关键词: 毕赤酵母GS115, 鼠李糖代谢, LRA3基因, 鼠李糖诱导型启动子PLRA3, 顺式作用元件

Abstract: Pichia pastoris has become a commonly used eukaryotic expression host due to its multiple advantages including easily genetic manipulation, efficient secretion of recombinant proteins, high-density fermentation and post-translational modification. And P. pastoris expression system based on the methanol-inducible promoter PAOX1 is widely used, however, this system is disadvantageous in production of food enzymes and biopharmaceutical proteins due to flammable and toxic methanol. Therefore, it is of great significance to develop non-toxic inducible expression systems. In previous study, the gene cluster, LRA1~LRA4, was predicted to associate with rhamnose metabolism in P. pastoris, and a rhamnose-inducible promoter PLRA3 was disclosed to drive efficient expression of recombinant proteins in P. pastoris. However, it is necessary to further improve the inducible strength and basal transcriptional profiles of PLRA3. Based on this situation, LRA3 was verified to involve in rhamnose metabolism in P. pastoris via gene disruption and gene complementation, simultaneously the transcription start site of PLRA3 was determined via circularized RNA reverse transcription PCR (CRRT-PCR)  and its cis-acting elements were predicted, which provided some clues for the rational design of PLRA3.

Key words: Pichia pastoris GS115, rhamnose metabolism, LRA3 gene, rhamnose-inducible promoter PLRA3, cis-acting elements