A new Yarrowia lipolytica expression system: An efficient tool for rapid and reliable kinetic analysis of improved enzymes

Protein expression level control is always a great challenge in the field of enzyme evolution. The performance of a dedicated strain of the yeast Yarrowia lipolytica was evaluated. This strain contains a zeta docking platform forcing the integration of the expression cassette at this locus. Activities of 102 transformants expressing the enzyme Lip2p, a lipase from Y. lipolytica, were analysed. Only 1 transformant presented an abnormal activity (57% increase). The others followed a normal distribution with a coefficient of variation of 9.1% and the interval mean ± two standard deviations accounts for 95% of the transformants. This strain was finally used to compare activities of a constructed library of Lip2p single-variants at position 232, located in the active site and crucial for enantiomer discrimination. A simple, non-time-consuming methodology is proposed. It consists in testing 3 transformants to select one representative for each variant. The use of 95% confidence intervals around the mean enables variant activities to be statistically compared. It is the first expression system which enables direct comparison of activities between enzymes or variants from the supernatant. The high reproducibility in expression levels avoids protein purification and quantification steps for false positive variants and avoids real positives from being discarded.