Explored The Relationship Between Resistance Of Saccharomyces Cerevisiae To Vanillin And Antioxidant Systems

Bioethanol has become a favourite biofuel.It can be blended easily with gasoline,can be stored and transported over long distances,and can be produced cheaply in very large quantities by a well-established industrial process.It is regarded as one of the most potential renewable and clean energy.Therefore,there is great interest in producing bioethanol with so-called second-generation substrates,i.e.cellulosic waste streams and bioenergy crops,which do not compete with food production.The yeast strains currently used for bioethanol production.It has high efficient utilization hexose,high ethanol tolerance,good resistance and other excellent properties.Lignocellulosic feedstock for ethanol production in Saccharomyces cerevisiae have two important problems.First,lignocellulose hydrolysis fermentable sugars,and the inhibition of yeast fermentation by the high levels of inhibitors that are present in lignocellulose hydrolysates.Second,There are 30%xylose in ligno cellulose hydrolysate.It is one of the key factors that realize the economic feasibility of lignocellulosic ethanol production.At present,xylose reductase and xylitol dehydrogenase,or xylose isomerase is usually introduced in Saccharomyces cerevisiae which produce xylulose after phosphorylation enter the pentose phosphate pathway,and then go through a series of transketolase and transaldolase reaction,to fructose 6-phosphate and phosphate 3 glyceraldehyde that return to the glycolytic pathway,producing ethanol.But there is a more simple way for xylose metabolism in some bacteria and archaea,that composed by D-xylose dehydrogenase,D-xylonate dehydratase,and 2-keto-3-deoxy-D-xylonate aldolase.Now,two research groups including my group,has been successfully expressed xylose dehydrogenase in Saccharomyces cerevisiae.The work of this paper mainly includes two aspects:1.Explored the relationship between resistance of Saccharomyces cerevisiae to vanillin and antioxidant systemsThe presence of high levels of inhibitors severely slows down the yeast fermentation process and limits the percentage of ethanol ccumulate.The type of phenolic compounds in the hydrolysate are material dependent,vanillin is a commonly used example.It was reported that the vanillin acts at a low concentration.The genes encoding the ergosterol biosynthetic enzymes were screened out using a set of diploid yeast deletion mutants,and were proposed to be associated with vanillin tolerance.This is because ergosterol is important for maintaining the fluidity and stability of the membrane.Saccharomyces cerevisiae with high tolerance to the inhibitors present in steam-exploded corn stover were obtained by ethyl methanesulfonate(EMS)mutation and adaptive evolution in preliminary work.In this work,transcriptional analysis of strain EMV-8 and of its parent strain NAN-27 was performed using RNA sequence technology to understand the high vanillin tolerance mechanism of EMV-8 further.From a molecular function viewpoint,the genes in EMV-8 that were up-regulated compared with the parent strain NAN-27 were enriched in several energy-generating pathways,redox enzymes,and antioxidant metabolism.Furthermore,we enhance ROS metabolism gene expression level and increase the intracellular amount of reduced glutathione in Saccharomyces cerevisiae.ROS concentration in Saccharomyces cerevisiae can be reduced by overexpressing Gpxl glutathione peroxidase gene.glucose consumption rate,vanillin consumption rate and ethanol yield reached in Saccharomyces cerevisiae that increase catalase(Cttlp)expression level were higher than that of the control strain.the maximum growth rate of Saccharomyces cerevisiae that increased glutathione peroxidase(Gpxlp)expression level in the presence of vanillin medium is higher than the control strain.Improve the peroxisome catalase(Ctalp),glutathione reductase expression level(Glrlp),co expression of catalase,superoxide dismutase expression level did not affect the strain of Saccharomyces cerevisiae glucose consumption rate and ethanol yield,vanillin consumption rate.Improved gamma glutamyl cysteine reductase(Gshlp)superoxide dismutase(Sod1p)expression level would seriously reduce glucose consumption rate,vanillin consumption rate and ethanol yield in Saccharomyces cerevisiae in the presence of vanillin medium.2.Expressing D-xylonate dehydratase in Saccharomyces cerevisiaeCurrently,xylose acid dehydratase gene in nature is very limited,we express xylose acid dehydratase gene from Pseudomona,Burkholderiasp,Achromobacter xylosoxidans in Saccharomyces cerevisiae.Unfortunately,enzyme assay experiment showed that,these xylonic acid dehydratase gene are not expressed in Saccharomyces cerevisiae.The establishment of this pathway still needs a lot of subsequent work.