Study On The Xylose Metabolic Efficiency Decisive Factors Relevant To The Post-glucose-effect In Saccharomyces Cerevisiae

Lignocellulosic ethanol,that is,second generation fuel ethanol technology,is an important direction of diversified energy strategy in the future.Saccharomyces cerevisiae(S.cerevisiae)is the most potential second-generation fuel ethanol production strain due to its outstanding characteristics of high yield of ethanol by glucose fermentation and food-grade safety.Glucose and xylose are the most important sugars in the lignocellulosic feedstock hydrolysate.Co-fermentation of glucose and xylose is one of the key technologies to make full use of raw materials and reduce production costs.In recent decades,recombinant S.cerevisiae strains constructed through metabolic and evolutionary engineering have achieved better xylose utilization capacity.However.it was found that the xylose consumption rate and ethanol production rate of strains during glucose-xylose co-fermentation were generally lower than that when xylose was the sole carbon source in the medium(referred to as the X stage).It is generally believed that the utilization of xylose is competitively inhibited by glucose in transport and metabolism.However,in the xylose consumption phase after glucose depletion during glucose-xylose co-fermentation(referred to as the GX stage),the growth rate(μ),specific xylose utilization rate(rxyose),and specific ethanol production rate(rethanol)of strain are much lower than those of strain when xylose was used as the sole carbon source,which seriously affected the fermentation efficiency of the mixed sugar,We call this phenomenon the post effect of glucose on xylose metabolism(referred to as"post-glucose effect").The post-glucose effect has been neglected for a long time.which seriously affects the fermentation efficiency of lignocellulosic hydrolysate raw materials.Here,the xylose metabolic efficiency decisive factors relevant to the post-glucose effect in S.cerevisiae were studied from the perspective of transcriptional regulation network,and part of the mechanism was explored.specifically as follows:(1)Deletion of thiamine biosynthesis-related transcription factor Thi2p or overexpression of cell cycle-related transcription factor Nrm1p can alleviate the post-glucose effect.Two independent xylose-fermenting S.cerevisiae strains derived from a haploid laboratory strain BSGX001(Ru-XI,XK,gre3::PPP,cox4△,AE)and a diploid industrial strain XH7(phol3::XI,gre3::PPP,XK,3δ::XI,AE)were used in the present study.Both strains showed lower μ and rxylose during the GX stage than during the X stage.Their common differentially expressed genes(DEGs)of both strains in GX stage and X stage were compared,and then the GO terms and pathways of the common DEGs in both strains were analyzed.The transcriptome results indicated that glycolysis and pentose phosphate pathway related gene expression were reduced;tricarboxylic acid cycle and glyoxylic acid cycle related genes were increased;ribosomes and translation-related gene expression were reduced,during the GX stage than during the X stage.Compared with the X stage,the activity of Snf3p/Rgt2p pathway in the GX stage is closely related to glucose metabolism,while the Snflp-Miglp and cAMP-PKA pathways are less active.These changes in transcriptional levels combined with physiological phenomena in which GX stage cell growth is almost stagnant indicate that cells in the GX stage enter the fermentation lag phase.Then,we verified the effects of significantly regulated transcription factors and some metabolic enzymes on the xylose utilization of the strain.The results showed that overexpression of the cell cycle-related gene NRM1 altered the cell cycle,and increased the μ,rxylose and rethanol of strain by 8.7%,30.0%and 76.6%,respectively,in the GX stage.Furthermore,knockout of ribosome synthesis related gene THI2,the μrxylose and rethanol of strain increased by 17.4%,26.8%,and 32.4%,respectively,in the GX stage.(2)Revealing the regulatory network of thiamine biosynthesis-related transcription factor Thi2p in the GX stage.In view of the previous reports that xylose isomerase,which is the first step of xylose metabolism,has an important influence on the metabolic efficiency of xylose,and as a heterologous expressed gene,information cannot be obtained from the transcriptome.Therefore,we first confirmed that the transcription level of XylA and xylose isomerase(XI)activity of the thi2△ strain were not significantly different compared with the control strain,indicating that increased xylose metabolism ability of the THI2 deletion strain is not related to xylose isomerase activity.Then the transcriptional difference of the thi2△ strain and the control strain in the GX stage was compared in the present work.The regulation of Thi2p differs from a previous regulatory network that functions when glucose is the sole carbon source.which suggests that the function of Thi2p depends on the carbon source.Follow-up experiments indicate that deletion of THI2 regulates xylose metabolism in the GX stage by affecting the expression involing in cell cycle,stress tolerance,and cell viability-associated functional proteins and transcription factor-encoding genes.Some of these effects were very notable.For example,overexpression of the stress tolerance related genes MID2,STT4 and BDH2,cell viability TFC3,and function unknown gene RG12 increased the rxylose of strain by 45.9%,49.2%,26.2%,42.6%and 41.0%,respectively;Deletion of the cell cycle related gene CIPI and the stress tolerance related gene IXR1 increased rxylose by 26.2%and 36.1%,respectively.In addition,deletion of THI2 or overexpression of MID2,STT4,and CDC42 significantly reduced the proportion of dead cells in the GX stage,which may be one of the reasons that THI2 deletion alleviated the post-glucose effect.The anaerobic fermentation results of five of the above-mentioned most significant variants in the bioreactors showed that overexpression of STT4,TFC3 and RGI2 increased the rxylose of the strain by 36.9%and 42.8%,and 29.7%,respectively,in the GX stage under such tightly controlled conditions.