Upgrading yeast-based low-carbon biomanufacturing by harnessing a universal in vivo evolution system.

Authors

Shirui Duan, Zhihui Shi, Xinru Wang, Weihe Rong, Guoqing Peng, Lili Yun, Shuang Liang, Qian Chen, Aixuan Xiao, Songqi Liu, Hailin He, M D Abuhena, Wenxi Liu, Hongfei Ma, Yuping Lin, Xiaoping Liao, Guokun Wang

Published in

Metabolic engineering. Pages 102579. Nov 10, 2026. Epub Nov 10, 2026.

Abstract

Non-conventional yeasts are emerging as key cell factories in low-carbon biomanufacturing, but unlocking their full potential requires optimizing complex traits, such as substrate utilization and high-level protein expression, through in vivo genome-wide evolution. To address this, we developed a universal plasmid vector deploying a DNA Helicase-Activation-Induced Cytidine Deaminase (Helicase-AID) for genome-wide base editing. This system enables cross-species mutagenesis in diverse yeasts, including Yarrowia lipolytica, Pichia pastoris, and Kluyveromyces marxianus. We demonstrated that MCM5-AID base editing efficiency in Y. lipolytica is time-dependent, achieving a cumulative mutation rate of 3.13 × 10^-5 mutations per base pair (bp^-1) over 432 hours. Using this system, we evolved Y. lipolytica variants exhibiting a 37.77% increase in maximum specific growth rate on acetate, alongside altered starch production ranging from -92.96% to 17.45%. Crucially, we utilized this platform to activate the silent expression of heterologous proteins, overcoming a persistent bottleneck in microbial strain engineering. Evolutionary, transcriptomic and genomic analyses showed that evolved Y. lipolytica variants exhibited enhanced accumulation of Cas13x, a naturally difficult-to-express protein, via reduced proteolytic degradation. In contrast, evolved P. pastoris variants gained the ability to express previously silent thaumatin through significant transcriptional upregulation, enhanced protein folding, and increased amino acid biosynthesis. Ultimately, this universal in vivo evolution system provides a robust platform for genome-directed evolution across diverse industrial yeast species, accelerating the systematic exploration of their potential in low-carbon biomanufacturing.

PMID:
43009044
Bibliographic data and abstract were imported from PubMed on 11 Nov 2026.

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