| There are a few sugars that can not be effectively used by Saccharomyces cerevisiae in thedistillate of ethanol fermentation, mos of which are melibiose and cellobiose. During ethanolfermentation from cellulose, 'feedback inhibition action' of cellulase will be engendered bycellobiose accumulation. Cellobiose is the dominant product of the cellulase. So cellulasewith β-glucosidase of high activity is necessarily for effectively hydrolyzed cellulose.α-galactosidase and β-glucosidase were directly added or immobilized in the process of VHGethanol fermentation and cellulose hydrolyzation in order to use up melibiose (cellobiose) andrelease the feedback inhibition action. Therefore manufacturing cost would be influenced bythe price of supplied α-galactosidase and β-glucosidase.In this paper, the industrial strain S. cerevisiae Y was used as the precursor, theα-galactosidase gene mel and β-glucosidase gene bgl were integrated into GPD1 generespectively which was the key enzyme gene in the glycerol pathway of yeast. And therecombinants with improved properties of substrate utilization and ethanol productivity wereconstructed.Different types of chromatography columns and their separation conditions were comparedand carbohydrate analysis column was used in HPLC to analyze distillate ingredient of VHGethanol fermentation from starch materials. The HPLC method for qualitative and quantitivedetermination of cellubiose, melibiose and glycerol in the distillate of VHG ethanolfermentation were developed for the first time. Distillates of VHG ethanol fermentation fromdifferent starch materials were analyzed with this method, and it was shown that the contentof melibiose and cellobiose was respectively more than 500 mg/L, the content of glycerol was4,000-10,000 mg/L.Primers were designed according to α-galactosidase gene sequence of S. pombe, and mel genewas cloned by polymerase chain reaction with genomic DNA of S. pombe as the template. Arecombinant plasmid pYX-MEL was constructed by inserting mel gene into the downstreamof TPI promoter of pYX212. mel gene was expressed in S. cerevisiae W303-1A withelectroporation. Transformants were screened by YNBG medium and α-galactosidase activityof that was 0.81u/mL. It was proved by further experiment that the recombinants can growwell on the medium with melibiose as the sole carbon source.Primers were designed according to β-glucosidase gene sequence of Trichoderma reesei. Thetotal RNA of T. reesei was extracted with guanidine thiocyanate-phenyl-chloroform extractionmethod, by which the poly A~+ mRNA was purified. The gene encoding β-glucosidase wasamplified by RT-PCR technique using purified mRNA as the template. The recombinedplasmid pYX-BGL was constructed and expressed in S. cerevisiae W303-1A. Transformantswere screened by YNBG medium and β-glucosidase activity of that was 0.47u/mL. It wasproved by further experiment that the recombinants can be grow well on the medium withcellobiose as the sole carbon source.Based on the recombinant plasmids pYX-MEL and pYX-BGL as the precursor, kmr genesegment was respectively inserted into the downstream of mel gene and bgl gene as thedominant selective marker and recombinant plasmids pYX-MEL::?Km,pYX-BGL::?Kmwere successfully constructed. Then gpd1'::(PTPI-mel-?Km) and gpd1'::(PTPI-bgl-?Km) wereamplified by PCR, and gpd1 segment was introduced into the both end of PTPI-mel-?Kmcluster and PTPI-bgl-?Km cluster. The PCR products were transformed into the industrial S.cerevisiae Y by electroporation. Then the transformants were screened by G418 medium.Yeast shape, multiplication capacity and other items were observed and examined to evaluatethe characteristics of recombinant S. cerevisiae MG1 and S. cerevisiae CG1. It was shown thatthe characteristics of yeasts were not obviously changed after introducing the extrinsic geneexcept for their shape changs.In the 500 mL conical flask and 30 L fermentation tank, VHG ethanol fermentation from cornpowder and wheat starch by engineered S. cerevisiae MG1 and VHG ethanol fermentationfrom corn powder by engineered S. cerevisiae CG1 were reviewed. It was shown that thecontents of melibiose, cellobiose and glycerol were decreased respectively, while the ethanolstrength was enhanced.The engineered strain S. cerevisiae CG1 was used to incorporate cellulase for simultaneoussaccharification and fermentation of microcrystalline cellulose to ethanol. It was shown thatthe cellobiose accumulation had decreased and the alcohol productivity increased. And it wasconcluded that the 'feedback inhibition action' engendered by cellobiose accumulation waspartially released.Preliminary analysis on the DDGS ingredients of VHG ethanol fermentation distillate byengineered strains was carried out. It was shown that the quality of DDGS from corn powderas ethanol fermentation substrates was close to or better than that of our country (JilinProvince) and overseas country (USA) by comparing their protein and amino acidsingredients, but the protein ingredient of DDGS from wheat starch was a little lower.Preliminary study on the feasibility and security of DDGS from VHG ethanol fermentationdistillate by engineered strains used as feed was also discussed. It was shown that the DDGSas feed was feasible and comparatively safe. But in view of the long-term use, the Kmr geneshould be knocked out before industrial utilization of recombinant yeasts in order to guaranteeits security.
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