Construction Of Multicopy Integration Vector In Yarrowia Lipolytica Yeast By Using The Method Of Biobrick And Studying Gene Dosage Effect

In the field of biological science of the21st century, Synthetic biology is a rapidly developing new interdisciplinary. It begins from the most basic element, first, set up parts step by step, then organize these parts to create different function natural biological systems. Recombinant DNA obtained by traditional molecular cloning techniques depends on PCR, restriction enzyme digestion and ligation reaction. This method can not satisfy the needs of synthetic biology research. When needing a part, it must be designed with a unique cloning, and the intermediate product generally can not be applied to other components, there is no doubt that this is a waste of resources.With the rapid development of genetic engineering techniques, how to improve the expression level and stability of the target protein in the host strain have already caused the people’s attention at home and broad, especially small molecule bioactive peptides. The method of constructing multiple copy number of a series of target gene reveals a broad prospect for the efficient expression of a small peptide. To some extent, this strategy can increase the amount of target protein expression level and lay a certain technical foundation for the industrial production of heterologous protein expression.On the basis of the biobrick method in this study, we have respectively introduced into the Hindlll and N heⅠ; the XbaⅠand BamHⅠ restriction site sequences in Yarrowia lipolytica yeast expression vector pINA1296promoter upstream and terminator downstream by PCR. Finally, recombinant vector was successfully constructed which was named pINA12961. In order to facilitate the screening of recombinants. we selected two kinds of mannanase genes. respectively from Aspergillus niger and Bacillus subtilis, and then the genes were cloned to the vector pINA1296I to build tandem multiple copies of the target gene in vitro through using biobrick approach. A series of different copy numbers of vectors which had been sucessfully constructed were transformed into the Yarrowia lipolytica yeast polg strains respectively, and then study the gene dosage effect relationship by measuring the target protein concentration and enzyme activity.The results of measuring enzyme activity and the protein concentration of shake flask expression supernatant show that along with the copy number increased, man-A protein amount and enzyme activity increased gradually. Protein and enzyme activity of the five copies reached the maximum, while the six copies began to decline. The optimal pH and temperature of man-A are about3.5and65℃. When dealt with10minutes at70℃. the residual activity only remain24%. When incubated at pH2.0-8.0. room temperature for1h, it showed high activity in the range of pH from3.0to6.5and retained95%of its activity. Man-B protein concentration and enzyme activity were positively related to copy number, the six copies reached the maximum. Therefore, to a certain extent, constructing tandem multiple copies vectors in vitro by using biobrick can increase the amount of target protein expression.