Phytases in Saccharomyces cerevisiae. Exploring genes and proteins for the improvement of strains

Doctoral thesis, 2006

Degradation of myo-inositol hexaphosphate (InsP6, phytate) in food or feed by InsP6-degrading enzymes (phytases) is known to increase the availability of minerals and phosphate, which leads to a higher nutritional value. The aim of this work was to explore the extracellular degradation of InsP6 by Saccharomyces cerevisiae. Furthermore, strains with improved InsP6-degradaing capacity were constructed and evaluated, both in batch cultures and an in vitro digestion model. When subjected to phosphate starvation, S. cerevisiae expresses and exports the so called repressible acid phosphatases (rAPs), Pho5, Pho11 and Pho12, to release inorganic phosphate from organic compounds in the surroundings. In this work, it was shown that S. cerevisiae readily uses InsP6 as a phosphate source in the absence of inorganic phosphate, and that it uses the rAPs to degrade it. However, the rAP previously reported to be the most important for extracellular phosphatase activity, Pho5, did not seem to be involved in InsP6 degradation, whereas the expression of PHO11 or PHO12 was indispensable for InsP6 degradation. Expression of any of the genes PHO5, PHO11, or PHO12 was sufficient for degradation of para-nitrophenyl phosphate (pNPP). Interestingly, a strain with deletions in all three genes was also able to hydrolyze the phosphate group from pNPP. This result implies that there might be an additional secreted phosphatase, readily degrading pNPP but not InsP6. Different approaches were taken to increase InsP6 degradation and to make it constitutive. The effect of deleting the negative regulators PHO80 or PHO85, as well as overexpressing PHO4, encoding a transcriptional activator, or PHO5 was investigated. The most effective strain, deficient in PHO80 in combination with overexpression of PHO5, had a biomass specific activity more than nine-fold higher than the derepressed wild-type. The pho80 and pho85 strains were further evaluated in an in vitro digestion system to assess their survival and InsP6 degradation capacity during a simulated gastrointestinal digestion. Addition of the pho85 strain gave promising results, with good survival and up to 60% degradation of the added InsP6 in the model meal. This thesis presents novel knowledge in the area of S. cerevisiae phytase that can be used for further development of phytase-producing strains for feed or food use. It also contributes new data for a better understanding of the roles of the repressible acid phosphatases of S. cerevisiae.