Studying On Construction Of Starch-degrading Yeast Engineering Strain、Expression Enzymes And Production Ethanol

Because of the overage relying on the non-recycling energy, crisis of energy sources is slowing our steps in developing global economy and also the polluted environment created by using of petrol and coal has been influencing people’s health. Development recycling energy is a hot topic now. Biological ethanol transferred mostly from cellulose and starchiness. which is one of the important green and recycling energies and possesses of huge requirement.Transforming starchiness to ethanol now is in business scale and chaining industry model, then it’s a necessary process to append of isoamylase for hydrolyze branch chain amylum and glucoamylase or aamylase for cutting off theα-1,4 indicant bond to generate glucose before the starting ethanol production process by Saccharomyces cerevisiae.P. pastoris is a successful host for foreign expression. we studied using P. pastoris to express isoamylase andaamylase here. The isoamylase andaamylase genes were amplified and amalgamated his6 by PCR technic and the p. pastoris expression vectors of pGAP9K-a-amy and pGAP9K-iso were constructed by the DNA operation. Then the linearized expressive vectors were transformed into Pichia pastoris GS115 with electroporation technique and using 500-1,000μg/mL of G418 to select high gene copies recombinants: GS115(pGAP9K-a-iso) and GS115(pGAP9K-a-amy). Expression those recombinants in the sharking flash and spear the fermented broth on the dissolved amylum plate or branch chain amylum plats to choice the high expressed strains as engineering strains. High-density cell culture of GS115(pGAP9K-a-amy) was progressed in a 50 L bioreactor with a 20 L working volume for 54h, the biomass is A600=186 and secreted 125 mg/L aim product in the broth which behaves the activity of hydrolyzing amylum.If Saccharomyces cerevisiae owing those enzymes, such as isoamylase and glucoamylase, it would metabolize amylum directly to product ethanol and the cost on starchiness ethanol production could be significative decrease then. Here, the Isoamylase and glucoamylase genes were recombined into Saccharomyces cerevisiae genome by DNA operation. After series selection, such as anti-G418, analyzing enzymes activity on dissolved amylum or branch chain amylum plats and expressing aim product, a recombinant was selected as engineering strain, Saccharomyces cerevisiae (YIpGI) to using in the test of ethanol producion.While inoculating the Saccharomyces cerevisiae (YIpGI) into two medium, one containing of 10%cassava amylum and another of 10%dissolved amylum,4.17%and 4.58%alcohol is obtained and 57.5%and 61.2%are the using rate of amylum in a 4d fermentation. This result indicated that this engineering Saccharomyces cerevisiae behave function of expressing isoamylase and glucoamylase, hydrolyzing amylum and directly transforming amylum to ethanol.