Ultrahigh‐Throughput FACS‐Based Screening for Directed Enzyme Evolution

Yang, Guangyu; Withers, Stephen G.

doi:10.1002/cbic.200900384

Cited by 161 publications

(131 citation statements)

References 96 publications

Supporting

Mentioning

123

Contrasting

Order By: Relevance

“…Larger increases in k cat may require modified selection or screening strategies that explicitly couple survival with multiple turnover kinetics, perhaps by integrating our system with in vitro compartmentalization. Despite the widespread use of yeast display in the evolution of binding interactions (18), to the best of our knowledge, sortase A is only the third enzyme to be evolved using yeast display, in addition to horseradish peroxidase (30,31) and an esterase catalytic antibody (32). Our results highlight the attractive features of yeast display that offer significant advantages for enzyme evolution, including quality control mechanisms within the secretory pathway that ensure display of properly folded proteins and compatibility with FACS (18).…”

Section: Discussionmentioning

confidence: 85%

A general strategy for the evolution of bond-forming enzymes using yeast display

Chen

Dorr

Liu

2011

Proc. Natl. Acad. Sci. U.S.A.

508

584

View full text Add to dashboard Cite

The ability to routinely generate efficient protein catalysts of bondforming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A-catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods.

show abstract

Section: Discussionmentioning

confidence: 85%

A general strategy for the evolution of bond-forming enzymes using yeast display

Chen

Dorr

Liu

2011

Proc. Natl. Acad. Sci. U.S.A.

508

584

View full text Add to dashboard Cite

show abstract

“…The stability of GSBs allows them to be handled and analyzed in aqueous solution by standard FACS equipment (rather than custom-made microfluidic on-chip emulsion sorters 25,[31][32][33][34] ). FACS can also be employed for cell-based selections, but they either rely on in vivo live/dead assays 35 or require that the reaction product must be captured in or on cells 36,37 .…”

Section: Discussionmentioning

confidence: 99%

Evolution of enzyme catalysts caged in biomimetic gel-shell beads

et al. 2014

View full text Add to dashboard Cite

Natural evolution relies on the improvement of biological entities by rounds of diversification and selection. In the laboratory, directed evolution has emerged as a powerful tool for the development of new and improved biomolecules, but it is limited by the enormous workload and cost of screening sufficiently large combinatorial libraries. Here we describe the production of gel-shell beads (GSBs) with the help of a microfluidic device. These hydrogel beads are surrounded with a polyelectrolyte shell that encloses an enzyme, its encoding DNA and the fluorescent reaction product. Active clones in these manmade compartments can be identified readily by fluorescence-activated sorting at rates >107 GSBs per hour. We use this system to perform the directed evolution of a phosphotriesterase (a bioremediation catalyst) caged in GSBs and isolate a 20-fold faster mutant in less than one hour. We thus establish a practically undemanding method for ultrahigh-throughput screening that results in functional hybrid composites endowed with evolvable protein components. Evolution of Catalysts Caged in Biomimetic Gel-Shell Beads AbstractNatural evolution relies on improvement of biological entities by rounds of

show abstract

“…It is an ultrahigh-throughput technique, capable of screening up to 10 8 mutants per day (Yang & Withers, 2009). FACS is also characterized by high sensitivity.…”

Section: Facs Screeningmentioning

confidence: 99%