| Fuel ethanol is recognized as the most promising alternative energy source due to its multiple advantages, such as clean, green and renewal. Lignocellulose materials, including crop straw, forestry by-products and municipal waste, are the most widely distributed and abundant renewable resources on the earth, which are the best raw materials for fuel ethanol production. The utilization of such lignocellulose biomass for the production of fuel ethanol involves dilute acidic or alkali pretreatment first The hydrolyzed sugars can then be fermented to ethanol by Saccharomyces cerevisiae. In the pretreatment process, numerous compounds are inevitability released as by-products, and some of them are toxic to yeast cells and will interfere with cell growth and subsequent ethanol fermentation. Among the inhibitory compounds, furfural is one of the most important inhibitors. It has been reported that expression of about 300 genes were up-regulated to enhance the detoxification and tolerance abilities of S. cerevisiae under furfural stress. Transcriptional regulation factors related to up-regulated expression of those genes have been preliminary predicted and analyzed in S. cerevisiae.In this study, we chose 23 transcriptional regulation genes most probably related to furfural detoxification and tolerance, according to their functional classification, as the research object. The haploid laboratory S. cerevisiae strain BY4742 was used as experimental material and host for gene expression. Firstly, the promoters used for expression of these genes were comparatively studied and then screened out Secondly, recombination strains genetically expressing each regulation gene under selected promoter control were constructed by engineering. Differences of tolerance to furfural in phenotype within those recombinant strains were comparatively studied and the key regulation genes related to furfural tolerance were screened out.Finally, transcriptional analyses of down-stream representative genes controlled by the key regulation factors were carried out The new foundlings obtained in this study were as follows:(1) To study the promoter suitable for expression of regulation genes related to furfural tolerance, YAP1, an identified regulation gene, was chosen as representative gene. Then, recombinant plasmids expressing YAP1 under different promoter control, including six commonly used and constitutively overexpressed promoters ADHlp, HXT1p, PGKlp, PDClp, TEFlp, TDHlp, and TPIlp and one inductively expressed promoter ADH7p under furfural stress, were constructed, and successfully transformed into host strain of S. cerevisiae BY4742. Phenotypic differences of the genetic engineering strains in response to furfural were comparatively studied. The results showed that tolerant abilities of recombinant strains were remarkably shorter lag-phase under furfural stress and displayed stronger tolerance to furfural than reference strain, especially in genetic strains constructed using TEF1p, ADH7p and PGK1p as promoter to express YAP1.(2) Recombinant plasmids for expression of the rest of 22 candidate regulation genes under screened TEFlp (PGKlp) and ADH7p promoter control were constructed and successfully transformed into S. cerevisiae BY4742 to obtained genetic engineering strains expressing individual regulation gene. Phenotypic differences of the genetic engineering strains in response to furfural were comparatively studied. Among those recombinant S. cerevisiae strains expressing each regulation gene using ADH7p as the promoter, the recombinant strains of YAP1, MSN2, PDR3, RDS1, AFT1, from different families, showed remarkably shorter lag-phase under furfural stress and displayed stronger tolerance to furfural than other recombinant strains. The MSA^-expression strain (AM01) showed more obvious advantages among them. Among those recombinant S. cerevisiae strains expressing each regulation gene using TEFlp as the promoter, we found the recombinant strains of YAP1, ATF1, PDR1, RPN4, RDS1, from different families, showed remarkably shorter lag-phase under furfural stress and displayed stronger tolerance to furfural than other recombinant strains. The RDS1-expression strain (TR01) showed remarkably faster recovery from furfural stress than other recombinant strains, displaying the strongest tolerance to furfural.(3) Transcriptional levels of MSN2 and its down-stream regulated representative genes in recombinant strain AM01, and RDS1 and its down-stream regulated representative genes in recombinant strain PR01 were quantitatively studied under furfural stress, respectively, using real-time fluorescent quantitative PCR method. The result showed that transcription of both MSN2 and RDSl were regulated by heterologous promoter and their transcription levels were significantly higher in recombinant strains than in the control strain BY4742. The transcription levels of MSN2 in strain AM01 and RDSl in strain PR01 increased by 12.7 and 3.8 folds, respectively, compared with the control strain BY4742. Transcription levels of the down-stream regulated representative genes were significantly enhanced, simultaneously, and some of them increased by more than 30 times in the recombinant strain.Overall, recombinant S. cerevisiae strains expressing 23 regulation genes possible related to furfural stress were constructed individually in yeast, their tolerant abilities to furfural were analyzed, and then the key regulation genes related to furfural were screened out in this study. Research achievements from this study can help us to better understand the mechanism of in situ detoxification of furfural in S. cerevisiae. The key regulation genes screened out in this study can contribute to the co-expression of multiple regulation genes from different functional family and the better understanding the tolerant mechanisms ofS. cerevisiae to furfural. This study will contribute to the industrial production of bioethanol from lignocellulose in both scientific and practical aspects. |
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