Abstract
BackgroundPichia pastoris is a widely used host organism for heterologous production of industrial enzymes and other proteins, such as cellulase applied in various industries. By developing rational and irrational engineering strategies, great progress has been achieved in improving protein expression in P. pastoris. However, due to unknown genomic impact factors, the potential of the P. pastoris expression system has not been fully explored. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high throughput screening (HTS), is considered as a promising strategy in strain improvement at the genome level.ResultsIn this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic high-throughput screening (HTS), was developed to improve heterogenous cellulase production. The droplet-based microfluidic HTS platform based on a cellulase-catalyzed reaction of releasing fluorescence was established to be suitable for methanol-grown P. pastoris. The validation experiment showed a positive sorting efficiency of 94.4% at a sorting rate of 300 drops per second. Through five rounds of iterative ARTP mutagenesis and microfluidic screening, cellulase production capacities of mutants in each round were gradually enhanced. Among them, the best mutant strain R5-2 exhibited the cellulase activity of 11,110 ± 523 U/L, an about 2-fold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region during iterative ARTP mutagenesis and screening. Intriguingly, the point mutation Rsc1V22G was observed in all the top-performing producers selected from each round, and gene deletion analysis confirmed that Rsc1, a component of the RSC chromatin remodeling complex, might play an important role in cellulase production. ConclusionsWe established a droplet-based microfluidic HTS system, thereby facilitating whole-cell directed evolution of P. pastoris for enhancing cellulase production, and meanwhile identified genomic alterations by whole-genome resequencing and analyzed. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.
High-throughput genotyping by whole-genome resequencing
