毕赤酵母基因编辑技术及其在生物制造中的应用进展

Advances in genome editing technologies in Komagataella phaffii and their applications in biomanufacturing

摘要:

毕赤酵母( Komagataella phaffii)因其内源蛋白分泌背景低、外源蛋白分泌能力强、在复杂发酵条件下具备稳健的生长代谢特征，已成为重组蛋白和代谢产物生产的核心宿主之一。近年来，基因编辑技术与合成生物学工具的迅猛发展，显著提升了毕赤酵母在基因功能解析、代谢途径重构及动态调控方面的精确度与效率，持续强化了其作为细胞工厂的性能。在此背景下，毕赤酵母不仅在重组蛋白、工业酶及疫苗抗原的生产中占据重要地位，更在天然产物、生物医药分子及新型生物材料的合成中展现出巨大潜力。本文系统总结了毕赤酵母基因编辑技术的发展历程，涵盖了从传统重组方法到新型精准编辑工具的演进，重点梳理了CRISPR/Cas等技术在该体系中的应用进展、优化策略及工程化实践。同时，结合最新研究趋势，探讨了在提升编辑效率、实现复杂代谢通路快速组装以及推动工业化应用过程中面临的挑战与机遇，为毕赤酵母的理性工程化改造及其在合成生物学与生物制造领域的深入应用提供了理论参考。

Abstract:

Komagataella phaffii is widely recognized as a premier host for the production of recombinant proteins and value-added metabolites, owing to its low background secretion of endogenous proteins, strong capacity for heterologous protein secretion, and robust growth and metabolic performance under industrially relevant fermentation conditions. In recent years, rapid progress in genome editing technologies and synthetic biology toolkits has markedly improved the precision and efficiency of gene function interrogation, metabolic pathway reconstruction, and dynamic regulation in K. phaffii, thereby continuously strengthening its performance as a microbial cell factory. Consequently, beyond its established roles in producing recombinant proteins, industrial enzymes, and vaccine antigens, K. phaffii has also demonstrated substantial potential for the biosynthesis of natural products, biopharmaceutical molecules, and emerging biomaterials. This review systematically summarizes the evolution of genome engineering technologies in K. phaffii, spanning the transition from conventional recombination-based methods to next-generation precision editing tools. We highlight recent advances, optimization strategies, and engineering practices of CRISPR/Cas and related systems in this host. Moreover, in light of emerging research trends, we discuss key challenges and opportunities associated with improving editing efficiency, enabling rapid assembly of complex metabolic pathways, and accelerating industrial translation, thereby providing a reference for the rational engineering of Komagataella phaffii and its broader applications in synthetic biology and biomanufacturing.