Optimizing Xylose Utilization By Disturbing The Different Steps In The Glucose And Xylose Metabolism Of Saccharomyces Cerevisiae

The growing demand for energy and environmental concerns is driving research and commercialization of fuels from renewable sources.Among them,producing automotive fuel ethanol from lignocellulosic materials instead of sugar and starch-based crops is a feasible solution,which avoids the disadvantages of compete for food with people.Saccharomyces cerevisiae is a traditional strain of ethanol production which can convert a variety of sugars to ethanol efficiently,especially glucose.However,native S.cerevisiae is unable to ferment xylose,the second most abundant sugar present in lignocellulose hydrolysate.Two different xylose metabolic pathways have been successfully introduced into S.cerevisiae by heterologous expression:the xylose reductase(XR)and xylitol dehydrogenase(XDH)(XR-XDH)pathway and the xylose isomerase(XI)pathway.After the above-mentioned pathway,xylose is transformed into xylulose.Xylulose is phosphorylated by xylulokinase and then converted to fructose-6-phosphate and glyceraldehyde-3-phosphate through the pentose phosphate pathway,then enter into glycolytic pathway,which two pathways are active when glucose is present.Xylose consumption rate of recombinant S.cerevisiae has greatly promoted by engineering and evolution in recent decade.However,it is still lower than that of the glucose.Improve global cell metabolism and improve the xylose fermentation efficiency of strains is important for ethanol production.Glucose shares common downstream metabolic pathways with xylose.With the activation of glucose signal system maintaining the high level of glycolysis and pentose phosphate pathway is very important for the metabolism of xylose.Based on this,we conducted the following research on the recombinant S.cerevisiae.(1)The glucose metabolism of S.cerevisiae is coordinated by several signaling systems and metabolic pathways.AMPK/Snf1p is an important transcriptional regulatory system for glucose effect of S.cerevisiae.The Migl p-Hxk2p pathway is important system in glucose metabolism.The functions of both proteins have been extensively studied,but there is limited information about possible interactions among them in the repression pathway.Hxk2p nucleocytoplasmic traffic is regulated by phosphorylation and dephosphorylating at serine 14.Protein kinase Snf1p and the protein phosphatase Glc7p-Reg1p as novel regulatory partners for the nucleocytoplasmic shuttling of Hxk2p.In high glucose conditions,Hxk2p translocation into the nucleus and interacts with Miglp to generate a repressor complex.In low glucose,Hxk2p is phosphorylated in vivo at Ser-14.The phosphorylated Hxk2p loses the interaction with Miglp and leaves the complex.Under these conditions,the repressor complex is disorganized,and phosphorylated Hxk2p and Miglp leave the nucleus.In theory,deleting protein kinase Snflp can directly prevent phosphorylation of Hxk2p in theory and remains the activation of this glucose signal pathway,therefore bring postive effect on xylose utilization.However,our result revealed that deleting SNF1 seriously affects cell growth on xylose,which may because there are too many regulatory targets of Snflp.Moreover,we studied the effect of disturbing the location of Hxk2p on xylose metabolism.We replaced HXK2 with HXK2S14A and HXK2S14D respectively.Nuclear import of the S14D mutant of Hxk2p is severely decreased but the export is significantly enhanced.Conversely,nuclear import of the S14A mutant of Hxk2p was significantly enhanced,although the exportation was severely decreased.We found that the xylose utilization of HXK2S14A mutant was significantly enhanced.HXK2S14A demonstrated a 25.6%higher specific consumption rate of xylose than wild type in aerobic condition.The specific ethanol production rate that HXK2S14A mutant achieved was approximately 59.0%higher than wide strain.Under the condition of oxygen limitation,special ethanol production rate was 12.2%higher than that of wild-type.HYK2S14D mutant had no significant effect on xylose metabolism of the strain.The results show that HXK2S14A is a better