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Ethanol Fermentation from Jerusalem Artichoke Tubers by the Genetically-modified Saccharomyces Cerevisiae Strains Secreting Inulinase

Author: LiZuoZuo
Tutor: YuanWenJie
School: Dalian University of Technology
Course: Biochemical Engineering
Keywords: Inulinase gene Integration and Expression Saccharomyces cerevisiae Jerusalem artichoke Ethanol
CLC: TS261.11
Type: Master's thesis
Year: 2011
Downloads: 75
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Abstract


Inulinase gene integrated into the chromosome of Saccharomyces cerevisiae by homologous recombination, screened to produce inulinase genetically engineered strain of Saccharomyces cerevisiae and its inulinase production and the Jerusalem artichoke crude feedstock simultaneous saccharification and fermentation to produce ethanol were studied. Strong ethanol tolerance of Saccharomyces cerevisiae recipient strain was constructed a single copy Inulinase genetically engineered bacteria and Jerusalem artichoke powder raw material fermentation. First, the genomic DNA to Kluyveromyces marxianus, Kluyveromyces marxianus as a template, PCR amplification inulinase encoding gene Inu respectively inulinase own promoter and the yeast glyceraldehyde phosphate kinase (Phosphoglycerate kinase, pgk) promoter construct recombinant expression plasmid pHO / p-inu and pHO / pgk-inu. After Not I linearized using electroporation to transform S. cerevisiae industrial strains of Saccharomyces cerevisiae 6525 were obtained inulinase gene the positive strains HI6/1-HI6/10 and HPI6/1-HPI6/3. Experimental results show that high viability HI6 / 6 HPI6 / 3 inulinase were 86.0 U / mL and 23.8 U / mL, 4.6 times and 1.5 times the original strain. Further Jerusalem artichoke powder crude raw material for the substrate for ethanol fermentation, when the concentration of 200g / L, the recombinant strain H16 / 6 and HPl6 / 3 of the end of fermentation ethanol concentration was 55g / L and 52g / L, the sugar alcohol is converted rate of 0.495 and 0.453, respectively, 96.9% and 88.6% of the theoretical value. When the results of the the Jerusalem artichoke batch batch fermentation experiments showed that when the crude Jerusalem artichoke powder concentration of 200g / L, after 24 h of fermentation and fill to 280g / L, fermentation is basically the endpoint is reached in 48 h, H16 / 6 and HPl6, / ethanol were 74.5g / L and 77.5g / L, were higher than the original strain the inulinase enzyme activity has been in the range of 10 ~ 25 U / mL. The Jerusalem artichoke fermenter Experimental results show that, in a 3-L fermenter, when crude Jerusalem artichoke powder total concentration of 280g / L, the two strains were achieved in about 60 h of the end of fermentation, the maximum ethanol concentration reached 80 g / L residual sugar down to about 25g / L, the concentration of reducing sugar in the fermentation broth is maintained at 5g / L, inulinase activity remained at 10 to 20 U / mL. Integration sites to rDNA construct multicopy integration inulinase genetically engineered bacteria and Jerusalem artichoke powder raw material fermentation. Was amplified by the PCR method to obtain a the pgk promoter gene without its own promoter the inulinase gene inu and the expression of genes rDNA for mediated recombination in K. marxianus, and which in turn is connected to a carrier pFA6a constructed a multi-copy integration of the expression vector the pF A6a-RDn A-pgk-Inu its linearized electroporated into the competent cells of the S.cerevisiae 6525, and screened to obtain a better performance of the two bacteria 6525/rDNA-13 and 6525/rDNA-14. The experimental results show that the maximum activity of 6525/rDNA-14 strains reached 23U/mL, 6525/rDNA-13 strains the highest enzyme activity more than 20 U / mL, were higher than the original strain. Batch batch fermentation experiment results show that the fermentation process is basically the endpoint is reached in 48 h, wherein the transformants 6525/rDNA-14 maximum ethanol concentration of 72.5 g / L, 6525/rDNA-13 the maximum ethanol concentration was 69.5 g / L, ethanol production are higher than the original strain 67g / L.

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