Improving heterologous expression of laccase by Pichia pastoris via vanillin-induced stress response and its application for removing inhibitors of lignocellulose hydrolysate†

Abstract

A novel strategy to increase laccase heterogeneous expression by Pichia pastoris was developed via exploring vanillin-sensitive promoters by culture of the yeast under vanillin stress followed by transcriptome analysis. Two endogenous promoters with significant response to vanillin were screened out with green fluorescent protein as a reporter protein. Subsequently, these promoters were combined with the laccase gene lacc 6 from Pleurotus ostreatus in single-promoter and double-promoter modes for enhancing laccase production. The laccase activity of the supernatant broth reached 285.7 U L⁻¹, being 18–60% higher than that of the control group. The enhancement of the laccase production was mainly ascribed to the increased transcription level of gene lacc 6 as revealed by transcriptome analysis. The recombinant yeast also could efficiently remove vanillin in the fermentation medium. Therefore, the strategy developed in this work could not only improve laccase production by Pichia pastoris, but also eliminate vanillin stress by the recombinant yeast. To improve the efficiency of laccase utilization and avoid the recovery and separation of laccase from the treated hydrolysate, a novel system was further developed based on the principle of a liquid flow fuel cell (LFFC), in which laccase was employed as a cathodic catalyst for the oxygen reduction reaction (ORR) with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a mediator and Ag₂O as a anode catalyst. The LFFC system could well eliminate aldehyde stress factors thus improving the fermentability of dilute acid hydrolysate of biomass. This work thus can provide new ideas for boosting the efficiency of biomass bioconversion to produce biofuels and chemicals.