Characterization ofYarrowia lipolyticaXPR2multi-copy strains over-producing alkaline extracellular protease - a system for rapidly increasing secretory pathway cargo loads

Abstract

Upon transfer to alkaline extracellular protease (AEP) induction medium, strain 773-2 (50 integrated copies of XPR2), derived from highly inbred strain E129, grew for at least 10 h before AEP production began, and then growth rate decreased before increasing again; by then, cells had lost copies of XPR2 (Le Dall et al., 1994). Slowing of growth following AEP induction suggested that increased secretory pathway cargo load was affecting cell growth and that such a system had potential for secretion stress studies. Development of W29-derived XPR2 multi-copy strains and improved AEP induction conditions realized this potential. AEP production was sixfold higher than for 773-2. Rapid AEP induction and slowing of growth by 3 h minimized loss of XPR2 gene copies. Two strains, examined in more detail, differed in initial AEP productivity, extent of slowing of growth during AEP induction, and subsequent recovery of growth rate and AEP productivity demonstrating that the system provides a range of secretion stresses and ensuing adaptations. W29-derived strains should be more 'wild type' than 773-2 for secretory pathway components and their regulation. They should provide an excellent system for kinetic analysis of gene expression responses to acute increases in secretory pathway cargo load.