Suicide-committing yeasts assist German scientists striving towards the perfect enzyme for catalysing asymmetric organic reactions.

Manfred Reetz and co-workers, at the Max Planck Institute for Coal Research, Mülheim, used directed evolution and implemented a novel selection strategy - where all non-desired mutant enzymes are automatically eliminated - in their quest to find a rapid way to identify enzymes with advantageous asymmetric catalytic activity.

Directed evolution is commonly used in molecular biology to evolve enzymes with properties not found in nature. Libraries of mutant enzymes are generated from host yeasts containing a gene that encodes a particular natural enzyme. The library is evaluated for its catalytic activity, and the mutated gene from the best mutant enzyme undergoes the same process again. This sequence is repeated until the desired degree of catalyst improvement has been reached.  However 'screening large libraries of mutant enzymes in order to fish out the best ones, is very time-consuming and costly,' explains Reetz.

'Our idea was to devise a selection system which responds to the degree of enantioselectivity of a given asymmetric chemical transformation,' Reetz says. It was designed so that the yeasts producing the enzyme catalysing the desired enantiomer, experience a growth advantage in a kinetic resolution - where one enantiomer undergoes a cleavage reaction to generate an acetate byproduct, which acts as an energy source for the yeast. However cleavage of the undesired enantiomer produces poisonous fluoroacetate. So, the yeasts effectively commit suicide if the enzyme their gene encodes for generates the toxic byproduct.

'Here the undesired enzyme mutants are not formed, which is of great advantage relative to screening systems in which mostly junk mutants are generated,' explains Reetz.

Stefan Lutz, who is a US expert on protein engineering and biomolecular chemistry, at Emory University, Atlanta, says the findings represent a significant advance in the field. 'It is a classic example of a carrot-and-stick idea, Reetz and colleagues have devised a system that uses easily synthesised substrates (acetate versus fluoroacetate esters of their alcohol of interest). It is hard to imagine that one could find something simpler.'

Kathleen Too