Study On Recombinant Expression Of Tll In Non-spore A. Niger SH-2 Via The Strain Reformation

The lipase from Thermomyces laguginosus(Tll) is an important industial enzyme, with distinct characteristics of thermostable and lipase properties, which is widely used in the fields of oil chemicals, food, biosurfactants and so on. Traditionally this enzyme was obtained through endogenous secretory, however, the low yield, high impurity and difficult purification limited its industrial applications. As its great capablity in extracellular protein secretion, filamentous fungi are used as excellent protein expression host to express improtant industial enzymes. Aspergillus niger, generally recognized as safe by U.S. Food and Drug Administration(FDA), becomes an ideal host for recombination protein production, due to its effective post-translational modifications and high efficient secretion expression system.In this study, the non-spore Aspergillus niger SH-2 was used as the start strain, which is originated from Aspergillus niger CBS 513.88, with the characteristics of thick and short mycelium and less viscosity during fermentation. The total amount of glucoamylase secreted by wild non-spore A.niger SH-2 is as much as 20 g/L, which suppress the the secretion, downstream purification and formulation of recombinant protein. Therefore, we used the pyr G gene as the double selective marker to consecutive knockout the multicopy gene of glucoamylase in Aspergillus niger SH-2, in order to improve the expression level of target protein and provided a foundation to make the strain as a host strain for the expression of homologous or heterologous protein.In order to improve the expression level of Tll in Aspergillus niger SH-2, several strategies were applied, including codon optimization, the usage of strong promoter, the depression protein feedback inhibition and constrution of different tll expression vectors, and transformed into the stain with glucoamylase deletion. After transformation, the most highest enzyme activity was 150 U/m L and the amount of protein was 0.83 g/L after chromatography, which is as much as twice times than the Tll expressed in wild-type A. niger SH-2. These data could provide important clues for the further large-scale fermentation.