Self-Cloning Baker's Yeasts That Accumulate Proline Enhance Freeze Tolerance in Doughs

Abstract

We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding ␥-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.Ordinary commercial baker's yeast (mostly strains of Saccharomyces cerevisiae) is generally susceptible to damage during the freeze-thaw process and frozen storage and does not retain sufficient leavening ability after thawing (8,11). Freeze tolerance is a necessary characteristic of baker's yeast used in frozen doughs because postthaw leavening activity is essential prior to baking. It is known that the laboratory yeast S. cerevisiae induces trehalose synthesis during various stresses (1,10,34) and that induced trehalose functions as a stress protectant (7,16,23).We previously found that proline and charged amino acids, such as arginine and glutamate, have cryoprotective activities nearly equal to that of glycerol or trehalose in baker's yeast (26). It was shown previously that yeast cells with higher levels of proline are more tolerant of freezing than wild-type cells (13,14,22,26,28,30). Proline and trehalose were reported to preserve membrane structure and function during freezing (20). It has been suggested that proline can prevent ice nucleation and dehydration by forming strong hydrogen bonds with intracellular free water. ␥-Glutamyl kinase (GK; the PRO1 gene product) is the key enzyme in proline biosynthesis in S. cerevisiae, and GK activity is subjected to feedback inhibition by proline (22,32). Proline oxidase (PO; the PUT1 gene product) catalyzes the first step of the proline degradation pathway in S. cerevisiae (36). Interestingly, the D154N and I150T mutant GKs are less sensitive to proline feedback inhibition than the wild-type enzyme, and yeast cells expressing these mutated GKs accumulate proline and show higher tolerance for freezing than wild-type cells (22). With respect to industrial yeast, we previously constructed novel proline-accumulating sake yeast by disrupting the PUT1 gene and introducing the pro1(D154N) mutant allele and found that the resultant strain accumulated proline and was more tolerant of ethanol stress than the control strain (29). Furthermore, self-cloning (SC) diploid sake yeasts that accumulate proline have been developed, and their fermentation profiles during sake brewing have been analyzed (27). For the application of recombinant yeasts for commercial use, an SC yeast, which has no foreign genes or DNA sequences except for yeast DNA, may be more acceptable to consumers than a genetically modified (GM) yeast.Our objectives in this study were (i) to construct SC diploid baker's yeast strains that accumulate proline and (ii) to determine if intracellular proline enhances the freeze tolerance of baker's yeast strains in do...

Molecular Mechanisms Modulating Glutamate Kinase Activity. Identification of the Proline Feedback Inhibitor Binding Site

“…9,24,[31][32][33] The effects observed upon mutating additional residues located in this area show that the binding sites of glutamate and proline overlap and support the binding conformation predicted by the docking calculations (Table 1). Asn134 and Asp137 are invariant in all bacterial GKs and plant and mammal P5CSs.…”

“…2 In other kingdoms of nature, proline overexpression by deregulated enzymes confers osmotolerance to plants, yeast, and bacteria. [4][5][6][7][8][9][10][11][12] This functional versatility of proline has drawn attention to the proline biosynthetic pathway and its key control enzyme, glutamate kinase (GK). [13][14][15][16][17][18] In bacteria, GK phosphorylates glutamate, which is reduced sequentially to proline by glutamyl phosphate reductase (GPR) and pyrroline-5-carboxylate reductase.…”

Improving industrial yeast strains: exploiting natural and artificial diversity

“…Hence, the development of more robust strains that are still capable of producing a high-quality end product is a prime focus in biotechnology (Kim et al ., 1996; Perez-Torrado et al ., 2002, 2010; Panadero et al ., 2007; Kaino et al ., 2008; Gomez-Pastor et al ., 2012). For example, using an inverse metabolic engineering approach, Perez-Torrado et al .…”

Environmental Stresses to Which Yeast Cells Are Exposed During Bioethanol Production from Biomass

“…Such techniques could improve a yeast's characteristics, such as fermentation ability or stress tolerance, and could improve the bioethanol production process (Hasunuma and Kondo 2012 ;Inaba et al 2013 ). Genetic engineering techniques produce two categories of yeasts: genetically modifi ed (GM) yeast, which contains a heterologous DNA segment derived from organisms taxonomically different from their host cells, and self-cloning (SC) yeast, which does not contain any DNA derived from other organisms and does not produce any additional proteins except for proteins originally produced in the yeast (Akada 2002 ;Kaino et al 2008 ;Ando et al 2005 ). SC processes are considered the same as naturally occurring gene conversion, such as recombination, deletion, and transposition, and thus SC yeast is not considered a GM organism.…”