Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.     

Novel Ree1 regulates the expression of ENO1 via the Snf1 complex pathway in Saccharomyces cerevisiae

Using cDNA microarray analysis, we found that the mRNA of YJL217W and several other genes related to cell wall organization and biogenesis were up-regulated by galactose in Saccharomyces cerevisiae early during the induction process. YJL217W is also known as REE1 (Regulation of Enolase I). Both the Gal4 regulatory region and the Mac1 binding domain were found on the upstream region of REE1, and the expression of REE1 was up-regulated by galactose but not by glucose. The up-regulation of REE1 by galactose was not observed in the Δgal4 strain. From the two-hybrid analysis, we found that Ree1 physically interacted with Gal83. Furthermore, from 2-D gel electrophoresis we found that the deletion of REE1 resulted in the up-regulation of Eno1. From Western blotting, we learned that the expression of Eno1 in the Δree1 strain was different from that in wild-type strains and that Eno1 expression was not changed by glucose stimulation. Taken together, these results suggest that Ree1p functions in the galactose metabolic pathway via the Gal83 protein and that it may control the level of Eno1p, which is also affected by the Snf1 complex, in S. cerevisiae.