Front. Synth. Biol. Frontiers in Synthetic Biology Front. Synth. Biol. 2813-818X Frontiers Media S.A. 1729790 10.3389/fsybi.2025.1729790 Mini Review Advances in genetic tools for metabolic engineering of non-conventional yeasts Frousnoon et al. 10.3389/fsybi.2025.1729790 Frousnoon Thasneem Banu 1 2 Conceptualization Investigation Writing - original draft Writing - review and editing Mondo Stephen 3 Funding acquisition Supervision Writing - review and editing Yoshikuni Yasuo 1 2 3 4 5 6 * Conceptualization Funding acquisition Supervision Writing - review and editing Center for Advanced Bioenergy and Bioproducts Innovation, Lawrence Berkeley National Laboratory, Berkeley, CA, United States Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, United States Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States Energy and Biosciences Institute, University of California, Berkeley, CA, United States Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, Hokkaido, Japan Correspondence: Yasuo Yoshikuni, yyoshikuni@lbl.gov 07 01 2026 2025 3 1729790 21 10 2025 13 12 2025 15 12 2025 Copyright © 2026 Frousnoon, Mondo and Yoshikuni. 2026 Frousnoon, Mondo and Yoshikuni https://creativecommons.org/licenses/by/4.0/ 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) and the copyright owner(s) 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.

Non-conventional yeasts are emerging as powerful alternatives to Saccharomyces cerevisiae for metabolic engineering, owing to their innate stress tolerance, broad substrate utilization, and distinctive metabolic capabilities. These attributes position them as promising chassis for producing biofuels, pharmaceuticals, and specialty chemicals. This review synthesizes recent advances in genetic toolkits for four such species—Pichia kudriavzevii (Issatchenkia orientalis), Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus—highlighting progress across plasmid architectures (episomal and integrative), identification of autonomously replicating sequences and centromeric elements, and the development of safe-harbor genomic loci. We summarize promoter and terminator libraries enabling tunable expression, the expansion of auxotrophic and antifungal selection markers with recycling strategies, and the rapid adaptation of CRISPR-based systems (Cas9 and Cas12a) with optimized guide RNA expression, multiplex editing, and approaches that enhance homologous recombination (e.g., KU70/80 disruption). We also review landing-pad platforms for modular, repeated integrations and transposon-based tools (e.g., piggyBac) that facilitate multigene pathway assembly. Collectively, these innovations are accelerating design-build-test-learn cycles and enabling precise, scalable engineering of non-conventional yeasts. Remaining challenges—including limited species-specific episomal systems, variable transformation efficiencies, genome-stability concerns, and alternative codon usage—define clear priorities for future toolkit development. Together, these advances and open needs chart a path toward robust, sustainable biomanufacturing using diverse non-conventional yeast chassis.

 Debaryomyces hansenii genetic tools genome engineering metabolic engineering non-conventional yeast Pachysolen tannophilus Pichia kudriavzevii Starmerella bombicola U.S. Department of Energy 10.13039/100000015 DE-AC02-05CH11231 The author(s) declared that financial support was received for this work and/or its publication. This work was funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number DE-SC0018420 and DE-AC02-05CH11231 [for TF and YY]). This work was supported by laboratory-directed research and development (LDRD). The work (https://orcid.org/0000-0002-8372-640X) conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231 [for YY]. section-at-acceptance Metabolic Engineering Introduction

Yeasts play a foundational role in biotechnology, supporting global food, beverage, and biomanufacturing industries. Saccharomyces cerevisiae has long been both a premier eukaryotic model organism and a dominant industrial chassis (Borodina and Nielsen, 2014). However, its usefulness is constrained by a relatively narrow substrate range, limited metabolic flexibility, and only moderate tolerance to industrial stresses such as high temperature, osmotic pressure, and inhibitory by-products. These limitations have fueled growing interest in non-conventional yeasts—phylogenetically diverse species that naturally exhibit robust metabolic bapabilities and superior stress tolerance (Radecka et al., 2015; Wagner and Alper, 2016; Löbs et al., 2017; Wang et al., 2025).

Non-conventional yeasts span multiple genera, including Pichia, Yarrowia, Kluyveromyces, Starmerella, Debaryomyces, Pachysolen, and Saturnispora (Figure 1). Each genus is adapted to different ecological niches and displays unique physiological traits with biotechnological potential (de Souza Varize et al., 2019). Despite these advantages, the broader industrial adoption of non-conventional yeasts has historically been limited by insufficient genetic tools, low transformation efficiencies, and poorly characterized regulatory parts (Moon et al., 2025). Recent advances in genome sequencing, synthetic biology, and CRISPR-based genome engineering, however, are rapidly overcoming these obstacles. An expanding genetic toolbox—including episomal and integrative plasmids, diverse promoters and terminators, and increasingly efficient genome-editing systems—is now enabling the systematic domestication of these species (Table 1; Figure 1a). As a result, non-conventional yeasts are emerging as competitive, and in some cases superior, platforms relative to S. cerevisiae for industrial biotechnology (Moon et al., 2025).

Genetic tools in non-conventional yeasts.

