Improving yield of industrial biomass propagation by increasing the Trx2p dosage

Abstract

The beneficial effect of improving yeast redox response by increasing thioredoxin levels has been shown. Decreased lipid and protein oxidation is reflected in an increased biomass yield. In addition, increased redox defenses like glutathione and ROS scavenging enzymes are observed. Furthermore, the wine produced with the modified strain presented more aromatic compounds than the control strain, and its organoleptic properties increased. Here, we hypothesize that reduced glycolytic enzyme carbonylation can increase not only the glycolytic flux but also, and consequently, the biomass yield in the industrial biomass propagation process. The commercial use of the thioredoxin bioengineered yeast as an antioxidant dietetic supplement is also discussed.

Genetic bioengineering of industrial yeasts has been used to enhance many aspects relating to bread, wine, beer or other alcoholic beverages. However, very few studies have focused on the industrial production of yeast biomass, whose main challenge is to obtain high yields, but this is a difficult issue for modern bioengineering methods.¹ In several studies, increased yield has been ascribed to a new or enhanced capability of sugar² or nitrogen assimilation.³ However, an increased yield during usual glucose metabolism has not yet been described.

We have recently shown⁴ that enhancing the stress response of industrial yeast by increasing cytosolic thioredoxin may be beneficial for several aspects of yeast physiology. An increased Trx2p dosage (TTRX2 strain), the main thioredoxin isoform, has proved to lower cellular damage, for instance lipid and protein oxidation, during the industrial biomass propagation of wine yeasts. This overexpression influenced the redox stress response, and increased the activity of ROS scavenging enzymes and the amount of the antioxidant molecule glutathione.

The biomass propagation process consists in two main phases: the batch phase, in which metabolism is mainly fermentative, and the fed-batch phase, in which energy is obtained from metabolic sugar oxidation. Since thioredoxins are small proteins that protect against cellular oxidation, an important role during the fed-batch phase can be easily explained. However, we have observed that the most critical oxidative response takes place during the batch phase when fermentable sugars are exhausted and oxidation of ethanol starts. Molecular oxidation was especially abundant during this metabolic transition, which is probably due to an unbalanced NAD⁺/NADH ratio, the cofactors of alcohol dehydrogenase. The presence of increased levels of cytosolic thioredoxin appears to decrease oxidation during this metabolic transition.

One of the most interesting phenotypes of the TTRX2 strain is the increased biomass yield if compared to the control strain. We hypothesize that TTRX2 strain-specific enzymes will be less carbonylated than in the control strain and that they will be more efficient in their metabolic tasks. In fact, several enzymes of the central carbon metabolism, such as Adh1p and Tdh3p, have been identified as thioredoxin functional targets by our group (unpublished results) and others.⁵ This increased glycolytic activity will increase glycolytic flux and favor yeast growth in glucose-limiting conditions like the fed-batch phase of the industrial biomass propagation process (Fig. 1). Many attempts have been made to increase the glycolytic flux rate by increasing enzyme concentration.⁶ However, these approximations were not very successful due to the tight regulation of carbon central metabolism. In contrast, our strategy does not affect the enzyme protein levels, but enhances their efficiency by decreasing protein oxidation damage. This suggests that preventing the oxidation of enzymes can prove an interesting way to improve the flux of highly regulated metabolic pathways, especially in those processes involving metabolic oxidations.

Figure 1.

Schematic representation of increasing thioredoxin levels in the activity of Tdh3p and Adh1p.

Given their high vitamin and protein contents, yeasts have been widely used as both food supplements and dietetic products. Furthermore, the yeast strain Saccharomyces boulardii has been used as a probiotic as it is suitable for fighting intestinal infections.⁷ On the other hand, thioredoxin has been proposed as a dietetic supplement because of its antioxidant capability which has been seen to decrease allergenicity.⁸ Thus, the availability of a yeast strain with high levels of thioredoxin may be used to produce a dietary supplement with antioxidant properties and decreased allergenicity. Furthermore, the use of this wine yeast opens up the possibility of producing wines with a higher antioxidant thioredoxin content.

Addenum to: Gómez-Pastor R, Pérez-Torrado R, Cabiscol E, Ros J, Matallana E. Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass. Microb Cell Fact. 2010;9:9. doi: 10.1186/1475-2859-9-9.