The Expression Of Kluyveromyces Marxianus INU1 Gene In Aureobasidium Melanogenum P10 Strain And Lipids Production

In recent years, biodiesel production has received great interests because the biodiesel as one of the biofuels has many advantages over any other biofuels. Biodiesel is renewable, non-toxic, and has much shorter formation time compared with fossil fuels. So it is regarded as an alternative to fossil fuels. In the previous studies we found that A.melanogenum P10 strain isolated from the mangrove ecosystems can accumulate 66.3 g oil per 100 g of cell dry weight, its cell mass was 1.3 g per 100 ml. The biodiesel produced from the extracted lipid could be burnt well (Wang et al.,2014). After the analysis of the genomic DNA of A. melanogenum,it was found that there were no genes encoding inulinase in this yeast. In addition, inulinase activity produced by A.melanogenum P10 strain was very low. Therefore, it is very important to overexpress the inulinase gene in A.melanogenum P10 strain in order to make the recombinant yeast strain efficently produce lipids from inulin directly.The expression vector pAPX13 for over-expression of a homologous or heterologous gene in A.melanogenium was constructed. The inulinase gene INU1 from Kluyveromyces marxianus KM was ligated into the expression vector pAPX13 and expressed in A.melanogenum strain P10. The transformant AP141 obtained in this study produced 28.5 U/mL of inulinase activity, while its wild type strain P10 yielded only 8.6 U/mL of inulinase activity. The optimal temperature and pH of the inulinase is 50℃ and 4.5, respectively. The inulinase was stable in the temperature range of 0℃-50℃ and in the pH range of 3.0-7.0.At flask level, cell mass of the transformant API41 strain was 1.29 g per 100 ml, while the cell mass of its wild type strain P10 was 1.0 g per 100 ml. And the single cell oil (SCO) content in strain API41 was 59.67 g per 100 g of cell dry weight, while the SCO content of its wild type strain P10 was 53.71 g per 100 g of cell dry weight. At the end of the cultivation, the residual sugars in the cultures containing strain API41 and strain P10 were 0.21g per 100 ml and 1.34 g per 100 ml, respectively. During 10-1 fermentation, the cell mass and SCO content of strain API41 were 1.43 g per 100 ml and 66.20 g per 100 g of cell dry weight, respectively.The compositions of the fatty acids in the lipids produced by strain AP141 and strain P10 were detected by gas chromatography. It was found that the wild type strain P10 and the transformant AP141 contained similar compositions of fatty acids. Nearly 95% of the fatty acids from both of the two strains was C16 0, C18 1 and C18 2, suggesting that the fatty acids could be used to produce biodiesel. The SCO obtained from strain AP141 was transformed into biodiesel. It was found that 76.3% of the SCO could be transformed into biodiesel in 25 minutes. The biodiesel obtained burnt well.