Screening for and characterization of phospholipase A(1) hypersecretory mutants of Tetrahymena thermophila

We have described a procedure for the isolation of mutants of Tetrahymena t hermophila with hyperscretion of phospholipase A(1) (PLA(1)). Using random chemical mutagenesis,. uniparental cytogamy, genetic crossing and a new, fa st and effective screening procedure, four PLA(1)-hypersecretory mutants we re isolated. The screening procedure is based on the formation of a halo ap pearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this proc edure in primary screening for hypersecretory features, of which 60 putativ e hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8-2.2 more PLA(1) acti vity in the cell-free supernatants compared to the wild-type strain CU 438. 1. Hypersecretion was only observable for PLA(1); no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutant s of T. thermophila can be very useful for increasing the yield of PLA(1) i n fermentation processes. This is particularly relevant because, in contras t to other phospholipases, PLA(1) is not available on the commercial market for fine chemicals and little is known about the role of PLA(1) in cell si gnaling and metabolism.