Genetic Tool Examples Notes References
Pichia kudriavzevii
Promoters FBA1, TEF1, PGK1, TDH3, PDC1, GAPDH, GPM1, SED1, indolepyruvate decarboxylase 6, inositol-3-phosphate synthase INO1, translation elongation factor EF-1 alpha, lipid-binding protein HSP12 Heterologous gene expression Xiao et al. (2014), Sohn et al. (2019), Lee et al. (2022)
tRNA promoter (Pol III), RPR1′-tRNALeu promoter, RPR1, 5S RNA-tRNALeu Drives sgRNAs for CRISPR applications Tran et al. (2019), Sun et al. (2020)
Terminators CYC1, PGK1, PDC1, GAPDH, MDH1, PDC1, INO1 Constitutively expressed Sohn et al. (2019), Cao et al. (2020), Lee et al. (2022)
Markers URA3, LEU2, TRP1, HIS3 Leu2 from S. cerevisiae Xiao et al. (2014), Tran et al. (2019), Cao et al. (2020), Sun et al. (2020)
Nourseothricin N-acetyl transferase (NAT) 120 μg/mL Lee et al. (2022)
Zeocin 200 μg/mL Zhang et al. (2023)
Aureobasidin A (AUR1-KP729614) 0.5 μg/mL Yoo and Kim (2015), Sohn et al. (2019)
Integration Sites 21 loci were identified for high efficiency integration Large pathways (18 kb) were integrated Fatma et al. (2023)
Starmerella bombicola
Promoters GPD, eno, pGAPD, pUGTA1 pUGTB1, ura3 Constitutively expressed, native Van Bogaert et al. (2007), 2013; Li et al. (2016a); Li et al. (2016b); Geys (2017), Jezierska et al. (2020) Lodens et al. (2020), Chatterjee et al. (2022)
pCYP52M1 Promoter of a biosynthetic gene cluster Geys, 2017; Lodens et al. (2020)
TEF1, GAPD, PGK1 Native, constitutively expressed, used for SbCas9 Shi et al. (2022), Zhang et al. (2022)
pGALK Inducible galactokinase promoter Shi et al. (2022), Zhang et al. (2022)
Terminators TK (HSV thymidine kinase), Trpc Non-native, TK is a viral terminator, Trpc is from Aspergillus nidulans Van Bogaert et al. (2013), Li et al. (2016a); Li et al. (2016b); Chatterjee et al. (2022), Qazi et al. (2022)
tGAL Constitutively expressed Lodens et al. (2018), 2020
PGK1 (phosphoglycerate kinase), tCYP52M1, ura3, tUGTA1, tUGTB1 Native Geys (2017), Chatterjee et al. (2022), Shi et al. (2022)
Trpl41b Strong terminator from the S. cerevisiae ribosome protein-encoding gene Chatterjee et al. (2022)
Tsyn7 Constitutively expressed, used for SbCas9 Shi et al. (2022), Zhang et al. (2022)
Markers URA3 Native Van Bogaert et al. (2007), Geys, 2017; Shi et al. (2022)
Nourseothricin acetyl transferase (NAT), Hygromycin B Hygromycin B- 500 μg/mL Li et al. (2016a); Geys, 2017; Shi et al. (2022)
GFP, SbGFP SbGFP is codon-optimized yeGFP Lodens et al. (2020), Shi et al. (2022)
FBP Can be used under oxygen-limitation Lodens et al. (2020)
Amylase system Can be used as a reporter for extracellular protein expression Lodens et al. (2020)
Integration Sites AT locus, Ura3 locus, CYP52M1 locus, UGTA locus, UGTB locus Large cassettes and biosynthetic clusters were integrated Saerens et al. (2011); Van Bogaert et al. (2013), Geys, 2017; Lodens et al. (2018), 2020; Zhang et al. (2022)
PXA1 locus, Sble locus Used for CRISPR-Cas9 and to integrate Cas12a expression cassette respectively Shi et al. (2022), Zhang et al. (2022)
Debaromyces hansenii
Promoters GPD1 (glycerol-3-phosphate dehydrogenase) Strong promoter from S. cerevisiae, constitutively expressed Maggi and Govind (2004)
GPD1d (Native GPD1), Dh_RNR2p and Dh_RHR2p GPD1d is an endogenous constitutive strong promoter, Dh - D. hansenii Maggi and Govind (2004), Strucko et al. (2021)
HSP12, SME1 Heat Shock protein, protein kinase from S. cerevisiae used for heterologous expression, constitutively expressed, moderate levels of expression Maggi and Govind (2004)
CYC1, Cl_TDH3, Ag_TEF1p Iso cytochrome C1 from S. cerevisiae, Cl - Candida lusitaniae, Ag - Ashbya gossypii Ricaurte and Govind (1999), Strucko et al. (2021)
Cl_SNR52p, Dh_SRC1p, DhTEF1p RNA pol III promoters for sgRNA expression cassette Strucko et al. (2021)
Terminators MF Mating factor Maggi and Govind (2004)
CYC1, Ag_TEF1t, Ca_ENO1t, A. adeninivorans-derived TEF1 CYC1 from S. cerevisiae, Ca - Candida albicans Ricaurte and Govind (1999), Terentiev et al. (2004), Strucko et al. (2021)
DhTEF1t Native Minhas et al. (2009); Strucko et al. (2021)
Markers URA3 Auxotrophic marker from S. cerevisiae Ricaurte and Govind (1999), Maggi and Govind (2004)
Hygromycin B 100 μg/mL, expression of the cassette is weaker with respect to URA3 Ricaurte and Govind (1999)
HIS4, ADE2, NAT NATCUG is used Minhas et al. (2009); Spasskaya et al. (2021), Strucko et al. (2021)
GFP, RFP GFP is from the jellyfish Aequorea victoria Maggi and Govind (2004), Terentiev et al. (2004), Minhas et al. (2009)
Integration Sites 25S rRNA gene locus A conserved A. adeninivorans-derived 25S rDNA sequence was used for targeting Terentiev et al. (2004)
Pachysolen tannophilus
Promoters GAPDH Native Riley et al. (2016)
Terminators GAPDH Native Riley et al. (2016)
Markers Hygromycin, Leu2 CUG-leu codons in hygromycin were changed to other Leu codons Riley et al. (2016), Mei et al. (2018)

(a) Summary of genetic tools in non-conventional yeasts. The tools currently available are denoted in green and the limitations/challenges are denoted in red. (b) Phylogenetic tree of selected non-conventional ascomycete yeasts belonging to the subphylum Saccharomycotina with Schizosaccharomyces pombe designated as the outgroup. The yeasts emphasized in this review, Pichia kudriavzevii, Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus, are highlighted in pink.

Diagram (a) shows a circular chart detailing the genetic engineering tools available for five yeast species, highlighting their capabilities for biofuel production, biomass transformation, and waste valorization. Each section notes their genetic tools and limitations. Diagram (b) is a phylogenetic tree of the Ascomycota phylum, showing evolutionary relationships among various yeast species within Taphrinomycotina and Saccharomycotina.

In this review, we highlight four Saccharomycotina yeasts, Pichia kudriavzevii, Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus. We focused on these species because they collectively encompass a wide range of industrially relevant phenotypes that are not well represented in other, more extensively studied non-model yeasts (e.g., Yarrowia lipolytica, Komagataella phaffii, Kluyveromyces marxianus, Schizosaccharomyces pombe), and because recent efforts have substantially expanded the genetic tools available for each. For example, P. kudriavzevii thrives in low pH environments (∼pH = 1.5) and is well suited for organic acid production, whereas S. bombicola is a natural producer of high-value sophorolipids (Tran et al., 2023; Wu et al., 2023; Frousnoon et al., 2025; Zhang et al., 2025). D. hansenii displays exceptional halotolerance and osmotolerance, allowing growth in high-salt environments (>1 M) ( Breuer and Harms, 2006; Xelhuantzi et al., 2024; Estrada et al., 2023; Fukuda et al., 2004 ). Meanwhile, P. tannophilus efficiently ferments both glucose and xylose, making it attractive chassis for lignocellulosic biomass conversion (Maleszka et al., 1982; Slininger et al., 1982; Slininger et al., 1987; Mei et al., 2018). Collectively, these yeasts provide a representative view of the physiological and genetic diversity now accessible for systematic metabolic engineering.

