| Energy, resources and environmental crisis has been the main obstactes for the development of human civilization, and it is hot topic to search for safe, clearner, and convenient renewable energy in this century. Biomass for fuel ethanol production has attracted widespread research due to its economic, environmental, practical, renewable and other advantages. Lignocellulose with high added value is an abundant and cheap resource in nature world. However, some cell growth inhibitors are generated during the pretreatment process, such as furfural, 5-HMF, weak acid and phenolic compounds, and they will impair fermentation process. Therefore, it is very important for industrial fermentation to get strains with high furfural tolerance through selecting or modificating.In this study, irrE gene(or named ppr I, inducer of pleiotropic proteins promoting DNA repair), which codes IrrE, a global regulator from Deinococcus radiodurans R1, was used to improve furfural tolerance of Saccharomyces cerevisiae AS2.489. Firstly, three rounds of sequential error prone PCR were applied to wild-type irrE gene, mutant libraries were constructed and the mutant strains with higher furfural torlerance were selected respectively. Secondly, the selected FR mutant was tested in glucose/furfural mixure for further resistance analysis. Thirdly, intracellular reactive oxygen species(ROS) levels and stress-related genes expression levels were determined for elucidating the mechanism of the furfural tolerance increased. Finally, we studied the fermentation performance of FR mutant using cassava residue hydrolysate.The major results were as follows. Compared with control group WT, p YPK, the growth of FR mutant was significantly higher in YPD medium containing 1.5 g/L or 2.0 g/L or 2.5 g/L furfural. The growth of OD600 value between muant FR and control group had significant difference after 40 hours cultivation at 200 g/L glucose and different concentrations of fufural(1.5 g/L, 2.0 g/L, and 2.5 g/L). Furthermore, the growth of OD600 value between FR mutant and control group had significant difference after 40 hours cultivation at 2.0 g/L furfural and different concentrations of glucose(20 g/L, 80 g/L, and 140 g/L). In summary, the tolerance of FR mutant improved obviously for fufural and glucose/furfural mixture.When the medium containing 200 g/L glucose and 3.5 g/L furfural, the ROS levels of WT, p YPK was 2.71- and 2.89- fold as much as that of FR mutant respectively, and it showed that FR mutant had a better ability to eliminate ROS. Meanwhile, the expression levels of fufural stress-related genes were measured. It showed that the expression levels of ald6, gnd1 and adh6 in WT, p YPK and FR muant were up-regulated by 12.18, 10.41, 56.90 folds, 13.18, 13.53, 217.81 folds and 33.58, 29.12, 64.75 folds, respectively. Therefore, increased tolerance of FR mutant to the fufural may be related to stress-genes at high express levels.The fermentation period of FR mutant was shortened than WT and p YPK by 24 h in simulated medium of lignocellulosic hydrolysates(containing 200 g/L glucose and 2.0 g/L furfural). All the strains including FR, WT and p YPK, which was 89.63 g/L, 88.12 g/L, 87.50 g/L respectively, but ethanol productivity was 1.18 g/L/h, 0.90 g/L/h, 0.86 g/L/h, respectively. Under dilute sulfuric acid hydrolysis of cassava residue conditons, the fermentation period of FR mutant was shorter than WT by about 5 h, the maximum difference of OD600 between two strains was about 2.30, and the ethanol productivity was 1.56 g/L/h, 1.44 g/L/h accordingly.In this study, the irrE gene from Deinococcus radiodurans R1 was applied to enhance the furfural tolerance of Saccharomyces cerevisiae and one mutant with high resistance to glucose/furfural was successfully obtained. It has great significance for ethanol production by cheap lignocellulose reducing production cost and protecting environment. |
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