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, 2 (10), e1092

Biosynthesis of Vitamin C by Yeast Leads to Increased Stress Resistance

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Biosynthesis of Vitamin C by Yeast Leads to Increased Stress Resistance

Paola Branduardi et al. PLoS One.

Abstract

Background: In industrial large scale bio-reactions micro-organisms are generally exposed to a variety of environmental stresses, which might be detrimental for growth and productivity. Reactive oxygen species (ROS) play a key role among the common stress factors--directly--through incomplete reduction of O(2) during respiration, or indirectly--caused by other stressing factors. Vitamin C or L-ascorbic acid acts as a scavenger of ROS, thereby potentially protecting cells from harmful oxidative products. While most eukaryotes synthesize ascorbic acid, yeast cells produce erythro-ascorbic acid instead. The actual importance of this antioxidant substance for the yeast is still a subject of scientific debate.

Methodology/principal findings: We set out to enable Saccharomyces cerevisiae cells to produce ascorbic acid intracellularly to protect the cells from detrimental effects of environmental stresses. We report for the first time the biosynthesis of L-ascorbic acid from D-glucose by metabolically engineered yeast cells. The amount of L-ascorbic acid produced leads to an improved robustness of the recombinant cells when they are subjected to stress conditions as often met during industrial fermentations. Not only resistance against oxidative agents as H(2)O(2) is increased, but also the tolerance to low pH and weak organic acids at low pH is increased.

Conclusions/significance: This platform provides a new tool whose commercial applications may have a substantial impact on bio-industrial production of Vitamin C. Furthermore, we propose S. cerevisiae cells endogenously producing vitamin C as a cellular model to study the genesis/protection of ROS as well as genotoxicity.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Ascorbic acid biosynthetic pathway.
Schematic representation of the pathway of L-AA production from D-glucose in plants. The following enzymes are involved: A, hexokinase (2.7.1.1), B, glucose-6-phosphate isomerase (5.3.1.9), C, mannose-6-phosphate isomerase (5.3.1.8), D, phosphomannomutase (5.4.2.8), E, mannose-1-phosphate guanylyltransferase (2.7.7.22), F, GDP-mannose-3,5-epimerase (5.1.3.18), G, GDP-L-galactose phosphorylase (E.C.C not assigned), H, L-Galactose 1-phosphate phosphatase (3.1.3.25), I, L-galactose dehydrogenase, J, L-galactono-1,4-lactone dehydrogenase (1.3.2.3).
Figure 2
Figure 2. Conversion of D-Glucose into L-ascorbic acid (milligrams/liter/OD) by transformed S. cerevisiae GRF18U and BY4742 cells.
All strains were grown on mineral medium (2% w/v glucose, 0.67% w/v YNB), starting with an initial OD660 of 0.05 for 18 h, when samples were taken and the concentration of L-ascorbic acid inside the cells was determined (GRF18U and BY4742 correspond to the parental strains transformed with the empty plasmids harboring in the productive strains the genes of the L-AA pathway). The control cells, as well as the cells expressing ScALO1 and AtLGDH can not accumulate L-ascorbic acid starting from D-glucose, therefore measured values correspond to the endogenous erythro-ascorbic acid. The standard deviation bars correspond to the data obtained from independent clones, and from independent growth and antioxidant determinations. Please note the different scale of the ordinate axes in the two graphs.
Figure 3
Figure 3. Growth curves of wild type and L-ascorbic acid producing yeasts under oxidative stress.
Kinetics of growth of wild type and engineered strains GRF18U (left panels) and BY4742 (right panels) as inoculated in minimal glucose media without H2O2 (3A and 3B) or in presence of H2O2 3.5 mM (3C and 3D). • GRF18U wild type; ▪ GRF18U[ScALO AtLGDH AtME AtMIP]; ▴ GRF18U[ScALO AtLGDH AtME AtMIP RnFGT]; ○ BY4742 wild type; □ BY4742[ScALO AtLGDH AtME AtMIP]; ▵ BY4742[ScALO AtLGDH AtME AtMIP RnFGT].
Figure 4
Figure 4. Growth curves of wild type and L-ascorbic acid producing yeasts under acidic stress.
Growth curves of wild type and engineered GRF18U and BY strains (upper and lower panel, respectively) inoculated in minimal glucose media at low inorganic pH (panel A) or at low pH plus organic acid (lactic acid, 45g/l, panel B) or at low pH plus organic acid with L-AA added exogenously (panel C). Panels 4A and 4B: • GRF18U wild type; ▪ GRF18U[ScALO AtLGDH AtME AtMIP]; ▴ GRF18U[ScALO AtLGDH AtME AtMIP RnFGT]; ○ BY4742 wild type; □ BY4742 [ScALO AtLGDH AtME AtMIP]; ▵ BY4742 [ScALO AtLGDH AtME AtMIP RnFGT]; Panel 4C: • GRF18U wild type; ○ BY4742 wild type; − GRF18U or BY4742 wild type added with 30mg/l ascorbic acid; * GRF18U or BY4742 wild type added with 60 mg/l ascorbic acid.
Figure 5
Figure 5. Flow cytometric analysis of wild type and vitamin C producing yeasts under oxidative stress.
Panel 5A: schematic representation of the different subpopulations that can be observed in the following panels, where wild type (5B) and recombinant strains (5C and 5D) grown in minimal glucose medium added with H2O2 3.0 mM were analyzed after DHR123 and PI staining (rodamine signal is reported in the abscissa and PI signal on the ordinate axes). Upper panels: GRF18U background. Lower panels: BY background. (B): wild type. (C): [ScALO AtLGDH AtME AtMIP] transformed cells. (D): [ScALO AtLGDH AtME AtMIP RnFGT] transformed cells

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References

    1. Bai Z, Harvey LM, McNeil B. Oxidative stress in submerged cultures of fungi. Crit Rev Biotechnol. 2003;23:267–302. - PubMed
    1. Mattanovich D, Gasser B, Hohenblum H, Sauer M. Stress in recombinant protein producing yeasts. J Biotechnol. 2004;113:121–135. - PubMed
    1. Ryter SW, Kim HP, Hoetzel A, Park JW, Nakahira K, et al. Mechanisms of cell death in oxidative stress. Antioxid Redox Signal. 2007;9:49–89. - PubMed
    1. Temple MD, Perrone GG, Dawes IW. Complex cellular responses to reactive oxygen species. Trends Cell Biol. 2005;15:319–326. - PubMed
    1. Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2004;55:373–399. - PubMed

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