 Synthetic biology tools Plasmids

Plasmids are naturally occurring extrachromosomal genetic elements capable of autonomous replication within host cells (Glass, 1982; Nora et al., 2019). In synthetic biology, engineered plasmids—commonly referred to as vectors—serve versatile molecular tools for applications such as cloning, heterologous gene expression, gene knockdown, reporter assays, and genome engineering. In yeast biology, vectors are generally categorized into episomal, centromeric, and integrative types, each offering distinct advantages and limitations (Nora et al., 2019).

Episomal and centromeric plasmids are circular DNA molecules that replicate independently of the host genome and are often maintained at high copy numbers (Gnügge and Rudolf, 2017; Nora et al., 2019). They are relatively easy to construct, introduce, and remove, making them ideal for rapid screening, transient expression, and high-level protein production. However, episomal and centromeric plasmids tend to be unstable without continuous selection pressure and can impose a metabolic burden on the host due to the energetic cost of maintaining multiple copies (Nevoigt, 2008). In contrast, integrative plasmids are typically linearized and stably integrated into the host genome via homologous recombination, often at defined chromosomal loci. Although they generally result in lower copy numbers and moderate expression levels compared to episomal systems, integrative plasmids offer superior stability across generations, even in the absence of selective pressure (Vještica et al., 2020). Despite being more time- and labor-intensive to construct, integrative plasmids are preferred for developing robust production strains suitable for industrial applications.

The functionality of a plasmid depends on several key genetic components that govern its replication and gene expression. These include promoters, terminators, and in the case of centromeric plasmids, autonomously replicating sequences (ARS) and centromeric (CEN) elements (Liachko and Dunham, 2014; Wagner and Alper, 2016; Vještica et al., 2020; Smith et al., 2024). Promoters, located upstream of the coding region, recruit RNA polymerase and transcription factors to initiate transcription. They can be constitutive, such as TEF1 promoter, or inducible, such as AOX1 promoter, which is activated only in response to specific stimuli. Terminators positioned downstream of the coding region ensure proper transcriptional termination and polyadenylation, contributing to mRNA stability and efficient expression.

The ARS enables autonomous plasmid replication within the host, typically resulting in high-copy-number maintenance. In contrast, the CEN element, derived from yeast centromeric DNA, allows plasmids to segregate similarly to chromosomes during cell division, resulting in stable inheritance at low copy number. CEN-based vectors are therefore useful for applications requiring controlled, low-level expression or stable maintenance without extensive selection pressure (Vještica et al., 2020).

 Markers

Markers are essential genetic elements used to verify successful transformation and to maintain plasmids or integrated constructs within host cells. They are broadly categorized into selection markers and reporter markers (Gnügge and Rudolf, 2017). Selection markers provide a growth advantage under specific conditions, allowing only transformed cells to proliferate. These are further divided into auxotrophic markers and antifungal-selection markers. Auxotrophic markers complement metabolic deficiencies in mutant host strains, enabling growth in selective media. For example, a ura-strain cannot grow in uracil-deficient media unless transformed with a plasmid carrying the URA3 gene. Other common examples include LEU2 and HIS3. Antifungal-selection markers, in contrast, confer resistance to antibiotics such as hygromycin B or nourseothricin, and can be used in prototrophic strains without the need for auxotrophic backgrounds (Gnügge and Rudolf, 2017; Nora et al., 2019; Vještica et al., 2020). Meanwhile, reporter markers facilitate the visual or quantitative identification of transformed cells. They often encode easily detectable proteins or enzymes that reflect gene expression or cellular activity. The green fluorescent protein (GFP), for instance, enables real-time monitoring and quantification of expression levels in living cells (Soboleski et al., 2005).

 Integration sites

Genomic integration sites are specific chromosomal loci within the host genome used for the stable insertion of foreign DNA. Integration at these sites ensures long-term maintenance and expression of the introduced construct without continuous selective pressure. Integration can occur either randomly or at defined loci that are known to tolerate insertions without disrupting essential host functions. Defined “safe harbor” sites are particularly valuable for achieving predictable expression and genetic stability in engineered yeast strains (Terentiev et al., 2004; Ronda et al., 2015; Babaei et al., 2021; Shi et al., 2022; Zhang et al., 2022; Fatma et al., 2023).

 Genome-editing tools

Genome editing tools enable precise and efficient manipulation of yeast genomes to engineer metabolic pathways and optimize strain performance. In S. cerevisiae, genome modification has traditionally relied on homologous recombination (HR), which occurs with high efficiency. However, non-conventional yeasts exhibit relatively low HR efficiency, promoting the development of alternative tools for targeted genome modification (Ji et al., 2020; Strucko et al., 2021). The advent of nuclease-based editing technologies, particularly CRISPR-Cas systems, has revolutionized genome engineering (Doudna and Charpentier, 2014). Enzymes such as Cas9 and Cas12a (Cpf1) introduce targeted double-strand breaks, enabling site-specific base editing, gene knockout, marker-free integration, and multiplex genome modification (Tran et al., 2019; Spasskaya et al., 2021; Strucko et al., 2021; Shi et al., 2022; Zhang et al., 2022). Beyond nuclease-based systems, landing pad approaches have been developed to facilitate repeated or modular genome engineering (Fatma et al., 2023). These systems introduce defined genomic docking sites (e.g., loxP or attP) that allow site-specific integration of genetic constructs via recombination (Santos et al., 2013). Additionally, transposon-based systems such as piggyBac allow random integration of genetic cassettes at TTAA sites without requiring homologous recombination or site-specific nucleases (Li et al., 2013). Such tools expand the flexibility and scalability of genome manipulation, accelerating the development of non-conventional yeast platforms for biotechnological applications.

