Yuan Huiling, Zhou Ying, Lin Yuping, Tu Ran, Guo Yufeng, Zhang Yuanyuan, Wang Qinhong
CAS Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
National Center of Technology Innovation for Synthetic Biology, Tianjin, 300308, China.
Biotechnol Biofuels Bioprod. 2022 May 14;15(1):50. doi: 10.1186/s13068-022-02150-w.
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.
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 Rsc1 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.
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.
巴斯德毕赤酵母是一种广泛用于异源生产工业蛋白（如纤维素酶）的宿主生物。尽管在提高毕赤酵母中蛋白质表达方面已取得了很大进展，但由于未知的基因组影响因素，毕赤酵母表达系统的潜力尚未得到充分探索。最近，全细胞定向进化采用了一轮又一轮的全基因组多样性生成和高通量筛选（HTS），被认为是在基因组水平上进行菌株改良的一种有前景的策略。
在本研究中，开发了采用常压室温等离子体（ARTP）诱变和基于液滴的微流控高通量筛选的毕赤酵母全细胞定向进化方法，以提高异源纤维素酶的产量。基于纤维素酶催化释放荧光反应建立的基于液滴的微流控平台被证明适用于以甲醇为碳源生长的毕赤酵母。验证实验表明，在每秒300个液滴的分选速率下，阳性分选效率为94.4%。经过五轮迭代的ARTP诱变和微流控筛选，获得了最佳突变株，其纤维素酶活性为11,110±523 U/mL，与出发菌株相比增加了约两倍。全基因组重测序分析进一步揭示了编码区的三个累积基因组改变。通过点突变重建和基因缺失验证了点突变和突变基因对纤维素酶产生的影响。有趣的是，在从每一轮中选出的所有表现最佳的生产者中都观察到了点突变Rsc1，基因缺失分析证实，作为RSC染色质重塑复合体的一个组成部分，Rsc1可能在纤维素酶产生中起重要作用。
我们建立了基于液滴的微流控高通量筛选系统，从而促进了毕赤酵母全细胞定向进化以提高纤维素酶产量，同时通过全基因组重测序和基因验证确定了基因组改变。我们的方法和发现将为加速具有高工业价值的宿主菌株和酶的全细胞定向进化提供指导。
