Screening for and characterization of phospholipase A₁ hypersecretory mutants of Tetrahymena thermophila

Abstract

We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hyperscretion of phospholipase A₁ (PLA₁). Using random chemical mutagenesis, uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA₁-hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8–2.2 more PLA₁ activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA₁; no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA₁ in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA₁ is not available on the commercial market for fine chemicals and little is known about the role of PLA₁ in cell signaling and metabolism.