| Production of biofuels and biochemicals from lignocellulosic biomass such as agricultural and forestry residues is important topic for biomass biorefinery.However,economic biorefinery is still challenging.There are two major challenges,one is inhibition of microbial growth and fermentation by various inhibitors present in lignocellulosic hydrolysate,and the other is the difficulty of native strains in utilizing abundant xylose present in the hydrolysate.Therefore,improving tolerance of microbial strains to the inhibitors,and developing strains that can efficiently ferment xylose are two important issues for efficient lignocellulosic biorefinery.The budding yeast Saccharomyces cerevisiae is widely used for lignocellulosic biorefinery studies,and it is of great importance to study the fine regulation of gene expression in S.cerevisiae,and to improve the efficiency of its genetic engineering,as well as optimization of the bioprocess.Therefore,this study includes the following sections:Firstly,it is important to fine tune gene expression in different metabolic pathways by controlling the strength of promoter.Constitutive promoters are commonly used for yeast strain development.However,it is not clear whether activities of these promoter are affected by stress conditions and the presence of xylose.Therefore,changes of promoter activity were investigated under the conditions related to cellulosic ethanol production.The activity of nine promoters,including eight commonly used native promoters and one synthetic hybrid promoter,were characterized in the presence of xylose and under different environmental stresses,with yEGFP as the reporter gene.It was found that PTDH3 and the synthetic hybrid promoter promoter P3ŚC-TEF1 exhibited the highest strength and stability under almost all the conditions.However,the activities of the commonly used "constutive" promoters,such as PADH1 and PPGK1 changed in a large range under a variety of environmental stresses."Inducible" promoters PHSP12 and PHSP26 showed induced activities under high temperature and acetic acid stresses,and these two promoters also showed higher expression levels when fermentation was performed in the presence of xylose as the sole carbon source or in a mixed sugar fermentation conditions after glucose was depleted.Secondly,CRISPR-Cas9-based genome editing technology is very effective for yeast strain construction,but optimization of the method is needed to efficiently construct S.cerevisiae strains.The construction strategy for the sgRNA expression vector was then optimized,allowing efficient and rapid construction of sgRNA expression vectors.In addition,new resistance screening marker was introduced into the Cas9 expression vector,and the plasmid elimination process was simplifed,allowing convenient and efficient applications of CRISPR-Cas9-based genome editing.Then the optimized CRISPR-Cas9 system was used for knockout of single and multiple genes in S.cerevisiae,in which the efficiency of single gene knockout was over 90%,while simultaneous knockout of two and three genes was achieved with the success rates of 83.3%and 66.7%,respectively.By using the optimized technique in this study,investigation on stress tolerance related gene can be carried out rapidly.In addition,the potential of artificial transcription factors based on dCas9 and the endogenous domains from S.cerevisiae in the transcriptional regulation was also explored.The results showed that the endogenous transcription factor domains of S.cerevisiae fusion to dCas9 could effectively regulate gene transcription,and the fusion of multiple transcription factor domains could further expand the regulatory scope.Acetic acid is ubiquitously present in lignocellulose hydrolysate,which seriously inhibit cell growth and ethanol fermentation of S.cerevisiae.Therefore,it is crucial to improve the tolerance of S.cerevisiae toward acetic acid for bioconversion of lignocellulosic biomass.Previous studies in our group found that elevated expression of the arginase-encoding gene CAR1 in stress tolerant S.cerevisiae.Overexpression of CAR1 improved acetic acid tolerance of S.cerevisiae,and it was found that CAR1 overexpression affected the global metabolism of intracellular amino acids in the presence of acetic acid stress.The content of most free amino acids in CAR1-overexpressing strain was significantly decreased under acetic acid stress,and changes in the transcription of key genes involved in amino acid metabolism and transport were also revealed.CRISPR-Cas9-mediated genomic editing technology was used to carry out point mutation on the transcription factor binding site in the CAR1 promoter region,and the regulatory mechanism of CAR1 was explored,which provided a basis for further studies on the mechanism of CAR1 regulated yeast stress tolerance.Finally,recombinant yeast strains that can ferment xylose were constructed,and cellulosic ethanol fermentation with Jerusalem artichoke stalk(JAS)as a feedstock was further studied and optimized.Simultaneous saccharification and fermentation with a short time pre-hydrolysis was performed with high solid loading of the pretreated JAS in a batch-feeding manner,which achieved 48.67 g/L(6.10%,v/v)and 1.01 g/L/h in ethanol titer and productivity,respectively.Separated hydrolysis and fermentation with feeding of solid biomass and cellulosic enzyme was carried out with NaOH-H2O2 pretreated JAS,which achieved 66.20 g/L ethanol,and ethanol yield based on the total sugar of the pretreated JAS was 0.317 g/g.The results in this thesis provide a basis for further construction of robust recombinant yeast strains and efficient biorefinery of lignocellulosic biomass. |
PDF Full Text Request