| Trichoderma reesei(T.reesei),which has been used in industry,is able to degrade lignocellulose(mainly cellulose,hemicellulose and lignin).Cellulase expression is influenced by many aspects,including induction of signal induction,signal transduction.and transcriptional regulation.The transcription factor is responsed to the external signals to activate or inhibit cellulase gene expression.The major transcription factors include Xyr1,Ace1,Cre1,Vib1,etc.,which bind to the promoter region of the cellulase gene to regulate the expression of cellulases.Crel,as the most important transcriptional repressor in T.reesei,plays an important role in the carbon catabolic repression(CCR).But the Crel deletion strain has a serious impact on the growth of the cells,therefore Crel is a bifunctional transcriptional factor.However,in the industrial production process,the release of CCR without affecting the growth of the cells is a very critical issue.However,there is no good method has been found,and the mechanism of Crel in CCR is not clear.In the second-generation ethanol,the last step is to ferment fermentable sugars into ethanol by yeast.Since the fermentable sugar cannot directly enter the cell by simple diffusion,the carrier is required to transport sugar from the extracellular to the intracellular.Therefore,the transport of sugar by sugar transporters is the first step to produce ethanol,and it is also a very important step.The most abundant of the lignocellulose raw materials produced by enzymatic hydrolysis is glucose and xylose.In Saccharomyces cerevisiae(S.cerevisiae),there is a lack of specific xylose transporters that can efficiently utilize xylose.The transport of xylose is mostly transported by non-specific hexose transporters.Therefore,there is a very serious glucose inhibition in the transport of xylose.Accordingly,finding a sugar transporter that can efficiently transport xylose has become a research hotspot for scientists in recent years.In this paper,Crel,a very important transcription factor in T.reesei,was studied in the alleviation of glucose carbon repression(CCR).At the same time,the sugar transporters derived from T.reesei are discovered and detailed analysis of its amino acid sites with key functions in the transport process was studied.Finally,a new green,efficient method to produces gluconic acid from potato pulp was developed.Specifically,it includes the following aspects:1)Study on the alleviation of carbon catabolite repression(CCR)by transcription factor Cre1 from Trichoderma reeseiCre1 in T.reesei is a global transcriptional regulator,which plays an important role in the growth of bacteria,spore germination and other physiological processes,and is involved in the inhibition of cellulase gene expression through the binding of the major cellulase gene cbh1 under glucose conditions.It was reported that Crel was also involved in the regulation of transcriptional activators XYR1 and ACE2.Phosphorylation of proteins plays an important role in life activities and the regulation of transcription factors.Crel of T.reesei is also a phosphorylated protein.It is reported that when the 241 amino acid of Crel is mutated from serine to alanine,mutated Cerl can bind to cbh1 gene without phosphorylation,resulting in permanent inhibition of CBH1 expression.When the 244 amino acid was mutated from glutamic acid to proline,Crel cannot be phosphorylated or bound to DNA,and it was found that CCR was significantly down-regulated.In the early from our lab,the effect of Crel from T.reesei on CCR was explored.It was noticed that compared with the Penicillium oxalicum 114-2 strain,the CreA had a frameshift mutation in the high-yield strain JU-A10 and therefore alleviated CCR.Therefore,an analogy was carried out in T.reesei,and the sequence after the Cre1 386 amino acid in T.reesei was truncated and prolonged.It was found that the transcription level of the main cellulase gene was significantly upregulated under glucose conditions,effectively alleviating CCR.We predicted the phosphorylation site of the amino acid after the 386 position of the Crel protein and found that there are five potential phosphorylation sites.Therefore,we hypothesized that the alleviation of the CCR may be the deletion of the phosphorylation site.In order to verify this hypothesis,we constructed a potential phosphorylation site mutation strain in the T.reesei strain,first studied its mycelial growth,phenotypic observation and sporulation determination under different carbon source culture conditions.However,no significant changes were found,indicating that the mutation had little effect on the growth of the strain.Next,we mainly studied the changes of major cellulase genes and transcription factors in the transcription level of glucose and its microcrystalline cellulose at different time points.After cultivated under glucose condition for 12h,the transcription level of the main cellulase gene(including cbh1,cbh2,egl1,egl2,and bgl1)was significantly upregulated,of which cbh2 and eg2 were the most significant,and the up-regulation level was about 150 times.This indicates that the transcriptional inhibition of the cellulase gene and regulatory factors under glucose conditions is alleviated.At the same time,we examined the transcription and enzyme activity of mutant strains under cellulose conditions.It was found no obvious change in the cellulase genes and transcription factors at the transcriptional level and in the enzyme activity level.Therefore,the Crel mutation relieves the inhibition of the cellulase gene and its regulatory factors under glucose conditions.2)Study on high efficient sugar transporter in Trichoderma reeseiThe transport of sugar was the first step from extracellular to intracellular which is of vital importance.In the process of degrading lignocellulose to produce ethanol,the hydrolysates obtained by enzymatic hydrolysis of lignocellulose contains a large amount of fermentable sugars such as glucose and xylose,while there is no xylose metabolic pathway for wild-type S.cerevisiae.After years