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KMID : 1094720160210010053
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Biotechnology and Bioprocess Engineering
2016 Volume.21 No. 1 p.53 ~ p.59
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Evaluation of copper-inducible fungal laccase promoter in foreign gene expression in Pichia pastoris
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Kim Sin-Il
Ha Byung-Suk
Kim Min-Seek
Park Min-Sa
Ro Hyeon-Su
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Abstract
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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 (PPPLCC1) was isolated and used to replace the methanol-inducible AOXI promoter (PAOX1) of pPICHOLI-2, an episomal expression vector for P. pastoris, to generate a new copper-inducible expression vector. The promoter activity of PPPLCC1 was compared with those of PAOX1 and PCUP1, 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 PPPLCC1 and PCUP1, respectively, after induction with 0.2 mM CuSO4 at OD600 = 1 and culture for 120 h at 15°C in complex medium containing 1% glucose. For PAOX1 activity, yeast cells harboring PAOX1-laccase plasmid were cultured for 120 h at 15°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, PPPLCC1 is a new copper-inducible promoter that shows superior performance in terms of efficiency of laccase production compared to commercial vectors. PPPLCC1 is additionally superior to PAOX1 since it does not require laborious feeding with a carbon source.
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KEYWORD
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copper-inducible, laccase, Pichia, promoter
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