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

Abstract

The ability to routinely generate efficient protein catalysts of bond-forming 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.

Fig. 1.

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

Fig. 2.

Validation of the enzyme evolution strategy. (A) FACS histogram of the reaction between cell surface–conjugated LPETGG and free GGGYK-biotin catalyzed by yeast-displayed WT S. aureus sortase A (WT srtA). Cells were stained with streptavidin-PE and an AlexaFluor488-anti-HA antibody. Negative control reactions with either the inactive C184A srtA mutant or without LPETGG are shown. (B) Dot plots comparing PE fluorescence (extent of reaction) vs. AlexaFluor488 fluorescence (display level) for two model screens. Mixtures of cells displaying either WT srtA or the inactive C184A srtA (1∶1,000 and 1∶100 WT:C184A) were processed as in A, then analyzed by FACS. Cells within the specified gate (black polygon) were collected. (C) Model screening results. Gene compositions before and after sorting were compared following HindIII digestion, revealing strong enrichment for active sortase.

Fig. 3.

Activity assays of mutant sortases. (A) Yeast pools recovered from the sorts were treated with TEV protease, and the cleaved enzymes were assayed for their ability to catalyze coupling between 5 μM CoA-LPETGG and 25 μM GGGYK-biotin. (B) Yeast cells expressing select individual clones were treated as described above. Error bars represent the standard deviation of three independent experiments.

Fig. 4.

Mutations in evolved sortases. (A) Highly enriched mutations are highlighted in black; other mutations are shown in blue. (B) Mapping evolved mutations on the solution structure of WT S. aureus sortase A covalently bound to its Cbz-LPAT substrate. The calcium ion is shown in blue, the LPAT peptide is colored cyan with red labels, and the side chains of amino acids that are mutated are in orange. The N-terminal Cbz group is shown in stick form in cyan.

Fig. 5.

Cell-surface labeling with WT and mutant sortases. Live HeLa cells expressing human CD154 conjugated at its extracellular C terminus to LPETG were incubated with 1 mM GGGYK-biotin and no sortase A (srtA), 100 μM WT srtA, or 100 μM P94S/D160N/K196T srtA. The cells were stained with AlexaFluor-conjugated streptavidin. (A) Flow cytometry analysis comparing cell labeling with WT sortase (blue) and the mutant sortase (red). Negative control reactions omitting sortase (black) or LPETG (green) are shown. (B) Live-cell confocal fluorescence microscopy images of cells. The YFP (transfection marker) and Alexa (cell labeling) channels are shown.