A novel gene MPR1 which was previously found in a yeast Saccharomyces cerevisiae S1278b strain by the present inventor (non-patent literature 1) codes for N-acetyltransferase (Mpr1) which detoxifies the toxic analogue of amino acid proline, azetidine-2-calboxylate (AZC), by the acetylation of AZC (FIG. 1) (non-patent literature 2).
AZC enters into cells via proline permease and is incorporated into proteins by competing with proline upon protein synthesis. As a result of the incorporation of AZC, proteins with abnormal structures and impaired functions accumulate in cells and inhibit the cell growth. In cells which express MPR1, AZC is subjected to the N-acetylation in cytosol and is not incorporated into nascent proteins and therefore, such cells are thought to acquire AZC resistance (non-patent literature 3).
As a result of homology search, homologous genes which have similar functions as MPR1 from fission yeast Schizosaccharomyces pombe and, sibling species of S. cerevisiae, S. paradoxus (ppr1+, Spa MPR1) were identified (non-patent literatures 4 and 5) and therefore, MPR1 is believed to be widely distributed among yeasts. However, interestingly, S. cerevisiae S288C strain which was used for the genomic analysis of yeasts and sake yeast lack MPR1. MPR1 is located near the subtelomeric region of 14th chromosome of S1278b strain. A homologous gene (MPR2) which differs from MPR1 only at the 85th residue is located near the subtelomeric region of 10th chromosome and there is no difference in the functions between MPR1 and MPR2 (non-patent literature 1). Comparing S288C strain and S1278b strain, though some genes show base substitutions and deletions, no gene other than MPR1 that is present only in S1278b strain and codes for a certain phenotype has been reported.
AZC is rare in nature and therefore, is probably not the intrinsic substrate of MPR1 product (Mpr1). Accordingly, the present inventors analyzed the biological function of Mpr1 and identified the intracellular substrate of the same (non-patent literature 6). Firstly, cells of MPR1•MPR2 disruptant were exposed to oxidative stresses such as hydrogen peroxide and heat shock. As a result, the MPR1•MPR2 disruptant showed decreased survival rate and increased intracellular reactive oxygen species (ROS) level compared to the wild-type strain. On the other hand, when multicopies of MPR1 were introduced into S. cerevisiae 288C strain which originally lacks MPR1 and MPR2, the strain exhibited increased survival rate under oxidative stress and decreased ROS level. Accordingly, it was confirmed that Mpr1 lowers the intracellular oxidative level. PUT2 disruptant which accumulates a metabolic intermediate of proline, Δ1-pyrroline-5-calboxylate (P5C), was confirmed to show growth impairment and increase in ROS level. Therefore, the role of Mpr1 in PUT2 disruptant was examined thereafter. The result showed that when MPR1 and MPR2 in PUT2 disruptant are disrupted, the growth is strongly inhibited and the ROS level is increased, whereas the overexpression of MPR1 decreased the ROS level. Further, the analysis using a recombinant enzyme showed that Mpr1 acetylates P5C or glutamate-gamma-semialdehyde (GSA) which is in equilibrium to P5C. From the above results, it is suggested that the intracellular accumulation of P5C leads to the generation of ROS which causes the cytotoxicity and that Mpr1 regulates the ROS level via the acetylation of P5C/GSA so as to alleviate the oxidative stress (FIG. 2) (non-patent literature 6).
Under the fermentative production environment, yeasts are subjected to various stresses such as cold temperature, freezing, drying, oxidation, high osmolarity, high ethanol concentration and biased nutrition. Long term exposure of yeast to such stresses as above brings about the cleavage of noncovalent bondings in the intracellular proteins which leads to exposure of hydrophobic amino acids on the surfaces of the proteins and generation of “abnormal proteins” which lost their normal structures and functions, and results in the restriction of the useful functions of the yeast. In particular, the oxidative stress is generated by various factors such as heat shock, hydrogen peroxide, freezing (freezing-thawing) damage, high ethanol concentration and the like and is a great cause of the inhibition of the growth of the yeast cells. In the fields of fermented food products and brewed food products, there is a desire for breeding a yeast which is highly resistant to the oxidative stress.
So far, the present inventors found that an amino acid proline has a property of protecting yeasts from stresses such as freezing, drying, oxidation and the like (Patent literature 1). Further, the present inventors found that a yeast strain whose gene encoding proline degradative enzyme was disrupted by the genetic engineering acquires the ethanol resistance by accumulating proline in the cells (Patent literature 2).    Patent literature 1: JP-A-9-234058    Patent literature 2: JP-A-2006-67806    Non-patent literature 1: H. Takagi et al., J. Bacteriol., 182, 4249-4256 (2000)    Non-patent literature 2: M. Shichiri et al., J. Biol. Chem., 276, 41998-42002 (2001)    Non-patent literature 3: C. Hoshikawa et al., Proc. Natl. Acad. Sci. U.S.A., 100, 11505-11510 (2003)    Non-patent literature 4: Y. Kimura et al., Yeast, 19, 1437-1445 (2002)    Non-patent literature 5: M. Nomura et al., J. Biochem., 133, 67-74 (2003)    Non-patent literature 6: M. Nomura et al., Proc. Natl. Acad. Sci. U.S.A., 101, 12616-12621 (2004)