 Non-conventional yeasts <italic>Pichia kudriavzevii</italic>

Pichia kudriavzevii (also known as Candida Krusei or Issatchenkia orientalis) is a ubiquitous non-conventional yeast known for its exceptional tolerance to high temperature, low pH, elevated salinity, and high concentrations of lignocellulosic inhibitors (Douglass et al., 2018; Lee et al., 2022; Chu et al., 2023; Wang et al., 2025). These robust traits make it an attractive platform for the bioproduction of organic acids and other value-added chemicals from renewable feedstocks (Sun et al., 2020; Tran et al., 2023; Wu et al., 2023; Tan et al., 2025). The species is predominantly diploid, although triploid and aneuploid variants have also been identified (Douglass et al., 2018; Hsieh et al., 2025).

Genetic modification of P. kudriavzevii has traditionally relied on linear integrative plasmids employing homologous recombination, commonly with the URA3 selection marker (Xiao et al., 2014; Wu et al., 2023). The introduction of episomal plasmids became feasible with the incorporation of an S. cerevisiae ARS element and a native centromere-like sequence (CEN-L), which together improved plasmid replication and segregation stability (Tran et al., 2019; Cao et al., 2020). Recent progress includes multicopy and multigene integrations using piggyBac transposon and landing pad systems targeting defined intergenic loci (Fatma et al., 2023; Wu et al., 2023). The development of CRISPR-Cas-based tools now allows efficient, marker-free genome editing without reliance on auxotrophic or antifungal selection markers (Tran et al., 2019). Transformation is most commonly achieved through the lithium acetate (LiAc) method, which remains the standard protocol for P. kudriavzevii.

 <italic>Starmerella bombicola</italic>

Starmerella bombicola (formerly Candida bombicola) is a non-pathogenic yeast renowned for its natural production of sophorolipids—biodegradable glycolipid surfactants with broad applications in pharmaceuticals, cosmetics, and environmental remediation (Kurtzman et al., 2010; De Graeve et al., 2018; Zhang et al., 2025). The species can metabolize both hydrophilic carbon sources (e.g., sucrose and fructose) and hydrophobic substrates (e.g., fatty acids, fatty alcohols, and long-chain alkanes), enabling valorization of inexpensive waste feedstocks for sophorolipid and long-chain dicarboxylic acid production (Li et al., 2016b; Chatterjee et al., 2022; Lee et al., 2025; Wang et al., 2019).

Genetic modification of S. bombicola has been challenging due to low HR efficiency (Shi et al., 2022; Zhang et al., 2022). Although extending homology arms to 1 kb can improve HR frequency, colony recovery on selective media remains slow (Saerens et al., 2011; Shi et al., 2022; Zhang et al., 2022). The species appears to rely minimally on non-homologous end joining (NHEJ) for DNA double strand break repair. Transformation is typically achieved via LiAc-mediated chemical transformation or electroporation, whereas Agrobacterium Tumefaciens-mediated transformation (ATMT) has proven ineffective (Lodens et al., 2018).

Plasmids have been derived from the pGEM-T and pJET backbones (Saerens et al., 2011; Geys, 2017; Lodens et al., 2018). The activities of 14 native promoters were characterized and shown to vary with carbon sources; among them, TEF1, GAPD, ENO, PGK1, TPI, and TDH1 promoters showed moderate to strong expression based on GFP assays (Shi et al., 2022). A codon-optimized Streptococcus pyogenes Cas9 (SbCas9) was developed by removing inhibitory secondary structures to enhance expression. Although RNA polymerase III promoters for sgRNA expression have not yet been identified in this species, a CRISPR-Cas12a (Cpf1) system employing a codon-optimized Acidaminococcus sp. Cas12a driven by the TEF1 promoter enabled efficient single and multiplex genome editing (Zhang et al., 2025). Using donor DNA with ∼300 bp homology arms further enhanced editing efficiency, outperforming both HR- and NHEJ-based approaches (Zhang et al., 2025).

Despite these editing strategies, further advances are limited by the lack of a functional episomal plasmid in S. bombicola. Attempts to identify ARS elements from Kluyveromyces lactis, S. cerevisiae (ARS/CEN4 and 2-micron plasmid), or its own genome have not yielded self-replicating constructs (Geys, 2017).

 <italic>Debaryomyces hansenii</italic>

D. hansenii is a non-pathogenic, halotolerant, osmotolerant, xerotolerant, and oleaginous, haploid yeast (Gunge et al., 1993; Maggi and Govind, 2004; Breuer and Harms, 2006). It secretes a NaCl-enhanced killer toxin that is lethal to other yeasts (Gunge et al., 1993; Breuer and Harms, 2006). Sodium ions appear to protect its growth even under extreme pH conditions (Prista et al., 2005; Navarrete et al., 2022). D. hansenii utilizes diverse carbon sources, including raffinose, xylose, and n-alkanes, and can thrive in highly saline (>1 M) or nutrient-limited environments, making it well suited for waste revalorization processes (Estrada et al., 2023; Navarrete et al., 2022; Breuer and Harms, 2006; Fukuda et al., 2004 ).

Stable plasmid replication in D. hansenii typically requires high salt conditions (∼2.1 g/L) (Gunge et al., 1993; Fukuda et al., 2004). Several episomal plasmids—pRGM, pMR95, pMR96, pDH4, and pDH11—derived from the pUC19 backbone contain D. hansenii ARS elements, while plasmids such as pDhARS2, pDhARS3, and pDhARS9 derived from pGEM7Z harbor C. famata ARS sequences for heterologous expression (Ricaurte and Govind, 1999; Maggi and Govind, 2004; Minhas et al., 2009). CRISPRCUG-tRNA plasmids (pDIV series) have also been developed, incorporating replication origins from Candida famata, K. lactis and S. cerevisiae (Strucko et al., 2021).

Because D. hansenii favors NHEJ over HR, a ΔKU70 mutant strain was engineered to enhance targeted gene integration (Minhas et al., 2009; Strucko et al., 2021; Navarrete et al., 2022). CRISPR-Cas9 transformation achieves approximately 3000 CFU/μg of DNA via electroporation (Ricaurte and Govind, 1999; Minhas et al., 2009). Improving transformation efficiency and toolkits for this species will expand functional genomics and pathway engineering. Furthermore, D. hansenii reassigns the CUG codon to serine instead of leucine, necessitating codon optimization for all heterologous genes (Strucko et al., 2021).

