Expression Of Different Inulinase Genes In Saccharomyces Sp. W0 For Ethanol Production

Inulin is composed of linear chains of β-(2-1) linked fructose residues terminated by a glucose residue. Inulin is a storage carbohydrate which exists in many plants, such as chicory, jerusalem artichoke and dahlia. Jerusalem artichoke with the characterwastic of drought-tolerant and wind resistance has been planted extensively in China. So the inulin can be used for producting the bioethanol, single cell oil, citric acid and so on. The inulinases can be divided into exo-inulinase and endo-inulinase. The exo-inulinase catalyzes the removal of the terminal fructose residues from the non-reducing end of the molecule. So the inulinase genes can be expressed in the Saccharomyces cerevisiae to produce bioethanol from hydrolysate of inulin.The INU1 gene encoding exo-inulinase from Candida membranifaciens subsp. flavinogenie W14-3 had an open reading frame (ORF) of 1,536 bp long. The coding region of the gene was not interrupted by any intron. It encoded 512 amino acid residues of a protein with a putative signal peptide of 23 amino acids. The exo-inulinase gene W14-3-INU1 with the signal sequence was subcloned into pMIDSC11 expression vector and expressed in Saccharomyces sp. WO. The recombinant yeast strain W14 could produce 20.3 U/ml of inulinase activity. The W14-3-INU1 gene relative express quantity of WO and W14 were detected by RT-PCR. It was found that W14-3-INU1 gene was expressed in transformant W14.In order to know if the transformant W14 constructed above could produce high concentration of ethanol from inulin, the 150-ml fermentation was performed. Fermentation parameters were optimized in the fermention. It can be observed that within 120h of the fermentation, ethanol concentration in the fermented medium was 12.8%(v/v), and 98.2% reducing sugar and 88.6% total sugar were used in the fermented medium. However, the WO produced only 9.8%(v/v) ethanol under the same condition and 96.2% reducing sugar and 83.5% total sugar were used in the fermented medium. The result means that the added inulin was efficiently converted into ethanol, cell mass and CO2 by the transformant W14 after completion of the fermentation. During the 2.0-L fermentation,13.4%(v/v,20 ℃) ethanol concentrarion was produced within 120h while 92.0% reducing sugar and 99.1% total sugar were utilized by the transformant W14 in the fermented medium.In order to get the transformant which has higher inulinase activity than the wild type strain, both the endoinulinse gene (EinuA) from A. niger strain F4 and the exoinulinase gene (W14-3-INU1) were expressed in the Saccharomyces sp. WO. First, the expression vector with the eno-inulinase gene EinuA without the signal sequence was contructed by using the vector pMIDSC31. Then, both the linear DNA fragments carrying the W14-3-INU1 gene from the expression vector pMIRSC11-W14-3-INU1 and the linear DNA fragments carrying the EinuA gene from pMIDSC31-EinuA were transformed into Saccharomyces sp. WO. The transformant WE85 which could produce 33.9 ± 0.2 U/mL of inulinase activity.was got. It suggested that the transformant WE85 which had both endo-inulinase gene and exo-inulinase gene could produce endo-inulinase and exo-inulinase together and had higher inulinase activity than transformant W14 which have only the exo-inulinase gene W14-3-INU1. In order to know if that both the two genes W14-3-INU1 and EinuA were expressed in the transformant WE85, the relative expression levels of these two genes were detected by RT-PCR. It was found that W14-3-INU1 gene and the EinuA gene in the transformant WE85 were expressed respectively. In order to know if the transformant WE85 constructed above could produce high concentration of ethanol from inulin, the 150-ml fermentation was performed. It can be observed that within 120h of the fermentation, ethanol concentration in the fermented medium was 13.8%(v/v), and 98.8% reducing sugar and 92.4% total sugar were used in the fermented medium.