(3)The effect of transcription factor Tec 1 p on post-glucose effect.Using the YEASTRACT,we predicted seven potential transcription factors(TFs)(Tec1p,Sfp1,Bas1p,Msn2p,Yapl,Ste12p,Ace2p),which regulated the expression of over 80%of these 11 differently expressed TFs between the GX and X stage mentioned in the above work(1).Then the effects of the seven TFs on the post-glucose effect were investigated.The results showed that overexpression of filamentous growth-associated transcription factor gene TECl(driven by TEF1 promoter)alleviate the post-glucose effect;expression of TECIT2T3M mutant variant in the chromosome,which can block of the degradation of Teclp caused by MAPK pathway signal,significantly alleviate the post-glucose effect,the rxylose of the strain in GX stage increased by 2.25 times compared with the control strain,and and the rethanol reached 0.071 g L-1 h-1 g-1 DCW higher than the wild type at GX stage;overexpression of TECl T273M(driven by TEF1 promoter)caused filamentous growth of the strain,which was not conducive to alleviating the post-glucose effect;expression of the TECl P411A P429A P448A(TEClAxY)mutant variant(driven by TEFl promoter),which can block of the degradation of Teclp caused by the TORCl pathway does notalleviate the post-glucose effect.Immunoblotting results showed that Tec1pwr≈Teclp AxY<Tec1pT273M<Tec1pT273M AxY,indicating that the increase of Teclp protein may be one of the reasons for promoting the xylose metabolism:the alleviation of the post-glucose effect is mainly related to the uncoupling of Tecl p and MAPK signaling pathways.Then we compared the transcriptome differences of the TECl T273M mutant variant in the chromosome and the control strain TEClWT at GX stage.Through RNA-seq analysis,we found that ribosome-related genes and iron-uptake-related genes were significantly regulated in TEClT273M mutant variant at GX stage.Combined with the reported view that the addition of iron ions to the medium promoted xylose fermentation,we speculated that TEClT273M enhanced the xylose metabolism may be related to the promotion of ribosome assembly to facilitate translation and the enhancement of iron ion absorption.The specific mechanism is still under exploration.(4)Preliminary study on inhibiting the xylose utilization by the medium after the end of glucose fermentation.Our recombinant strain was able to ferment xylose well to produce ethanol in a general medium with xylose as the sole carbon source(referred to as the xylose medium).which served as a positive control for this study.Then,we performed a batch of glucose/xylose co-fermentation,and after two hours of glucose depletion,the fermentation broth was centrifuged to collect the mud cells and supernatant,respectively.Some of the mud cells were inoculated into the fresh xylose medium as seeds,and it was found that the growth and xylose metabolism of strain were no significant difference,expect for the cell growth slightly delayed,compared with the positive control.However,the seeds in the logarithmic phase were added in the supernatant medium,it was found that the cells growth and metabolism were completely inhibited.This phenomenon indicates that there are factors limiting xylose fermentation in the fermentation broth after glucose fermentation.After that,we added nitrogen sources of different concentrations in the supernatant medium and treated the supernatant medium with protease,high temperature or freeze-thaw treatment,but the post-glucose effect was not alleviated.We hypothesized that small molecules secreted outside the cell after glucose deletion may influence the xylose metabolism.Glycerol/ethanol/acetate were added directly to the xylose medium in the same amount as the supernatant medium,but neither of them affected xylose metabolism.In order to explore the substances in the supernatant that inhibit xylose metabolism,we performed non-targeted metabolomics analysis on the supernatants of GX stage and X stage of BSGX001 strain,respectively.In the positive and negative ion mode,44 and 96 substances were identified,among which 41 substances were significantly different,and 20 and 23 substances is the positive and negative ion modes,respectively.By directly adding metabolites of different concentrations in the supernatant,or by genetically modifying related genes to regulate metabolic pathways,we try to change the metabolite concentration or screen genes that may affect xylose metabolism.The current work has not yet found a possible inhibitor,and the next step is to try to screen mutants that can alleviate the strong inhibitory effect in the supernatant by gene mutagenesis strategy,and the related work is still going on.In summary,this study clearly proposed the phenomenon of post-glucose effect for the first time,and tried to reveal the mechanism of the post-glucose effect from the transcriptional level and metabolic level,and to analyze some key metabolic nodes,metabolites,proteins and signaling pathways related to the regulation of xylose metabolism efficiency.Many new biological regulatory elements and genes have been discovered,different levels of gene regulatory networks have been drawn,effectively accelerate xylose metabolism and alleviate the post-glucose effect.Our work is of great significance in guiding the further transformation of engineering strains and improving the efficiency of lignocellulosic ethanol production.