of effort,scientists have done many metabolic engineering researches to modify the xylose metabolic pathway and introduced it into S.cerevisiae.Another problem is the efficient transport of xylose,which has become one of the main rate-limiting steps for xylose utilization.The xylose transport in S.cerevisiae is carried out by the endogenous hexose transporters.The most efficient one is Gal2,but it also has a low transport efficiency and the transport of xylose is severely inhibited by glucose.Therefore,the screening of heterologous sugar transporters with high transport capacity has become a hot topic for researchers.Microorganisms that naturally utilize xylose are considered to be good sources of transporters.As genomic level studies become clearer,many putative transporters are found to be derived from filamentous fungi,many of which can transport and utilize xylose.Therefore,bioprospecting of high efficient sugar transporters in T.reesei was conducted,and further studied on its efficient transport mechanism was also investigated.Based on the study of high and low system sugar transporters in Neurospora crassa,we have explored the T.reesei genome and found three putative sugar transporters that have not been studied before.Firstly,we analyzed its transmembrane structure by bioinformatic tools,and found that all three transporters have 12 transmembrane structures,belonging to the typical MFS family transporter.Next,we analyzed the expression and function of these three transporters in S.cerevisiae.We added a GFP fluorescent tag to the C-terminus of the protein.It was found all three transporters were correctly expressed on the cell membrane.Phenotypic analysis was then carried out under different carbon sources,and it was found that the Xltrlp expression strain FJTR3_Xltr1 can grow on various hexose conditions(including glucose,fructose,and mannose),while the other two transporters have no obvious growth.Next,we measured the xylose transport capacity of three proteins and found that only Xltrlp has the transport capacity of xylose,and the intracellular xylose accumulation at 120 min is 8.44 times higher than Gal2 from S.cerevisiae.In order to analyze the mechanism for its efficient transport,the transport type of Xltrlp was studied and found that it is a facilitator.Transporters belonging to this type generally have lower affinity for xylose transport,but with higher transport efficiency for xylose.At the same time,we analyzed the conserved amino acid sites of xylose transporters by bioinformatics analysis,and the structure of Xltrlp was predicted.Combined with the analysis,we selected 13 amino acid sites for mutation,so as to study their role in sugar transport.After mutation,it was found that most of the selected sites have a very important role in the transport of xylose.It was also found that some of these sites are specific for the transport of xylose or hexose,such as F300A causes the loss of xylose transport capacity but retains a higher level of glucose transport;while the N326F retains a relatively high xylose transport capacity but loses the transport capacity of glucose.What is more,the N434 amino acid site plays an important role in both transport of hexose and xylose.Hydrogen exists between Xltrlp and xylose molecule;and hydrophobic interaction exists between Xltrl p and glucose molecules.In addition,experiments on the transport of Xltrlp under different hexose conditions was also conducted.When different kind of hexose was used as a separate carbon source,the utilization rate of sugar is similar,but under mixed hexose,the sugar utilization rate is as follows:glucose>mannose>fructose.And mutations studies on hexose transport was also conducted to demonstrate the function of amino acid sites in the hexose transport process.It was found the transport capacity of hexoses(glucose,mannose,or fructose)was lost after mutating the following sites:D39A,R132A,Q292Y/W,F298A,I299A,Y301A,Y302A,N326F/Y/W,W407A and N434A.Since mannose has a high value in the industry,most of the current methods was using chemicals to prepare mannose from glucose using a dicarboxylic acid,and a mobile simulated bed was used to separate mannose.The traditional method is expensive and difficult to separate.Therefore,in the mutant strains that retain the transport capacity,we further measured the sugar transport and utilization of the mixed hexose conditions(glucose and mannose),and found that the FJTR3_Xltr1pQ291A strain achieved the separation of mixed sugars.Glucose was used up and about 70%of mannose was remaining,and a value-added product ethanol produced.Further,ethanol can be obtained by distillation,and finally mannose was obtained.The process is simple and accompanied by the production of high value-added products,and the separation of mannose from mixed sugars is achieved.3)Development of a method for producing high value-added gluconic acid from potato pulpPotato pulp is an agricultural waste generated in the starch production process.It is often discharged as garbage,which not only causes waste of resources,but also brings environmental pollution problems.However,potato production in China accounts for more than one-fifth of the world,so a large amount of potato pulp will be produced during the starch production process.Therefore,we tried to develop a new green,sustainable approach to producing high value-added products from potato waste.In this paper,we found that the main components of potato pulp are starch,pectin and cellulose.Therefore,we used mixed cellulase enzyme derived from T.reesei TX and P.oxalicum JUA10-1 and commercial pectinases to hydrolyze the potato pulp,and the hydrolysates were further fermented by Gluconobacter oxydans DSM 2003 to ferment the fermentable sugar in the hydrolysates to produce gluconic acid.The highest conversion rate from glucose to gluconic acid is 94.9.%.Therefore,we have successfully developed an efficient,green and sustainable method for producing gluconic acid from waste potato residue. |
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