 <italic>Pachysolen tannophilus</italic>

Pachysolen tannophilus is a haploid, non-conventional yeast with a broad substrate range, capable of metabolizing glucose, glycerol, galactose, and xylose, making it a promising host for industrial conversion of diverse hydrolysates (Maleszka et al., 1982; Slininger et al., 1982; Slininger et al., 1987; Yang and Jeffries, 1997; Wedlock et al., 1989). However, P. tannophilus is relatively sensitive to lignocellulosic inhibitors (Harner et al., 2014). Genome sequencing in 2012 revealed that its CUG codon is reassigned to encode alanine rather than leucine, placing it among the alternative-codon-usage yeasts (Liu et al., 2012; Riley et al., 2016; Muhlhausen et al., 2016).

Early transformation studies employed S. cerevisiae-derived plasmids such as YRp7, YEp13 and pACT containing 2-µm ARS sequences, supported limited replication in P. tannophilus (Mei et al., 2018; Wedlock et al., 1989). However, plasmids bearing S. cerevisiae ARS/CEN elements were not stably maintained, likely due to sequence divergence (Mei et al., 2018). Furthermore, previous work reported that successful transformation of P. tannophilus occurred only with plasmids bearing a codon-optimized hygromycin resistance gene, while plasmids containing the native version failed to produce transformants (Riley et al., 2016). Optimization of the LiAc-mediated transformation protocol improved transformation efficiency, yet the lack of native replication and centromeric elements remains a bottleneck for stable episomal maintenance.

The reassignment of the CUG codon further complicates heterologous expression, as genes encoding leucine via CUG may produce mistranslated proteins. Identifying native ARS/CEN sequences, together with species-specific selection markers and strong regulatory elements, will be critical for developing a reliable genetic toolkit. Establishing high-efficiency transformation and genome-editing systems will be key to advancing P. tannophilus as a robust microbial cell factory.

 Discussion

Despite significant progress in developing genetic tools for non-conventional yeasts, several challenges remain before these species can be fully established as robust industrial platforms. The lack of species-specific plasmid systems, low transformation efficiencies, and issues of genome stability continue to constrain efficient genetic manipulation and metabolic engineering. Although many non-conventional yeasts display exceptional stress tolerance and metabolic versatility, the underlying molecular mechanisms and regulatory networks governing these traits remain underexplored. Additional challenges are presented by species such as D. hansenii and P. tannophilus, which exhibit CUG codon reassignment. This deviation from the universal genetic code prevents the direct use of conventional plasmids and heterologous genes without codon optimization. Similarly, S. bombicola currently lacks a functional episomal plasmid, limiting its genetic tractability despite its natural capacity to produce biodegradable and non-toxic surfactants. Identifying native ARS/CEN elements and establishing species-specific expression systems will therefore be essential to expand their biotechnological potential. To unlock the full capabilities of these emerging yeast platforms, future research should prioritize the development of stable genomic integration loci, high-efficiency transformation methods, and tailored genome-editing strategies that accommodate species-specific characteristics. Such advancements will be critical to broaden the genetic toolbox and realize the full potential of non-conventional yeasts as sustainable microbial chassis for next-generation biomanufacturing.

 Author contributions

TF: Conceptualization, Investigation, Writing – original draft, Writing – review and editing. SM: Funding acquisition, Supervision, Writing – review and editing. YY: Conceptualization, Funding acquisition, Supervision, Writing – review and editing.

 Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author YY declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

 Generative AI statement

The author(s) declared that generative AI was used in the creation of this manuscript. Generative AI tools were used solely for proofreading and language polishing.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

 Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

 Author disclaimer

Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the U.S. Department of Energy.

