ASM News 2000 April, 66(4): 211-217
Bylina, E.J., Coleman, W.J., Dilworth, M.R., Robles, S.J., Tanner, M.A., Yang, M.M., and Youvan, D.C.
Excerpts from ASM News' feature article
Tailor-made enzymes are expected to become increasingly important in the synthesis of commercially valuable substances, including specialty chemicals and pharmaceuticals. Despite the growing popularity of directed evolution techniques to generate vast libraries of mutants expressing variant enzymes, identifying winners will require sophisticated approaches to high-throughput screening of such mutants. With one such technology, derived from the field of digital imaging spectroscopy, we now can follow enzyme kinetics in thousands of individual microcolonies-enabling us to screen for the proverbial needle in a haystack.
Why is There a Screening Bottleneck?
The "sequence space" encompassing enzyme variants that may have novel properties is immense. For example, there are approximately 201,000 possible protein sequences-many, many more than the 1080 particles estimated to exist in the universe! Thus, unless one believes the anthropic principle is at play in directed evolution (see box p.217), there is no reason to believe that favorable combinations of neutral mutations will randomly yield the particular enzyme variant one is seeking.
Yesterday's Relative Simplicity...
For some of us, an interest in large-scale screening mutants began almost 20 years ago. At that time, two of us (D.Y. and E.B.) were interested in identifying the genes encoding the photosynthetic reaction center and light-harvesting antennae from a purple nonsulfur bacterium, Rhodobacter capsulatus.
... Gives Way to Today's Increasing Complexity
The need to examine complex libraries of mutants (typically containing 104 to 106 members) to find enzymes with sequences that encode a desired function is further compounded in the case of directed evolution, where iterative rounds of mutagenesis and screening are usually required to find variants with appropriate properties.
A Model Enzyme for Evolution and Imaging
Our recent efforts using this solid phase assay to obtain an improved version of the Agrobacterium faecalis ß-glucosidase or "abg" help to demonstrate how this enzyme directed-evolution system works. This enzyme is relatively nonspecific, catalyzing the hydrolysis of many different substituted glucosides, galactosides, xylosides, fucosides, and arabinosides.
Imaging Spectroscopy and Software Analysis
Digital imaging spectroscopy (DIS) combines the advantages of digital image processing and optical spectroscopy so that each picture element (pixel) or group of pixels in a two-dimensional scene also includes a third dimension of spectral information. In developing instruments used in our Kcat system, we also added a fourth dimension corresponding to time. Thus, a single run of the Kcat instrument is functionally equivalent to having tens of thousands of spectrophotomers operating in parallel.
Gene Shuffling, Anthropic Principle Lead to a Huge Screening Problem
There seems to be a widespread misunderstanding among practioners of directed evolution. Some of them mistakenly believe that, when "gene shuffling" brings two neutral mutations together, this new combination necessarily proves beneficial. Another, even cruder example of this simplistic anthropomorphic notion, which some physicists call the anthropic principle, is the idea that elephants exist because of a "need for piano keys."
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