Engineered Trx2p industrial yeast strain protects glycolysis and fermentation proteins from oxidative carbonylation during biomass propagation

Abstract

Background: In the yeast biomass production process, protein carbonylation has severe adverse effects since it diminishes biomass yield and profitability of industrial production plants. However, this significant detriment of yeast performance can be alleviated by increasing thioredoxins levels. Thioredoxins are important antioxidant defenses implicated in many functions in cells, and their primordial functions include scavenging of reactive oxygen species that produce dramatic and irreversible alterations such as protein carbonylation.

Results: In this work we have found several proteins specifically protected by yeast Thioredoxin 2 (Trx2p). Bidimensional electrophoresis and carbonylated protein identification from TRX-deficient and TRX-overexpressing cells revealed that glycolysis and fermentation-related proteins are specific targets of Trx2p protection. Indeed, the TRX2 overexpressing strain presented increased activity of the central carbon metabolism enzymes. Interestingly, Trx2p specifically preserved alcohol dehydrogenase I (Adh1p) from carbonylation, decreased oligomer aggregates and increased its enzymatic activity.

Conclusions: The identified proteins suggest that the fermentative capacity detriment observed under industrial conditions in T73 wine commercial strain results from the oxidative carbonylation of specific glycolytic and fermentation enzymes. Indeed, increased thioredoxin levels enhance the performance of key fermentation enzymes such as Adh1p, which consequently increases fermentative capacity.

Figure 1

Protein oxidation of T73 strain during biomass propagation. (A) Anti-DNP western blots from 2D gels obtained from cells collected at 0 h, 15 h and 80 h of growth during industrial biomass propagation. A representative 2D-gel from triplicate experiments is shown. (B) Relative carbonylation levels of oxidized proteins grouped into different functional categories defined in Table 1. The ratio C_I (carbonyl intensity)/P_I (protein intensity) of each functional category was calculated as the summatory of C_I/P_I for each protein belonging to the respective functional category. Statistical comparisons with reference sample (Time 0 h) were made using a Student's t-test (* p < 0.05).

Figure 2

Differential carbonylation profiles using TRX2 gene modified strains. (A) Anti-DNP western blots from the 2D gels obtained from cells collected at 15 h and 80 h of growth under industrial conditions. Marked proteins present significant oxidative damage variation compared to the T73 control strain among the three biological replicates. (B) Silver-stained gels as a control of protein amount for each sample.

Figure 3

Enzyme activities of oxidative damaged proteins. The activities of ADH (alcohol dehydrogenase), PDC (pyruvate decarboxylase), ENO (enolase) and CAT (catalase) were determined as described in Materials and Methods for the T73 and TTRX2 strains. All the data are expressed as means ± standard deviation of three biological replicates. Statistical comparisons between T73 and TTRX2 strains were made using a Student's t-test (* p < 0.05).

Figure 4

Adh1p aggregation resulting from oxidative carbonylation. Anti-Adh1p western blot (upper panel) for the T73 and TTRX2 strains from dehydrated cells and cells grown in YPGF medium for 5 h (A) or from fresh YPGF growing cells, treated or not treated with carbonylation inductor glyoxal (GO) (B). The bottom panel shows the Coomassie-stained membrane used as a control of protein amount. (C) Anti-DNP western blot of Adh1p immunoprecipitated samples: ¹ T73 and ² T73+5 mM glyoxal. Marked bands correspond to the dimer (74 KDa) and tetramer (148 KDa) forms of Adh1p.