Construction Of Levoglucosan-utilizing Saccharomyces Cerevisiae And The Study Of Strain Tolerance To Lignocellulosic Materials

The production and development pattern dominated by fossil resources has brought economic and environmental problems.Utilization of abundant renewable lignocellulosic biomass to produce various chemical products and biofuels has attracted more and more attention.Three main processes:pretreatment,saccharification and fermentation,are necessary to use lignocellulosic materials.In the process of pretreatment,some weak acid,furan and phenolic compounds,which inhibit the growth and metabolism of microorganisms,are generated along with the release of hemicellulose sugars,which are xylose,glucose,and so on.Then hydrolyze the cellulose by cellulase and obtain D-glucose is the most mainstream strategy to prepare the sugars for subsequent fermentation to date.In addition,pyrolysis of cellulose,which is a very rapid reaction,has also been widely studied in recent years.The main product of cellulose pyrolysis is high concentrate levoglucosan,which can be convert to glucose-6-phosphate by levoglucosan kinase(LGK)or the 1,6-anhydro-N-acetylmuramic acid kinase(AnmK)that has LGK activity.Then the glucose-6-phosphate enter the glycolysis pathway.Saccharomyces cerevisiae,which is generally recognized as safe(GRAS),is widely used in the production of food,beverage,biofuel,drugs,and other important chemicals.As a model organism,its genetic background and physiological characteristics are widely studied.However,S.cerevisiae native strains cannot use levoglucosan due to lacking the initial levoglucosan metabolic pathway.In the context of the background that we mentioned above,this thesis works from two aspects.First,we selected the LGKs or AnmKs that well expressed in S.cerevisiae;modified Gal2p to enhance its levoglucosan transport capacity;and then constructed levoglucosan-utilizing S.cerevisiae recombinant strain.Second,we mined the transcription factors,which affect the tolerance of strain to the toxic compounds in lignocellulose hydrolysate,basing on the transcriptomic and genomic difference of strain LF1,a lignocellulose hydrolysate resistant strain,and its parent strain XH7.More details are as follows.1.Introduced the levoglucosan metabolic pathway into Saccharomyces cerevisiaeSix genes encoding the LGKs or AnmKs that have levoglucosan kinase activity were synthesized and respectively introduced into S.cerevisiae CEN.PK113-5D.The recombinant strain CEN-Rho,which expresses the AnmK gene from R.toruloides showed the best growth capacity in the plate contains the medium with levoglucosan as sole carbon source.The six genes were also introduced into strain B2-1,which is derived from an industrial strain BSIF.After adaptive evolution,the strain BE-Sch,which expressed the AnmK gene from S.stipitis,showed the better growth than other strains.In the medium with 5 g L-1 levoglucosan,BE-Sch consumed 21.93%levoglucosan after 70 hours fermentation.Consider the results we got in both two series of strains,the AnmKs from both R.toruloides and S.stipites were used in the following work.Their activity of levoglucosan kinase were compared.The activity of levoglucosan kinase in CEN-Rho was 10.65 U/mg of protein and that in CEN-Sch was 6.96 U/mg of protein.2.Optimize the transporter protein to improve the absorption of levoglucosan of the strainsWe determine the levoglucosan transport capacity of strain CEN.PK113-5D and hexose transporters null strain EBY.VW4000.The result showed that the levoglucosan transport capacity of strain CEN.PK113-5D was 4.022 mg levoglucosan g DCW-1,which was stronger than that of EBY.VW4000.This indicated that the hexose transporters could transport levoglucosan,however,the transport capacity of levoglucosan is much weaker than other sugars.Then we built the molecular model of Ga12p and docked with levoglucosan,the amino acid residues with a distance of less than 5A from levoglucosan were selected and changed to alanine,respectively.The mutants were expressed in EBY.VW4000 with AnmKs from R.toruloides or S.stipitis.The results showed that the mutations of F85A,Q341 A,N346A.Y446A and W455A positively affected the levoglucosan transportation of Ga12p.Further fermentation results in the medium with 5 g L-1 levoglucosan as the sole carbon source showed that strains expressing Gal2pQ341A and Ga12pW455A utilized 73.3%and 73.7%Ievoglucosan after 48h of fermentation.These are respectively 3.70 and 3.72 times of the strain expressing wild type Gal2p.and 12.86 and 12.93 times that of the blank control strain.Further molecular structural analysis showed that Q341A and W455A mutations of Gal2p may enhance the ability of levoglucosan transport by reducing the barrier to its entry into cells.3 Mining of transcription factors related to lignocellulosic hydrolysate toleranceIn our previous work,a strain LF1 with significantly improved tolerance to lignocellulosic hydrolysate was obtained by evolving a diploid strain XH7 in lignocellulosic hydrolysate.In this work,we compared the transcriptome and genome of LF1 and XH7.The transcription factors related to the significantly up-and down-regulated genes(LF1 vs XH7)were figured out by the tools supplied by website Yeastract.Then the mutations in these seven transcription factors were selected,and the effect of these mutations to the strain tolerance was studied in an easy-to-operate laboratory strain BY4741.Here is an example to illustrate the construction process of each series of strains.The gene encoding transcription factor X in the genome of BY4741 were knocked out.After that,the X genes of BY4741,XH7,and LF1 were respectively cloned and ligated into a centromere plasmid pJFE1.Then the result plasmids were transformed into the strain without X gene,and the fermentation characteristics of strains with these recombinant plasmids were investigated in the medium contains acetic acid,furfural,5-hydroxymethylfurfural(5-HMF)and vanillin(For the sake of simplicity,well call them inhibitors).With the process,we found that the maximum specific growth rate of strain pdr1Δ+pJFE1 increased by 66.1%compared with the control strain BY4741+pJFE1 in the liquid medium contains inhibitors.Consistent with this,the lag phase of pdr1Δ+pJFE1 was much shorter than that of BY4741+ pJFE1.Furthermore,the grow test on the plate with the medium contains separate inhibitors suggested that deletion of PDR1 mainly increased the strain tolerance to furfural and 5-HMF.The HPLC analysis of fermentation broth confirmed that the disappearance rate of furfural and 5-HMF in BY4741(pdr1Δ)were faster than that in the control strain BY4741.To study whether the results of deleting PDR1 are universal,we also deleted the PDR1 of CEN.PK113-5D and W303,respectively.It was found that knockout of the PDR1 gene also improved the inhibitor tolerance of W303 but not CEN.PK113-5D.While expressing the PDR1BY4741 in CEN.PK113-5D reduce the inhibitor tolerance of CEN.PK113-5D.The sequences blast result revealed that compared to BY4741 and W303,five consecutive asparagine residues from 1011 to 1015 were absent in CEN.PK113-5D.These results suggested that these five asparagine residues are important for the function of Pdr1p.