| Lignocellulosic hydrolysates consist of 60-70%glucose and 30-40%xylose,and the rapid co-fermentation of both sugars is essential to improve the utilization of lignocellulosic biomass feedstock and reduce the cost of fuel ethanol production.Saccharomyces cerevisiae is the superior organism for ethanol production,however,the wild-type strain lacks the pentose assimilation pathway.By introducing heterogenous xylose metabolic genes and optimizing relevant pathways endows S.cerevisiae strains with the capacity to utilize xylose.However,the xylose metabolic rates of the recombinant strains were only about 50%of the rate that they metabolize glucose when single sugar was used as the carbon source.Moreover,glucose significantly inhibits the use of xylose in glucose-xylose co-fermentation.The rate of xylose utilization is much lower than that when xylose as the sole carbon source,even after glucose is depleted(We defined the inhibition of xylose utilization after glucose depletion as "postglucose effect"),which greatly limits the utilization of lignocellulosic material.Metabolic engineering and directed evolution are used to improve xylose utilization.Reverse metabolic engineering strategies supported by omics are performed to reveal the key factors that affect xylose utilization,recently.Our previous work suggested that beside glucose there are signal molecules inhibiting xylose metabolism in the supernatant of fermentation broth of glucose-xylose co-fermentation when glucose depleted.Moreover,overexpression of Tec1 or a Tec1 mutants that will not be degraded with MAPK signals facilitated the metabolism of xylose in glucose-xylose co-fermentation during the stage after glucose depleted.Based on this,this paper starts from the external and internal factors,studying the relationship between MAPK signaling pathways and xylose metabolism;exploring possible global regulators of xylose metabolism by reverse metabolic engineering based on omics data;and developing genetic modification techniques to express exogenous genes in industrial strains.In addition,in order to develop a simple and efficient yeast genetic manipulation method,the integration method of heterogenous genes in the rDNA region was improved and optimized in this paper.The details are as follows:(1)Exploring the effect of extracellular potential signaling molecules and MAPK signaling pathways on xylose metabolismAim to get more clues about the signal molecules that inhibit xylose metabolism,we extracted the supernatant of fermentation broth by organic solvents with different polarities,and each phase was collected,evaporated,and added to fresh medium.The results showed that the evaporation and organic solvents extraction decreased the inhibition of the supernatant of fermentation broth,but the aqueous phase still strongly inhibited the cell growth using xylose as the carbon source.This suggested that the inhibiting factors mainly exist in the aqueous phase,and more than one factors affect the xylose metabolism in the supernatant of fermentation broth.Aim to reveal the effect of extracellular signals and the MAPK pathways on xylose metabolism,we knocked out the signal-receiving proteins of pheromone pathway and filamentous growth pathway,which are the MAPK signaling pathways related to Tec1.The results showed that knocking out the three membrane proteins genes Msb2,Sho1,and Opy2 in the filamentous growth pathway decreased the xylose ratio utilization by 45.9%,52.9%,and 36.5%,respectively.Msb2,Opy2,and Sho1 are also involved in the highly permeable glycerol(HOG),another MAPK pathway.Meanwhile,it is reported that the HOG1 mutant increased the xylose utilization.Therefore,it is reasonable to believe that the signals received by Msb2,Opy2,and Sho1 may be important influencing factors on the post-glucose effect/xylose utilization,and their effects may be realized through the regulation of Tec1 and Hog1,respectively.(2)To explore the transcription factors that influence the post-glucose effectTo dig transcription factors that may affect xylose metabolism,transcriptomic and genomic data of the wild-type diploid strain BSIF and its evolved strain XH7 were compared.We initially screened out three transcription factor genes(CST6,STB4,and SOK2)with InDels mutations and five genes(GIS1,ADR1,RSF2,RLM1,and SKN7)with SNPs mutations,and their effects on xylose utilization were studied by knocking out and overexpressing mutants,respectively.The results showed that knocking out the Cst6,which is involved in the stress response regulatory network and non-optimal carbon source utilization,increased the xylose specific utilization rate by 17.0%and ethanol yield increased by 10.5%.The mutation with Adr1 A49T brings negative effect on xylose metabolism during glucose-xylose co-fermentation.Overexpression of Skn7 R41P,S335N,a transcription factor that be required for optimal induction of heat-shock genes in response to oxidative stress and involved in osmoregulation,cell wall integrity,significantly reduced the ability of the strain to metabolize xylose,while knocking out SKN7 increased the specific utilization rate of xylose during glucose-xylose co-fermentation,and promoted cell growth.(3)Optimization of rDNA-mediated expression method for multicopy integration of genes in Saccharomyces cerevisiaeS.cerevisiae is a good kind of cell factory,and improving genetic manipulation tools can speed up the construction of engineered strains.Highly repetitive rDNA sequences are one of the two main loci for gene multicopy integration.In this study,a simple and efficient gene multicopy integration strategy was developed based on the dynamic balance of rDNA copy number.First,we pretreated the cells with hydroxyurea(HU)to reduce the copy number of rDNA,then integrated the heterologous gene into the rDNA sequence,and released the selection pressure at the end.The copy number of heterologous gene increased with the recovery of rDNA copy number of the strain.Our results showed that HU pretreatment almost doubles the copy number of heterologous gene integrated into the genome.Moreover,compared with removing HU stress during transformation,selecting the transformants on the medium containing 20,000 μg/mL G418 and 60 mM HU first,and then culture cells in HU free condition,the average copy number of integrated genes in the recombinant strains was higher,and the possibility to obtain the recombinant strains with high-copy numbers was higher.Finally,using this approach,we obtained a recombinant yeast with xylose isomerase gene(Ru-xylA)of 18 copies that integrated in S.cerevisiae genome by a single transformation process.This novel rDNA-mediated multicopy genome integration strategy provides a convenient and efficient tool for metabolic engineering of S.cerevisiae. |
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