Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Microbial Cell Factories (Impact Factor: 4.22). 04/2012; 11(1):40. DOI: 10.1186/1475-2859-11-40
Source: PubMed


During the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed.
We constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO plays an important role in baking-associated stress tolerance.
In this work, we clarified the importance of Put1- and Mpr1-mediated NO generation from proline to the baking-associated stress tolerance in industrial baker's yeast. We also demonstrated that baker's yeast that enhances the proline and NO synthetic pathway by expressing the Pro1-I150T and Mpr1-F65L variants showed improved fermentation ability under multiple baking-associated stress conditions. From a biotechnological perspective, the enhancement of proline and NO synthesis could be promising for breeding novel baker's yeast strains.

Download full-text


Available from: PubMed Central · License: CC BY
  • Source
    • "It would be interesting to investigate a gal4,put3 double deletion and assess for weak acid sensitivity. Previous work has shown that yeast strains with higher intracellular proline concentrations are osmotically and oxidatively tolerant (Sasano et al. 2012a, b). It was observed that these strains were weak acid tolerant when compared with BY4741; these strains displayed no increased tolerance to other inhibitory compounds. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fermentation of sugars released from lignocellulosic biomass (LCMs) is a sustainable option for the production of bioethanol. LCMs release fermentable hexose sugars and the currently non-fermentable pentose sugars; ethanol yield from lignocellulosic residues is dependent on the efficient conversion of available sugars to ethanol, a side-product of the process is acetic acid production. Presence of acetic acid reduced metabolic output and growth when compared with controls; however, it was observed that incubation with proline had a protective effect, which was proline specific and concentration dependent; the protective effect did not extend to furan or phenolic stressed yeast cells. Proline accumulating strains displayed tolerance to acetic acid when compared with background strains, whereas, strains with a compromised proline metabolism displayed sensitivity. Sensitivity to weak acids appears to be reduced with the addition of proline; proline is an imino acid freely available as a nitrogen source in the aerobic phase of fermentations. Yeast strains with higher intracellular proline concentrations would be desirable for industrial bioethanol fermentations.
    Full-text · Article · Feb 2014 · Antonie van Leeuwenhoek
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hyperprolinemia is an inherited disorder of proline (Pro) metabolism and patients affected by this disease may present neurological manifestations. However, the mechanisms of neural excitotoxicity elicited by hyperprolinemia are far from being understood. Considering the pivotal role of cytoskeletal remodeling in several neurodegenerative pathologies and the potential links between cytoskeleton, reactive oxygen species production and cell death, the aim of the present work was to study the effects of Pro on astrocyte and neuron cytoskeletal remodeling and the possible oxidative stress involvement. Pro induced a shift of actin cytoskeleton in stress fibers together with increased RhoA immunocontent and ERK1/2 phosphorylation/activation in cortical astrocytes. Unlike astrocytes, results evidenced little susceptibility of neuron cytoskeleton remodeling, since Pro-treated neurons presented unaltered neuritogenesis. We observed increased hydrogen peroxide production characterizing oxidative stress together with decreased superoxide dismutase (SOD) and catalase (CAT) activities in cortical astrocytes after Pro treatment, while glutathione peroxidase (GSHPx) activity remained unaltered. However, coincubation with Pro and Trolox/melatonin prevented decreased SOD and CAT activities in Pro-treated astrocytes. Accordingly, these antioxidants were able to prevent the remodeling of the actin cytoskeleton, RhoA increased levels and ERK1/2 phosphorylation in response to high Pro exposure. Taken together, these findings indicated that the cytoskeleton of cortical astrocytes, but not of neurons in culture, is a target to Pro and such effects could be mediated, at least in part, by redox imbalance, RhoA and ERK1/2 signaling pathways. The vulnerability of astrocyte cytoskeleton may have important implications for understanding the effects of Pro in the neurotoxicity linked to inborn errors of Pro metabolism.
    Full-text · Article · Nov 2012 · Experimental Cell Research
  • [Show abstract] [Hide abstract]
    ABSTRACT: Yeasts rarely encounter ideal physiological conditions during their industrial life span; therefore, their ability to adapt to changing conditions determines their usefulness and applicability. This is especially true for baking strains of Saccharomyces cerevisiae. The success of this yeast in the ancient art of bread making is based on its capacity to rapidly transform carbohydrates into CO rather than its unusual resistance to environmental stresses. Moreover, baker's yeast must exhibit efficient respiratory metabolism during yeast manufacturing, which determines biomass yield. However, optimal growth conditions often have negative consequences in other commercially important aspects, such as fermentative power or stress tolerance. This article reviews the genetic and physiological characteristics of baking yeast strains, emphasizing the activation of regulatory mechanisms in response to carbon source and stress signaling and their importance in defining targets for strain selection and improvement.
    No preview · Article · Feb 2013 · Review of Food Science and Technology
Show more