Encapsulation Speeds Up Directed Evolution Of Enzymes | July 28, 2014 Issue - Vol. 92 Issue 30 | Chemical & Engineering News
Volume 92 Issue 30 | p. 35
Issue Date: July 28, 2014 | Web Date: July 23, 2014

Encapsulation Speeds Up Directed Evolution Of Enzymes

Gel-shell beads could make directed evolution of biomolecules easier to carry out
Department: Science & Technology | Collection: Life Sciences
News Channels: Biological SCENE, Materials SCENE
Keywords: directed evolution, enzymes, genetic engineering, high-throughput screening
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CIRCLE GAME
In GSB-based directed evolution, an enzyme is expressed in bacteria (top). The enzyme and its gene sequence (in plasmid) are incorporated into droplets. The substrate is modified so the enzymatic reaction yields a fluorescent product. Droplets form gels that are enclosed in GSBs, and FACS screens the GSBs for activity. Plasmids from the most active enzymes are randomized and entered into additional cycles to optimize enzyme activity.In GSB-based directed evolution, an enzyme is expressed in bacteria (top). The enzyme and its gene sequence (in plasmid) are incorporated into droplets. The substrate is modified so the enzymatic reaction yields a fluorescent product. Droplets form gels that are enclosed in GSBs, and FACs screens the GSBs for activity. Plasmids from the most active enzymes are randomized and entered into additional cycles to optimize enzyme activity.
Credit: Adapted from Nat. Chem.
Diagram illustrating gel-shell directed-evolution technique.
 
CIRCLE GAME
In GSB-based directed evolution, an enzyme is expressed in bacteria (top). The enzyme and its gene sequence (in plasmid) are incorporated into droplets. The substrate is modified so the enzymatic reaction yields a fluorescent product. Droplets form gels that are enclosed in GSBs, and FACS screens the GSBs for activity. Plasmids from the most active enzymes are randomized and entered into additional cycles to optimize enzyme activity.In GSB-based directed evolution, an enzyme is expressed in bacteria (top). The enzyme and its gene sequence (in plasmid) are incorporated into droplets. The substrate is modified so the enzymatic reaction yields a fluorescent product. Droplets form gels that are enclosed in GSBs, and FACs screens the GSBs for activity. Plasmids from the most active enzymes are randomized and entered into additional cycles to optimize enzyme activity.
Credit: Adapted from Nat. Chem.

Enzymes are usually pretty fast workers, but through a technique known as directed evolution, they can be made to work even faster—and in some cases be given completely new jobs. This iterative modification-and-selection technique could someday yield commercial enzymes with improved catalytic activity and therapeutic proteins with refined target selectivity.

Directed evolution mimics biological evolution by mutating biomolecules, selecting mutants that best fit a desired activity profile, and repeating the process multiple times to optimize the improvement.

Florian Hollfelder of Cambridge University and coworkers have now developed a method that could make directed evolution more accessible and allow screening of larger numbers of variants (Nat. Chem. 2014, DOI: 10.1038/nchem.1996).

The method uses microfluidics to make gel-shell beads (GSBs), which encapsulate evolving biomolecules and the DNA sequences that encode them, and it uses fluorescence-activated cell sorting (FACS) to select the most active variants.

The researchers used the technique to make a phosphotriesterase work 20 times as fast as a starting version in less than an hour.

Hollfelder says he hopes the technique will help make directed evolution into “a pervasive technology that is easily implemented.” His team is collaborating with drug and biotech companies to commercialize the technology.

Biocatalysis expert Uwe T. Bornscheuer of the University of Greifswald, in Germany, comments that the GSB approach “allows one to identify improved enzymes within a very short time and appears to be rather simple and robust.” However, he notes, “it stills needs to be demonstrated that one can use it not only for phosphotriesterases but for other enzyme classes too.”

Chemical biologist and biocatalysis specialist Nicholas J. Turner of Manchester Institute of Biotechnology adds, “The ability to screen enzymes in a high-throughput manner has become a bottleneck in the discovery of new biocatalysts. Methods such as GSB will undoubtedly accelerate this process, particularly if they turn out to be applicable to a broad range of different enzyme activities.”

In the technique, bacteria are genetically engineered to harbor a plasmid encoding a modified version of the gene for the enzyme to be improved. A microfluidic droplet generator incorporates plasmids and expressed enzymes from single bacteria into droplets containing a liquid gel. Separately, the enzyme’s substrate is modified so the enzymatic reaction will produce a fluorescent product. After the reaction takes place, cooling solidifies the gel, turning the droplets into beads. The beads are then coated with polyelectrolyte layers, forming GSBs. Enzymes with better activity produce more intense fluorescence, and FACS is used to identify the most highly fluorescent GSBs. Plasmids in the selected GSBs are extracted and entered into subsequent rounds of directed evolution until an optimized enzyme is obtained.

GSBs are much simpler to sort than in previous directed-evolution experiments because the transformation of droplets into gel beads immortalizes droplet compartmentalization, making it possible to use widely available FACS instruments to screen many more of them than has generally been feasible.

The approach could be used to evolve not only single enzymes but also individual components in sequential enzyme cascades, tandem reactions, enzymatic pathways, or synthetic gene circuits, the researchers predict. Hollfelder suggests it might also be used to cage and recycle enzymes, as an alternative to using surface-bound enzymes.

 
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