Edited by: Ahsan Islam, Loughborough University, United Kingdom

Reviewed by: James Robertson, Middle Tennessee State University, United States

James Grissom, Lenoir–Rhyne University, United States

 References Babaei M. Sartori L. Karpukhin A. Abashkin D. Matrosova E. Borodina I. (2021). Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites. FEMS Yeast Research 21 (4), foab027. 10.1093/femsyr/foab027 33893795 Borodina I. Nielsen J. (2014). Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals. Biotechnol. Journal 9 (5), 609–620. 10.1002/biot.201300445 24677744 Breuer U. Harms H. (2006). Debaryomyces hansenii-an extremophilic yeast with biotechnological potential. Yeast Chichester, Engl. 23 (6), 415–437. 10.1002/yea.1374 16652409 Cao M. Fatma Z. Song X. Hsieh P. H. Tran V. G. Lyon W. L. (2020). A genetic toolbox for metabolic engineering of Issatchenkia orientalis. Metab. Engineering 59, 87–97. 10.1016/j.ymben.2020.01.005 32007615 Chatterjee M. Patel J. B. Stober S. T. Zhang X. (2022). Heterologous synthesis and secretion of ricinoleic acid in Starmerella bombicola with sophorolipid as an intermediate. ACS Synthetic Biology 11 (3), 1178–1185. 10.1021/acssynbio.1c00457 35157794 Chu Y. Li M. Jin J. Dong X. Xu K. Jin L. (2023). Advances in the application of the non-conventional yeast Pichia kudriavzevii in food and biotechnology industries. J. Fungi Basel, Switz. 9 (2), 170. 10.3390/jof9020170 36836285 De Graeve M. De Maeseneire S. L. Roelants S. L. K. W. Soetaert W. (2018). Starmerella bombicola, an industrially relevant, yet fundamentally underexplored yeast. FEMS Yeast Research 18 (7). 10.1093/femsyr/foy072 29982357 de Souza Varize C. Maria Christofoleti‐Furlan R. de Souza Miranda Muynarsk E. Vinícius de Melo Pereira G. Dantas Lopes L. Carlos Basso L. (2019). “Biotechnological applications of nonconventional yeasts,” in Yeasts in biotechnology. IntechOpen. 10.5772/intechopen.83035 Doudna J. A. Charpentier E. (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Sci. (New York, N.Y.) 346 (6213), 1258096. 10.1126/science.1258096 25430774 Douglass A. P. Offei B. Braun-Galleani S. Coughlan A. Y. Martos A. A. R. Ortiz-Merino R. A. (2018). Population genomics shows no distinction between pathogenic Candida krusei and environmental pichia kudriavzevii: one species, four names. PLoS Pathogens 14 (7), e1007138. 10.1371/journal.ppat.1007138 30024981 Estrada M. Navarrete C. Moller S. Quirós M. Martínez J. L. (2023). Open (non-sterile) cultivations of Debaryomyces hansenii for recombinant protein production combining industrial side-streams with high salt content. New Biotech. 78, 105–115. 10.1016/j.nbt.2023.10.005 37848161 Fatma Z. Tan S. I. Boob A. G. Zhao H. (2023). A landing pad system for multicopy gene integration in Issatchenkia orientalis. Metab. Engineering 78, 200–208. 10.1016/j.ymben.2023.06.010 37343658 Frousnoon T. B. Pham N. N. Wu Z. Y. Hsieh P. H. Yoshikuni Y. (2025). Comparison of stress tolerance mechanisms between Saccharomyces cerevisiae and the multistress-tolerant Pichia kudriavzevii. FEMS Yeast Research 25, foaf024. 10.1093/femsyr/foaf024 40343780 Fukuda K. Jin-Shan C. Kawano M. Sudo K. Gunge N. (2004). Stress responses of linear plasmids from Debaryomyces hansenii. FEMS Microbiology Letters 237 (2), 243–248. 10.1111/j.1574-6968.2004.tb09702.x 15321668 Geys R. (2017). Engineering the metabolism of Starmerella bombicola for the production of tailor-made glycolipids. Available online at: https://core.ac.uk/download/pdf/84043262.pdf. Glass R. E. (1982). “Plasmids,” in Gene function (Boston, MA: Springer US), 159–192. 10.1007/978-1-4684-6689-8_4 Gnügge R. Rudolf F. (2017). Saccharomyces cerevisiae shuttle vectors. Yeast Chichester, Engl. 34 (5), 205–221. 10.1002/yea.3228 28072905 Gunge N. Fukuda K. Morikawa S. Murakami K. Takeda M. Miwa A. (1993). Osmophilic linear plasmids from the salt-tolerant yeast Debaryomyces hansenii. Curr. Genetics 23 (5-6), 443–449. 10.1007/bf00312632 8391396 Harner N. K. Bajwa P. K. Habash M. B. Trevors J. T. Austin G. D. Lee H. (2014). Mutants of the pentose-fermenting yeast Pachysolen tannophilus tolerant to hardwood spent sulfite liquor and acetic acid. Antonie Van Leeuwenhoek 105 (1), 29–43. 10.1007/s10482-013-0050-y 24122119 Hsieh P.-H. Sasaki Y. Yang C. I. Wu Z. Y. Steindorff A. S. Haridas S. (2025). MAESTRO uncovers tandem paralog dynamics as a core driver of fungal stress adaptation. bioRxiv. 10.1101/2025.08.29.673034 Jezierska S. Claus S. Van Bogaert I. N. A. (2020). Identification and importance of mitochondrial citrate carriers and ATP citrate lyase for glycolipid production in Starmerella bombicola. Appl. Microbiology Biotechnology 104 (14), 6235–6248. 10.1007/s00253-020-10702-z 32474798 Ji Q. Mai J. Ding Y. Wei Y. Ledesma-Amaro R. Ji X. J. (2020). Improving the homologous recombination efficiency of Yarrowia lipolytica by grafting heterologous component from Saccharomyces cerevisiae . Metab. Engineering Communications 11 (e00152), e00152. 10.1016/j.mec.2020.e00152 33294367 Kurtzman C. P. Price N. P. J. Ray K. J. Kuo T. M. (2010). Production of sophorolipid biosurfactants by multiple species of the starmerella (candida) bombicola yeast clade: sophorolipids from yeasts. FEMS Microbiology Letters 311 (2), 140–146. 10.1111/j.1574-6968.2010.02082.x 20738402 Lee Y.-G. Kim C. Kuanyshev N. Kang N. K. Fatma Z. Wu Z. Y. (2022). Cas9-based metabolic engineering of Issatchenkia orientalis for enhanced utilization of cellulosic hydrolysates. J. Agricultural Food Chemistry 70 (38), 12085–12094. 10.1021/acs.jafc.2c04251 36103687 Lee J. Cornet I. De Sitter K. Noëlle Adrienne Van Bogaert I. (2025). Turning the non-pathogenic yeast Starmerella bombicola into a powerful long-chain dicarboxylic acid production host. Bioresour. Technology 419 (132006), 132006. 10.1016/j.biortech.2024.132006 39733811 Li X. Burnight E. R. Cooney A. L. Malani N. Brady T. Sander J. D. (2013). piggyBac transposase tools for genome engineering. Proc. Natl. Acad. Sci. U. S. A. 110 (25), E2279–E2287. 