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bioRxiv · 10.64898/2026.05.17.725789

Repurposing native non-homologous end joining for multicopy random integration in Wickerhamomyces ciferrii

Lee, S.-R.·Seo, Y.·Lee, P. C.
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Abstract

Wickerhamomyces ciferrii is a non-model diploid yeast that naturally produces tetraacetyl phytosphingosine (TAPS), a sphingoid base used in cosmetic and dermatological applications. However, its strong preference for non-homologous end joining (NHEJ) over homologous recombination (HR) limits conventional genome editing, while disruption of LIG4, a core NHEJ gene, compromises cellular fitness. Here, we repurposed native NHEJ activity to develop a homology-independent multicopy genome integration platform for W. ciferrii. The platform combines three optimized donor-design features: telomeric end-shielding with two tandem copies of an 11 bp repeat to improve linear donor persistence, a defective URA5 auxotrophic marker to enrich multicopy integrants, and 5'-phosphorylated donor termini to enhance transformant recovery and integration output. These features were consolidated into the platform vector pTdmVU5. As a metabolic engineering demonstration, multicopy integration of LCB1 and LCB2, encoding the two subunits of serine palmitoyltransferase, increased TAPS titer by 2.7-fold. This work converts the native NHEJ bias of W. ciferrii from a barrier to precise genome editing into a practical tool for pathway amplification and establishes a framework for engineering NHEJ-dominant non-model yeasts.

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Lee, S.-R., Seo, Y., Lee, P. C.. 2026-05-18. Repurposing native non-homologous end joining for multicopy random integration in Wickerhamomyces ciferrii. https://doi.org/10.64898/2026.05.17.725789

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