Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris

Abstract

Background Pichia pastoris is a widely used host organism for heterologous production of industrial proteins, such as cellulases. Although great progress has been achieved in improving protein expression in P. pastoris, the potential of the P. pastoris expression system has not been fully explored due to unknown genomic impact factors. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high-throughput screening (HTS), has been considered to be a promising strategy in strain improvement at the genome level. Results In this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic HTS, was developed to improve heterogenous cellulase production. The droplet-based microfluidic 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 droplets per second. After five rounds of iterative ARTP mutagenesis and microfluidic screening, the best mutant strain was obtained and exhibited the cellulase activity of 11,110 ± 523 U/mL, an approximately twofold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region. The effects of point mutations and mutant genes on cellulase production were verified using reconstruction of point mutations and gene deletions. Intriguingly, the point mutation Rsc1G22V 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. Conclusions We 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 genetic validation. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

“…To improve heterologous cellulase production in P. pastoris, a novel approach was developed based on the use of atmospheric and room-temperature plasma (ARTP) mutagenesis and a droplet-based microfluidic HTS system in whole-cell directed evolution. 82 The results indicated the best mutant strain R5-2 with a twofold increase in cellulase activity compared to the wild strain. Whole genome sequence analysis showed that a point mutation (Rsc1 V22G) was found in all topperforming variants.…”

“…Whole genome sequence analysis showed that a point mutation (Rsc1 V22G) was found in all topperforming variants. 82 From Chaetomella raphigera, the D2-BGL (⊎-glucosidase) gene was isolated via directed evolution and expressed in P. pastoris. Modified D2-BGL carried three novel mutations provided to have greater tolerance to substrate inhibition, higher V max value, enhanced specific activity, and higher protein titer as compared to wild-type enzyme.…”

Deep‐qGFP: A Generalist Deep Learning Assisted Pipeline for Accurate Quantification of Green Fluorescent Protein Labeled Biological Samples in Microreactors

“…[29] The agarose digital PCR original images in Figure 6c were provided by collaborators Dr. Xuefei Leng and Prof. Chaoyong Yang (College of Chemistry and Chemical Engineering, Xiamen University). [30] The original images in Figure 6d,e were captured from previously reported journal articles [31,32] respectively.…”

Current advances of Pichia pastoris as cell factories for production of recombinant proteins

“…Compared with traditional microplate screening, the reagent cost was reduced by millions of times ( Lu et al, 2019 ). With atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic high-throughput screening, it is possible to explore the potential of protein production by employing iterative rounds of genome-wide diversity generation ( Kenzom et al, 2015 ; Ma et al, 2019 ; Yuan et al, 2022 ). For example, compared to the beginning strain, the best mutant strain showed a twofold increase in cellulase activity in one research ( Yuan et al, 2022 ).…”