Metabolic Engineering Research Of Direct Ethanol Production From Cassava By Recombinant Saccharomyces Cerevisiae

In order to solve problems such as complex processes and large energy consumption in theindustrial alcohol fermentation from cassava, recombinant Sacharomyces cerevisiae strainsco-expressing α-amylase and glucoamylase genes were constructed. Taking advantage of thisengineered amylolytic yeast, the direct production process established showed good effect.Firstly, genes encoding α-amylase and glucoamylase were obtained by RT-PCR respectivelyfrom the total RNA of Aspergillus oryzae CICC40344and Aspergillus niger CICC40179. Theamplified genes were cloned into expression vector pScIKP in sequence to generate theco-expression plasmid pSc-ga-amy. It was then transformed into S.cerevisiae by pulse celltransfection. Amylolytic transformants were screened with clear halos by iodine staining onstarch plates, and showed α-amylase and glucoamylase activities of0.865U/ml and34.79U/ml.The mixed enzymes tests were carried out, and the cocktails with α-amylase andglucoamylase activity ratio of1:5showed the best starch hydrolysis effects. In order to increasethe starch hydrolysis capacity of the recombinant strain, using isocaudamer enzyme of NheI andXbaI ligation, the glucoamylase gene expression cassette was repeatedly inserted to pScIKPvector to make the α-amylase and glucoamylase expression cassettes reach a ratio of1:5. But theresults showed the recombinant yeasts with one α-amylase gene and five glucoamylase geneswere not of high starch hydrolyzing ability. It may be due to the mutual interference of eachpromoter, randomness of integration, restrain of rDNA function, position effect, translationpolarity and other factors.The recombinant strain AS2.489/pScAG with the highest starch hydrolyzing ability wasselected to study its growth characteristics, enzyme properties and genetic stability, and directstarch fermentation conditions were optimized by orthogonal test. Direct ethanol fermentationwas also conducted in a5L jar fermenter with200g/l of cassava using this engineered yeast.The ethanol concentration could reach8.68%(v/v), approximate to80.9%of theoretical yield.This result indicated that the strain AS2.489/pScAG could sufficiently produce ethanol directlyfrom cassava without addition of any commercial enzymes. It would significantly simplify theproduction process and reduce energy consumption.