10.1073/pnas.1305987110 23723351 Li J. Li H. Li W. Xia C. Song X. (2016a). Identification and characterization of a flavin-containing monooxygenase MoA and its function in a specific sophorolipid molecule metabolism in Starmerella bombicola. Appl. Microbiology Biotechnology 100 (3), 1307–1318. 10.1007/s00253-015-7091-2 26512005 Li J. Xia C. Fang X. Xue H. Song X. (2016b). Identification and characterization of a long-chain fatty acid transporter in the sophorolipid-producing strain Starmerella bombicola. Appl. Microbiology Biotechnology 100 (16), 7137–7150. 10.1007/s00253-016-7580-y 27183996 Liachko I. Dunham M. J. (2014). An autonomously replicating sequence for use in a wide range of budding yeasts. FEMS Yeast Research 14 (2), 364–367. 10.1111/1567-1364.12123 24205893 Liu X. Kaas R. S. Jensen P. R. Workman M. (2012). Draft genome sequence of the yeast Pachysolen tannophilus CBS 4044/NRRL Y-2460. Eukaryot. Cell 11 (6), 827. 10.1128/EC.00114-12 22645232 Löbs A.-K. Schwartz C. Wheeldon I. (2017). Genome and metabolic engineering in non-conventional yeasts: current advances and applications. Synthetic Systems Biotechnology 2 (3), 198–207. 10.1016/j.synbio.2017.08.002 29318200 Lodens S. De Graeve M. Roelants S. L. K. W. De Maeseneire S. L. Soetaert W. (2018). Transformation of an exotic yeast species into a platform organism: a case study for engineering glycolipid production in the yeast Starmerella bombicola. Methods Molecular Biology Clift. N.J. 1772, 95–123. 10.1007/978-1-4939-7795-6_5 29754224 Lodens S. Roelants S. L. K. W. Luyten G. Geys R. Coussement P. De Maeseneire S. L. (2020). Unraveling the regulation of sophorolipid biosynthesis in Starmerella bombicola. FEMS Yeast Research 20 (3), foaa021. 10.1093/femsyr/foaa021 32329773 Maggi R. G. Govind N. S. (2004). Regulated expression of green fluorescent protein in Debaryomyces hansenii. J. Industrial Microbiology and Biotechnology 31 (7), 301–310. 10.1007/s10295-004-0150-9 15258828 Maleszka R. Wang P. Y. Schneider H. (1982). Ethanol production from d-galactose and glycerol by Pachysolen tannophilus. Enzyme Microbial Technology 4 (5), 349–352. 10.1016/0141-0229(82)90059-x Mei G.-Y. Bajwa P. K. Dashtban M. Ho C. Y. Lee H. (2018). Transfer of plasmid into the pentose-fermenting yeast Pachysolen tannophilus. J. Microbiological Methods 148, 97–103. 10.1016/j.mimet.2018.03.013 29596958 Minhas A. Biswas D. Mondal A. K. (2009). Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hansenii. FEMS Yeast Research 9 (1), 95–102. 10.1111/j.1567-1364.2008.00457.x 19076242 Moon S. Y. An N.-Y. Lee J. Y. (2025). Transforming non-conventional yeasts into key players in biotechnology: advances in synthetic biology applications. Front. Microbiology 16, 1600187. 10.3389/fmicb.2025.1600187 40384783 Mühlhausen S. Findeisen P. Plessmann U. Urlaub H. Kollmar M. (2016). A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes. Genome Res. 26 (7), 945–955. 10.1101/gr.200931.115 27197221 Navarrete C. Estrada M. Martínez J. L. (2022). Debaryomyces hansenii: an old acquaintance for a fresh start in the era of the green biotechnology. World J. Microbiol. Biotechnol. 38 (6), 99. 10.1007/s11274-022-03280-x 35482161 Nevoigt E. (2008). Progress in metabolic engineering of Saccharomyces cerevisiae . Microbiol. Molecular Biology Reviews MMBR 72 (3), 379–412. 10.1128/MMBR.00025-07 18772282 Nora L. C. Westmann C. A. Martins-Santana L. Alves L. d. F. Monteiro L. M. O. Guazzaroni M. E. (2019). The art of vector engineering: towards the construction of next-generation genetic tools. Microb. Biotechnology 12 (1), 125–147. 10.1111/1751-7915.13318 30259693 Prista C. Loureiro-Dias M. C. Montiel V. García R. Ramos J. (2005). Mechanisms underlying the halotolerant way of. FEMS Yeast Research 5 (8), 693–701. 10.1016/j.femsyr.2004.12.009 15943004 Qazi M. A. Wang Q. Dai Z. (2022). Sophorolipids bioproduction in the yeast starmerella bombicola: current trends and perspectives. Bioresour. Technology 346 (126593), 126593. 10.1016/j.biortech.2021.126593 34942344 Radecka D. Mukherjee V. Mateo R. Q. Stojiljkovic M. Foulquié-Moreno M. R. Thevelein J. M. (2015). Looking beyond saccharomyces: the potential of non-conventional yeast species for desirable traits in bioethanol fermentation. FEMS Yeast Research 15 (6), fov053. 10.1093/femsyr/fov053 26126524 Ricaurte M. L. Govind N. S. (1999). Construction of plasmid vectors and transformation of the marine yeast Debaryomyces hansenii. Mar. Biotechnology (New York, N.Y.) 1 (1), 15–19. 10.1007/pl00011745 10373605 Riley R. Haridas S. Wolfe K. H. Lopes M. R. Hittinger C. T. Göker M. (2016). Comparative genomics of biotechnologically important yeasts. Proc. Natl. Acad. Sci. U. S. A. 113 (35), 9882–9887. 10.1073/pnas.1603941113 27535936 Ronda C. Maury J. Jakočiunas T. Jacobsen S. A. B. Germann S. M. Harrison S. J. (2015). CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae . Microb. Cell Factories 14 (1), 97. 10.1186/s12934-015-0288-3 26148499 Saerens K. M. J. Saey L. Soetaert W. (2011). One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola. Biotechnol. Bioengineering 108 (12), 2923–2931. 10.1002/bit.23248 21702032 Santos C. N. S. Regitsky D. D. Yoshikuni Y. (2013). Implementation of stable and complex biological systems through recombinase-assisted genome engineering. Nat. Communications 4 (1), 2503. 10.1038/ncomms3503 24056574 Shi Y. Zhang L. Zhang M. Chu J. Xia Y. Yang H. (2022). A CRISPR-Cas9 system-mediated genetic disruption and multi-fragment assembly in Starmerella bombicola. ACS Synthetic Biology 11 (4), 1497–1509. 10.1021/acssynbio.1c00582 35294186 Slininger P. J. Bothast R. J. Van Cauwenberge J. E. Kurtzman C. P. (1982). Conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus. Biotechnol. Bioengineering 24 (2), 371–384. 10.1002/bit.260240210 18546309 Slininger P. J. Bolen P. L. Kurtzman C. P. (1987). Pachysolen tannophilus: properties and process considerations for ethanol production from d-xylose. Enzyme Microbial Technology 9 (1), 5–15. 