotic (see references). By applying sequential generations of random mutagenesis, recombination, and screening, the enzyme's catalytic efficiency has been increased more than 100-fold. While yielding a powerful new catalyst for important synthetic reactions, this work demonstrates that enzymes can acquire capabilities not found in naturally occurring organisms. It also shows that these capabilities may be conferred without conventional breeding or rational protein design.
Mutations identified in a pNB esterase optimized by directed evolution. Both activity and temperature stability improved in mutants compared with the natural enzyme.
Molecular Breeding, DNA Shuffling (Back to Top)
Anyone who attends a dog show marvels at the enormous diversity achieved by breeding. Selective breeding within a single species has produced a wide array of dogs, ranging from chihuahuas to wolfhounds. In plants, just a few generations of breedingwithin or between speciescan produce substantial variation. Now such focused diversity can be achieved with amazing speed at a molecular level. Molecular breeding is a simple and efficient technology for functional optimization of DNA sequences, including plasmids, viruses, genes, and partial genomes.
Molecular breeding is based on the same processes as classical breeding, but on the molecular vs. the organism level. As in traditional breeding, the process only requires being able to detect incremental improvements and does not require the information necessary for improvement by rational design.
Molecular breeding consists of taking hereditary material (DNA) of one or several individuals, the sequences of which need not be known, and recursively applying a process of mutation, selection, and amplification, until no further improvement can be achieved. DNA shuffling is a powerful method used for molecular breeding, and forms the proprietary foundation of Maxygen's technology.
DNA shuffling is essentially sexual recombination at a molecular level. Successful DNA shuffling often depends upon the ability to select or screen for a desired improvement in function of the target sequence. Weak activity can be improved to strong activity by shuffling, if a selectable phenotype is available.
Interest from Industry (Back to Top)
Molecular breeding certainly works in the lab, but its success will be measured in how easily the process can be scaled up and adapted to industrially significant proteins. Maxygen has three commercial partnerships in place to demonstrate that its technology will work for large-scale processes: with Novo Nordisk (for industrial enzymes), DuPont/Pioneer Hi-Bred (ag-bio products), and DSM Anti-Infectives/Gist Brocades (improved manufacturing of penicillin-type antibiotics).
References (Back to Top)
- Moore and Arnold, Nature Biotechnology, 14:458467 (1996).
- Moore et al., J. Molecular Biol., 272, 336347 (1997).
For more information: Simba Gill, Vice President, Business Development, Maxygen Inc., 515 Galveston Dr., Redwood City, CA 94063. Tel: 650-298-5300. Fax: 650-364-2715.
By Angelo DePalma