Studying On Cloning, Expression And Bioactivity Analysis Of Ai、rk And Rpe

In view of rising prices of crude oil due to increasing fuel demands,The need for alternative souces of bioenergy is expected to increase sharply in the coming years.Among potential alternative bioenergy resources lignocellulosics have been identived as the prime source of biofuels and other value-added peoducts. The major glucose and pentose sugars from hydrolysis of Cellulose ahd Hemicellulose of lignocelluloses has been extensively investigation. L-arabinose is one of the composition of Hemicellulose. because Saccharomyces cerevisiae strains are lack of L-arabinose isomerase,L-ribulokinase and L-ribulose-5-phosphate-4-epimerase, they cannot convert L-arabinose into ethanol directly. Our laboratory Study on Cloning, Expression in yeasts, and Bioactivity Analysis of ai, rk and rpe.The series Escherichia coli araBAD operon of ai、rk and rpe were cloned and amalgamation his6 by PCR technic. Then constructed inducible expressive vectors (pPIC9K-ai\pPIC9K-rk and pPIC9K-rpe)and constitutive expressive vectors (pGAP9K-ai and pGAP9K-rpe).Linearized expressive vectors with BglⅡwere transformed into Pichia pastoris G S115 with electroporation technique. The positive transformants were first determined b y PCR technique.G418 method was used to screen the high copy integrative transform ants. Transformants GS115(pPIC9K-ai),GS115(pPIC9K-rk), GS115(PPIC9K-rpe),GS115 (pGAP9K-ai), GS115(pGAP9K-rpe) had been obtained.All of those strain can resist G418 in 700μg/mL.The foreign genes had been expressed in shaking flask. GS115(pPIC9K-ai),GS115 (pPIC9K-rk),GS115(pPIC9K-rpe),can utilize Methanol as a carbon and GS115(pGAP9K-rpe) and S115(pGAP9K-ai) can utilize Glycerol as a carbon.SDS-PAGE analysis revealed that L-arabinose isomerase,L-ribulokinase and L-ribulose-5-phosphate-4-epimerase can Secretory expression Successfully in P. pastoris.The 1-arabinose isomeras activity test with sugar fermentation tube show that recombination protein With activity.Linearized pGAP9K-ai plasmid with BglⅡwere transformed into Saccharomyces cerevisiae JL1 by electroporation technique. G418 method was used to screen the hig h copy integrative transformants. Engineered strain JL1(pGAP9K-ai)had been expressed in shaking flask and Enzyme activity analysis with sugar fermentation tube show that recombination protein With activityWe describe here the cloning and expression of gene of L-arabinose isomerase,L-ribulokinase and L-ribulose-5-phosphate-4-epimerase from E.coli DH5a in Pichia Pasto ris and expression of gene of L-arabinose isomerase in Saccharomyces cerevisiae. This research had built a good foundation for construction engineered S. cerevisiae strain which utilize and metabolic 1-arabinose from Hemicellulose directly product bioethanol and production recombination L-arabinose isomerase,L-ribulokinase and L-ribulose-5-phosphate-4-epimerase which used production of ethanol from Hemicellulose.