Optimized expression and high-density fermentation of Aspergillus fischeri lipase in Pichia pastoris

Abstract:

[Background] Lipase is widely used in textile, food, medicine, leather and other industrial fields. The study on the heterologous expression of lipase in microorganisms further promotes the production and application of lipase products. [Objective] To perform the efficient heterologous expression of lipase from Aspergillus fischeri in Pichia pastoris. Screening suitable strategies for expression and fermentation of the engineering strain to increase yield and reduce production cost of lipase. [Methods] After codon optimization of the lipase coding gene of Aspergillus fischeri, plasmid pPIC9k was used to integrate the codon optimized gene (lip605) into the genome of Pichia pastoris GS115 to construct an engineering strain for high yield of the recombinant lipase. The lipase expression was further increased by optimizing the fermentation conditions, screening the optimal chaperone protein and performing high-density fermentation, successively. [Results] The optimal fermentation conditions for the engineering strain with high lipase production in shake flask were determined as 3.103% (v/v) methanol, 0.4 mg/L biotin, 11.5 g/L yeast powder, and 13.4 g/L YNB; the initial pH was 6.4, the filling volume was 50 mL/250 mL, the speed was 220 r/min, the temperature was 24 °C, and the culture time was 40 h. After optimization, the activity of extracellular lipase reached 72.34 U/mL, which was 5.8 times higher than that of initial fermentation. Furthermore, 12 chaperones were selected for co-expression with Lip605, among which the co-expression of chaperone Rpl10 (pPICZA-RPL10) had the best effect, which further increased the expression of Lip605 by 46.8%. On this basis, after 142 h high-density fermentation with batch feeding in a 10 L fermenter, the maximum extracellular lipase activity reached 680 U/mL, with a protein concentration of 15.89 g/L. [Conclusion] In this paper, compound strategies were used to effectively improve the fermentation yield of lipase Lip605 in Pichia pastoris, which laid a good foundation for its further industrial production.

Key words:Pichia pastoris, Lipase Lip605, Response surface, Co-expression, Chaperone proteins, High-density fermentation