The Role Of Nisin In Ethanol Production With Yeast And Study On Nisin Genes Heterogeneous Expression

In recent years bioethanol has attracted worldwide interest as a non-conventionalbioenergy source. Fuel ethanol was produced by low pH to inhibit bacterial contaminationrather than high temperature sterilization, whist this can be influenced by the quality of rawmaterial and local climate. In the fuel ethanol industry, control of bacterial contamination wasachieved by using antibiotics. However, it was prohibited because disproportionate use ofantibiotics for industrial processes would promote antibiotic resistance in contaminatingbacterial populations and cause environmental problem. Eco-friendly nisin was chosen as analternative to investigate its effect on ethanol production, subsequently two nisin-productingstrains were isolated and one of them was explored for nisin genes expression in E. coliBL21（DE3） and Saccharamyce cerevisiae w303-1A.First, it was indicated that2IU mL^-1nisin could partly inhibit the growth andmetabolism of L. brevis but not sangnificant to L. plantarum by the investigation of effects ofnisin on L. plantarum and L. brevis contamination of yeast during ethanol fermentation. Thelowest concentration of nisin (5IU mL^-1) effectively controlled contamination of YP/glucosemedia with106CFU mL^-1lactobacilli; Ethanol productions were increased from65.6g L^-1and68.8g L^-1to72.3g g L^-1and72.7g L^-1respectively, while the productions of lactic acidwere reduced from4.9g L^-1and5.3g L^-1to2.1g L^-1and2.3g L^-1which were very close tothe control1.9g L^-1.Second, a total of34lactic acid bacteria （LAB） resistant to nisin were isolated by M17medium and two strains L2/L3with strong antibacterial activity to antibiotic sensitiveindicator （Micrococcus luteus） were tested by Oxford plate assay system. Sequencing16SrDNA genes showed that the two stains were Lactococcus lactis, preliminarily designed asLactococcus lactis L2and Lactococcus lactis L3. The antibacteriocins produced by the twostrains were heat-stable at low pH, insensitive to pepsin and trypsin, and inhibited somegram-positive strains as well as some microbial contamination of ethanol production. Thenisin structure genes of L2and L3were identical to nisin Z translation gene.NisZ, nisB, nisC and nisP fragments of Tn5276were transformed in E. coli BL21（DE3）and Saccharamyce cerevisiae w303-1A, respectively. Four E. coli BL21（DE3） recombinantsDE3-nisZ, DE3-nisB, DE3-nisC, DE3-nisP were screened by resistant plate and gene PCRamplification; IPTG was used to induce expression of nisin genes under the control of lacpromter. Cell lysate supernatant of the four recombinants was mixed together by1:1:1:1andkept warm at37oC for1h. The antibacterial activity of the mixture was tested by Oxford plateassay system on fuel ethanol lactobacilli contamination （L. plantarum and L. brevis）. Theinhibition zones of L. plantarum and L. brevis were1.10±0.05cm and1.27±0.04cm. Celllysate supernatant of Escherichia coli expressing NisB, NisC, and NisP proteins were able tomodify and cleave the nisin precursor peptide. At last four Saccharamyce cerevisiaerecombinants SC-nisZ1, SC-nisB1, SC-nisC1and SC-nisP1were constructed and investigatedfor heterologous expression.