Evaluation of Copper-inducible Fungal Laccase Promoter in Foreign Gene Expression in Pichia pastoris

Abstract

The promoter region of copper-inducible laccase gene, LCC1, from Pycnoporus coccineus was explored in the heterologous expression of foreign protein in Pichia pastoris. The promoter region (P-PPLCC1) was isolated and used to replace the methanol-inducible AOXI promoter (P-AOX1) of pPICHOLI-2, an episomal expression vector for P. pastoris, to generate a new copper-inducible expression vector. The promoter activity of P-PPLCC1 was compared with those of P-AOX1 and P-CUP1, a copper-inducible promoter of a commercial vector pPICHOLI-C, using a laccase gene as a reporter gene in P. pastoris GS115. Reporter laccase activity of the culture broth reached 182 and 43 units/L for P-PPLCC1 and P-CUP1, respectively, after induction with 0.2 mM CuSO4 at OD600 = 1 and culture for 120 h at 15A degrees C in complex medium containing 1% glucose. For P-AOX1 activity, yeast cells harboring P-AOX1-laccase plasmid were cultured for 120 h at 15A degrees C in complex medium with intermittent feeding with 1% methanol every 12 h to avoid methanol toxicity. Laccase activity of culture broth was 124 units/L. Conclusively, P-PPLCC1 is a new copper-inducible promoter that shows superior performance in terms of efficiency of laccase production compared to commercial vectors. P-PPLCC1 is additionally superior to P-AOX1 since it does not require laborious feeding with a carbon source.