Bioethanol Production By Heterologous Expression Of Two Individual 1-FEH Genes From Helianthous Tuberosus In Saccharomyces Cerevisiae

The world is facing energy crisis and bioethanol is a promising renewable source of energy.Bioethanol could be produced by Saccharomyces cerevisiae.Since there are no enzymes in Saccharomyces cerevisiae can hydrolyze fructan from inulin,inulinases from microorganisms was heterologously expressed in the yeast Saccharomyces cerevisiae and improved ethanol production in fermentation was extensively reported.Fructan exohydrolases(FEHs)from fructan-rich plants hydrolyze fructofuranosyl units in inulin to produce fructose.And there are no such reports that FEHs from plants were heterologously expressed in yeaststo promot ethanol fermentation.Here,we examined whether the heterologous expression of FEHs could also improve ethanol production in yeast.Currently,the HtFEH ? and HtFEH ? gene from Helianthus tuberosus were individuallycloned and expressed,but there still exsit third FEH gene in Helianthus tuberosus according to the analysis of transcriptome of Helianthus tuberosus.The main results are below:1.We expressed two Jerusalem artichoke(Helianthus tuberosus)FEH genes(Ht1-FEH? and ?)in Pichia pastor is yeast X-33 to examine the biochemical properties of the encoded enzymes.Htl-FEH I was relatively stable at pH 4-8 and 4-35? and Ht1-FEH ?was relatively stable at pH 4-8 and 4-40?.Li+?Ca2+?K+?Na+?Mg2+ and Co2+ activated Ht1-FEH ?/? activities in different levels,and Fe2+ and Cu2+ inhibited Htl-FEH ?/?acitivities.The activities of Htl-FEHs were inhibited by SDS instead of EDTA or PMSF.The Km and Vm values of Ht1-FEH ? were 0.68 mg/ml and 0.00129 mg/min,while those of Ht1-FEH ? were 0.92 mg/ml and 0.0048 mg/min,respectively.2.The activities of heterologous expressed Htl-FEH ? and Htl-FEH ? respectively were 2.44±0.069(U/ml)and 2.98±0.009(U/ml)which were both higher than the activity of wild-type yeast,indicating Ht1-FEH ? and Ht1-FEH ? were both expressed in S.cerevisiae.The transgenic expression of Htl-FEH ? and Htl-FEH ? in S.cerevisiae 6525 at pH 6,30°Cresulted in 25%and 27%increases in ethanol production compared to the non-FEH-transformed control(CK),respectively.Furthermore,the conditions of pH5 and 35? and some metal irons inhibited the fermentation.3.ThirdFEH genenamed Htl-FEH ? from Helianthus tuberosus was identified by blasting FEHs from plants in transcriptome of Helianthus tuberosus.We cloned the gene and induced the enzyme and found no activity of FEH.The first conseverd region in amino acid sequneces ADPNG was different with Htl-FEH ? and Htl-FEH ?(NDPNG),then point mutation were made to the first amino acid N,but there was still no activity.We compared this FEH ? sequence in another transcriptome genome of Helianthustuberosusfrom Korea and found there are 200bp sequences absent which maybe the reason the induced enzyme without activity.