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ORIGINAL RESEARCH article
Front. Bioeng. Biotechnol.
Sec. Synthetic Biology
Volume 13 - 2025 | doi: 10.3389/fbioe.2025.1586218
This article is part of the Research TopicSynthetic Biology Approaches for Biocatalytic Production of Value-Added ChemicalsView all articles
A Versatile Genetic Toolkit for Engineering Wickerhamomyces ciferrii for Tetraacetyl Phytosphingosine Production
Provisionally accepted- Ajou University, Suweon, Republic of Korea
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Wickerhamomyces ciferrii, a non-conventional yeast with significant industrial potential for tetraacetyl phytosphingosine (TAPS), remains underutilized due to the lack of a comprehensive genetic toolbox. In this study, we developed a modular genetic system tailored for W. ciferrii to enable strain engineering and metabolic pathway optimization. This toolkit includes episomal plasmids incorporating multiple selectable markers, replication origins, and fluorescent reporters. Systematic evaluation of four antibiotic resistance markers demonstrated that nourseothricin, geneticin, and zeocin effectively confer resistance, whereas hygromycin B did not support selection in this host. Among three tested replication origins, 2μ and CEN6/ARS4 enabled stable episomal maintenance, whereas panARS failed to replicate.Expression analysis of six fluorescent proteins under the endogenous PGK1 promoter revealed significant variability in transcript levels, which correlated with codon adaptation index values, emphasizing the importance of codon optimization for heterologous expression. Additionally, characterization of the endogenous TDH3, PGK1, and PDA1 promoters using two highly expressed fluorescent proteins confirmed that promoter strength is largely independent of the downstream coding sequence. To demonstrate the functional application of this toolkit, we overexpressed a phosphorylation-insensitive mutant of acetyl-CoA carboxylase (ACC1 S26A- S1161A ), resulting in a 2.4-fold increase in TAPS production. Collectively, this study establishes a versatile genetic platform for W. ciferrii, providing a robust foundation for future synthetic biology and metabolic engineering applications.
Keywords: genetic toolkit, Non-model yeast, Wickerhamomyces ciferrii, Episomal plasmid system, tetraacetyl phytosphingosine
Received: 02 Mar 2025; Accepted: 16 Apr 2025.
Copyright: © 2025 Lee, Kang and Lee. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Pyung Cheon Lee, Ajou University, Suweon, Republic of Korea
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