10.1016/0141-0229(87)90043-3 Smith J. Sislak C. Fernandez Mendoza P. Carmichael L. Lewis A. Chen A. (2024). Auxotrophy-independent plasmid shuttle vectors for applications in diverse yeasts. Appl. Microbiology Basel, Switz. 4 (1), 453–469. 10.3390/applmicrobiol4010031 Soboleski M. R. Oaks J. Halford W. P. (2005). Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. FASEB Journal Official Publication Fed. Am. Soc. Exp. Biol. 19 (3), 440–442. 10.1096/fj.04-3180fje 15640280 Sohn J. H. Park H. J. Lee S. H. Bae J. H. Sung B. H. (2019). Pichia kudriavzevii NG7 microorganism and uses thereof. Available online at: https://patentimages.storage.googleapis.com/ca/a5/36/53bd36f0d2fb8b/US10443078.pdf (Accessed October 12, 2025). Spasskaya D. S. Kotlov M. I. Lekanov D. S. Tutyaeva V. V. Snezhkina A. V. Kudryavtseva A. V. (2021). CRISPR/Cas9-mediated genome engineering reveals the contribution of the 26S proteasome to the extremophilic nature of the yeast Debaryomyces hansenii. ACS Synthetic Biology 10 (2), 297–308. 10.1021/acssynbio.0c00426 33501828 Strucko T. Andersen N. L. Mahler M. R. Martínez J. L. Mortensen U. H. (2021). A CRISPR/Cas9 method facilitates efficient oligo-mediated gene editing in Debaryomyces hansenii. Synth. Biology 6 (1), ysab031. 10.1093/synbio/ysab031 34746438 Sun W. Vila-Santa A. Liu N. Prozorov T. Xie D. Faria N. T. (2020). Metabolic engineering of an acid-tolerant yeast strain Pichia kudriavzevii for itaconic acid production. Metab. Engineering Communications 10 (e00124), e00124. 10.1016/j.mec.2020.e00124 32346511 Tan S.-I. Liu Z. Tran V. G. Martin T. A. Zhao H. (2025). Issatchenkia orientalis as a platform organism for cost-effective production of organic acids. Metab. Engineering 89, 12–21. 10.1016/j.ymben.2025.02.003 39954846 Terentiev Y. Pico A. H. Böer E. Wartmann T. Klabunde J. Breuer U. (2004). A wide-range integrative yeast expression vector system based on arxula adeninivorans-derived elements. J. Industrial Microbiology and Biotechnology 31 (5), 223–228. 10.1007/s10295-004-0142-9 15175929 Tran V. G. Cao M. Fatma Z. Song X. Zhao H. (2019). Development of a CRISPR/Cas9-Based tool for gene deletion in Issatchenkia orientalis. mSphere 4 (3), e00345-19. 10.1128/mSphere.00345-19 31243078 Tran V. G. Mishra S. Bhagwat S. S. Shafaei S. Shen Y. Allen J. L. (2023). An end-to-end pipeline for succinic acid production at an industrially relevant scale using Issatchenkia orientalis. Nat. Communications 14 (1), 6152. 10.1038/s41467-023-41616-9 37788990 Van Bogaert I. N. A. De Maeseneire S. L. De Schamphelaire W. Develter D. Soetaert W. Vandamme E. J. (2007). Cloning, characterization and functionality of the orotidine-5’-phosphate decarboxylase gene (URA3) of the glycolipid-producing yeast Candida bombicola. Yeast Chichester, Engl. 24 (3), 201–208. 10.1002/yea.1448 17351910 Van Bogaert I. N. A. Holvoet K. Roelants S. L. K. W. Li B. Lin Y. C. Van de Peer Y. (2013). The biosynthetic gene cluster for sophorolipids: a biotechnological interesting biosurfactant produced by starmerella bombicola: the sophorolipid gene cluster. Mol. Microbiology 88 (3), 501–509. 10.1111/mmi.12200 23516968 Vještica A. Marek M. Nkosi P. J. Merlini L. Liu G. Bérard M. (2020). A toolbox of stable integration vectors in the fission yeast Schizosaccharomyces pombe . J. Cell Science 133 (1), jcs240754. 10.1242/jcs.240754 31801797 Wagner J. M. Alper H. S. (2016). Synthetic biology and molecular genetics in non-conventional yeasts: current tools and future advances. Fungal Genetics Biology FG and B 89, 126–136. 10.1016/j.fgb.2015.12.001 26701310 Wang H. Roelants S. L. K. W. To M. H. Patria R. D. Kaur G. Lau N. S. (2019). Starmerella bombicola: recent advances on sophorolipid production and prospects of waste stream. J. Chem. Technol. Biotechnol. 94 (4), 999–1007. 10.1002/jctb.5847 Wang Y. Wang Y. Cui J. Wu C. Yu B. Wang L. (2025). Non-conventional yeasts: promising cell factories for organic acid bioproduction. Trends Biotechnology 43 (7), 1609–1626. 10.1016/j.tibtech.2024.12.004 39799011 Wedlock D. N. James A. P. Thornton R. J. (1953). Glucose-negative mutants of Pachysolen tannophilus. Microbiology 135 (7), 2019–2026. 10.1099/00221287-135-7-2019 Wu Z.-Y. Sun W. Shen Y. Pratas J. Suthers P. F. Hsieh P. H. (2023). Metabolic engineering of low-ph-tolerant non-model yeast, Issatchenkia orientalis, for production of citramalate. Metab. Engineering Communications 16 (e00220), e00220. 10.1016/j.mec.2023.e00220 36860699 Xelhuantzi M. S. C. Ghete D. Milburn A. Ioannou S. Mudd P. Calder G. (2024). High-resolution live cell imaging to define ultrastructural and dynamic features of the halotolerant yeast Debaryomyces hansenii. Biol. Open 13 (7), bio060519. 10.1242/bio.060519 39078271 Xiao H. Shao Z. Jiang Y. Dole S. Zhao H. (2014). Exploiting Issatchenkia orientalis SD108 for succinic acid production. Microb. Cell Factories 13 (1), 121. 10.1186/s12934-014-0121-4 25159171 Yang V. W. Jeffries T. W. (1997). Regulation of phosphotransferases in glucose- and xylose-fermenting yeasts. Appl. Biochem. Biotechnol. 65 (1), 97–108. 10.1007/bf02920416 9170243 Yoo B.-H. Kim M.-D. (2015). Isolation of the inositol phosphoceramide synthase gene (AUR1) from stress-tolerant yeast Pichia kudriavzevii. J. Microbiology Biotechnology 25 (11), 1902–1907. 10.4014/jmb.1508.08019 26323269 Zhang M. Shi Y. Zhang L. Zhu S. Yang H. Shen W. (2022). A CRISPR–Cas12a system for multi-gene editing (CCMGE) and metabolic pathway assembly in Starmerella bombicola. Syst. Microbiology Biomanufacturing 2 (4), 665–675. 10.1007/s43393-022-00093-9 Zhang B. Li R. Yu L. Wu C. Liu Z. Bai F. (2023). L-Lactic acid production via sustainable neutralizer-free route by engineering acid-tolerant yeast Pichia kudriavzevii. J. Agricultural Food Chemistry 71 (29), 11131–11140. 10.1021/acs.jafc.3c03163 37439413 Zhang M.-M. Chen W. Z. Zhang L. P. Lu P. (2025). Knockout mutation to enhance sophorolipid production in Starmerella bombicola based on computational structural analysis of fatty acid oxidation enzymes. Int. Journal Biological Macromolecules 307 (Pt 4), 142104. 10.1016/j.ijbiomac.2025.142104 40112970