The extracellular Staphylococcus hyicus lipase was expressed under the control of different promoters in Lactococcus lactis and Bacillus subtilis. Its expression at high and moderate levels is toxic for the former and the latter hosts, respectively. In L. lactis, the lipase was expressed at a high level, up to 30% of the total cellular proteins, under the control of the inducible promoter PnisA. About 80% of the lipase remained associated with the cells. Close to half of this amount remained associated with the inner side of the cytoplasmic membrane as unprocessed pre-pro-lipase. The other half was trapped by the cell wall and partially degraded at the N-terminal end. This result suggests that extracellular proteases degrade the lipase. Surprisingly, the kinetics and the pattern of lipase degradation were different in the two L. lactis subspecies, L. lactis subsp. cremoris and L. lactis subsp. lactis. The extracellular proteolytic systems that degrade lipase are thus different in these closely related subspecies. The incorrect export of the lipase is not due to an inappropriate leader peptide but may be due to an inefficiency of several steps of lipase secretion. We propose that (i) the S. hyicus lipase may require a special accessory system to be correctly exported or (ii) the kinetics of lipase synthesis may be a critical factor for proper folding.Lactic acid bacteria are widely used in the production and preservation of foodstuffs. Since these bacteria are generally regarded as safe, their use as delivery vehicles for foreign proteins in the field of medicine can be envisaged. With recent advances in the field of molecular biology, efficient expression vectors have been developed and allow the expression of heterologous proteins in Lactococcus lactis (53,57,64,68). The aim of this work was to express a bacterial lipase in L. lactis in order to use lactococci as a lipase delivery vehicle. This goal could alleviate lipase deficiency in the digestive tract during digestion (steatorrhea) or improve flavor development in some cheese-making processes (23,35,56).Among the best-characterized bacterial lipases are those of several Pseudomonas and Staphylococcus species (24). The genes encoding these lipases have been tested for heterologous expression in a variety of potential industrial production hosts. It appears that the Pseudomonas lipase cannot be overexpressed in heterologous hosts to commercially acceptable levels because it requires a helper protein to fold properly (15,25). Moreover, the high GC content of Pseudomonas genes may require resynthesis of a gene to fit with the codon bias of the new host. On the other hand, the lipase of Staphylococcus hyicus already has been expressed successfully in Staphylococcus carnosus (66), Escherichia coli (16), and Lactobacillus curvatus (67). No chaperone or specialized helper proteins were reported to be required for the proper folding and secretion of this lipase (24). The GC content of the lip gene, 37%, is similar to that of the lactococcal genes and is comparable t...