regulatory element than SNF1 deletion.Our physiological investigation of strain with HXK2S14A mig1? genotype revealed that MIG1 is necessary to the optimizing of HXK2S14A in xylose metabolism.The revealed transcriptional targets of HXK2S14A and mig1? were only HXT5 and XKS1,which may affect xylose utilization.There are six Diff-Expressed Gene contains the combined sequence of Miglp.Breathing-related genes AI1,AI2,COX2 and COX3 are down regulated in BSHA01 indicated that reduce the respiratory.Besides,thirty percent transcriptome difference genes between the HXK2S14A strain and the wild-type genes are related to the synthesis of ribosomes.In HXK2S14A strain,the key regulatory gene TOD6 for ribosome synthesis was significantly higher than in HXK2WT strain,indicating that the synthesis of ribosomes is released from inhibition by HXK2S14A.Moreover,HXK2 plays a key role in the localization of Ras protein,and the synthesis of ribosome is regulated by Ras-cAMP pathway.We conduced that the optimizing of xylose about HXK2S14A may be involved in the regulation of Ras-cAMP pathway in addition to the interaction with Mig1p.(2)Xylose can enter into glycolytic pathway by F-6-P and GAPD,then the part of glucose-6-phosphate is one of the most obvious difference between xylose and glucose metabolic.Enolase enzyme catalyzing glycerin acid phosphate form pyruvic acid is one of the key enzymes in glycolysis.Transcriptome studies have shown that ENO2 transcription in xylose metabolic is only 59.5%of glucose Glucose-6-phosphate is the important inducer of ENO2 expression.Concentration studies revealed that glucose-6-phosphate in the cells was equivalent between two kind of carbon source.The enzyme activity of enolase in xylose fermentation is only 29%of that in glucose fermentation.In contrast to the wild-type strain,ENO2 deletion and over-expression ENO1 had no significant effect on recombination strain,but over-expression ENO2 reduced the growth of the strains.Xylose utilization within 72 hours was reduced by 28.8%than the wild type.The production of ethanol in the glycolysis was seriously inhibited.The proportion of two enolase enzymes and its expression may have been meet the needs of xylose metabolism.(3)Critical cellular metabolite pyruvate is the end product of glycolysis and has two major metabolic fates.At the pyruvate branch point,there are the fermentative and oxidative metabolic routes diverge.How pyruvate is processed has a major influence on the overall metabolism of the cell.Mitochondrial pyruvate carrier(MPC)is encoded by MPC1,MPC2,and MPC3 in yeast.MPC is necessary for up taking of pyruvate into the mitochondria.The expression of MPC1 is not dependent on the carbon source.The expression of MPC2 and MPC3 is specific to fermentative or respiratory conditions respectively.This gives rise to two alternative carrier complexes that we have termed MPCFERM and MPCox.MPCox has a higher transport activity than MPCFERM.In this work the distribution of pyruvate was altered by constitutively expressing the two alternative complexes in yeast.The results showed that MPC3 deletion did not cause a great disturbance to metabolism.In 2%xylose aerobic fermentation,the recombinant strains of MPC1 deletion and wild type have the same specific rate of growth,but the dry cell weight accumulation of recombinant strains was 17.2%(0.29/0.24,p-value?0.05)lower than the wild type.And the accumulation of ethanol increased by 12.45%(p-value?0.05)in recombinant strains.Destroying pyruvate transport carrier could improve the production of ethanol in the xylose fermentation without affect the special growth rate.In conclusion,by changing the phosphorylation state of hexokinase 2 partly maintain the activated state of glucose signaling system in xylose fermentation.The accumulation of ethanol under xylose metabolism was significantly improved.On the other hand,knocking out MPC1 resulted in completely destruction of the mitochondria pyruvate carrier and increasing the accumulation of ethanol.Adjusting the state of cell metabolic not only optimize xylose metabolic obviously but also has important reference significance to improve the other substrate utilization.