ABSTRACT The auxin-inducible degron (AID) technology is a convenient and powerful tool for protein functional characterization in a broad array of eukaryotic species. We recently demonstrated that the original AID and improved AID2 systems are very effective at rapid protein depletion in Candida albicans, and described a limited set of reagents for their use in certain auxotrophic lab strains. With an eye toward broader applicability with improved flexibility, we report here a new series of template vectors suitable for employing AID2 technology in prototrophic C. albicans strains, such as clinical isolates and the reference strain SC5314. We adapted a common recyclable antibiotic marker system for the required genome editing steps, and developed a strategy for simultaneous CRISPR/Cas9-mediated tagging of both target alleles. We also developed a composite all-in-one tagging cassette that combines the degron tag and the OsTIR1F74A gene for single-step strain engineering. We added a fluorescent protein tag option and designed and validated an approach for N-terminal tagging that retains natural promoter control. We also compared the effectiveness of the two commonly used synthetic auxins, 5-phenyl-indole-3-acetic acid and 5-adamantyl-indole-3-acetic acid, and the two common OsTIR1 variants, F74A and F74G, and provide guidelines for using the new AID2 system. Finally, using the novel all-in-one cassette, we demonstrate that the AID2 system also works in Candida auris, albeit less effectively under some conditions. The new reagents should enhance the convenience and accessibility of the AID2 system for the Candida research community. IMPORTANCE Invasive fungal infections, including those caused by Candida species, are a persistent global health problem, and their treatment is hindered by limited antifungal options and the emergence of drug resistance. There is an urgent need for tools and methods to accelerate the discovery of novel therapeutic targets. The expanded and optimized auxin-inducible degron system described herein provides a versatile platform for characterizing protein function and dissecting pathways governing important traits like virulence, stress tolerance, and antifungal resistance. The new reagents make AID technology applicable to any strain. Ultimately, this enhanced toolkit has the potential to help identify and validate new high‑value drug targets and deepen our understanding of molecular mechanisms that drive pathogenicity of Candida and other fungal pathogen species. Invasive fungal infections, including those caused by Candida species, are a persistent global health problem, and their treatment is hindered by limited antifungal options and the emergence of drug resistance. There is an urgent need for tools and methods to accelerate the discovery of novel therapeutic targets. The expanded and optimized auxin-inducible degron system described herein provides a versatile platform for characterizing protein function and dissecting pathways governing important traits like virulence, stress tolerance, and antifungal resistance. The new reagents make AID technology applicable to any strain. Ultimately, this enhanced toolkit has the potential to help identify and validate new high‑value drug targets and deepen our understanding of molecular mechanisms that drive pathogenicity of Candida and other fungal pathogen species.
A new series of template vectors suitable for employing AID2 technology in prototrophic C. albicans strains, such as clinical isolates and the reference strain SC5314 are reported, and it is demonstrated that the AID2 system also works in Candida auris, albeit less effectively under some conditions.
E. Danzeisen, Michelle V. Lihon, Kedric L. Milholland et al.· bioRxiv· 0 citations
Aspergillus
features numerous species with diverse characteristics, and efforts to identify the genes responsible for these traits are ongoing worldwide. Advanced multi-gene knockout technologies are required to analyze the functions of these genes. Although CRISPR/Cas9-based gene disruption methods have become widely used in many organisms, conventional homologous recombination remains the most reliable method for multiple-gene disruption in
Aspergillus
species. Here, we report the development of a novel marker recycling cassette for
Aspergillus
spp. This system enables highly efficient gene disruption and marker recycling by combining a doxycycline-inducible Cre/
loxP
system with a uracil biosynthesis pathway and counterselection using uracil analogs. Using the model filamentous fungi
Aspergillus nidulans
and the human pathogen
Aspergillus fumigatus
, we disrupted genes involved in conidial pigment biosynthesis and amino acid and vitamin biosynthesis. This system is expected to facilitate and accelerate functional genomic analyses in
Aspergillus
spp.
Genetic engineering of non-conventional yeasts is frequently limited by the lack of robust dominant selection systems that function across phylogenetically diverse hosts. These organisms are increasingly important platforms for sustainable bioproduction due to their unique metabolic capabilities.
Candida famata
, an industrial riboflavin overproducer belonging to the CTG clade with alternative codon decoding, represents a particularly relevant model for evaluating cross-species selection strategies. Many commonly used antibiotic resistance markers exhibit strong host dependence, especially in yeasts with non-standard genetic codes, thereby restricting strain construction and metabolic engineering. Aureobasidin A (AbA) is a potent antifungal compound that inhibits inositol phosphorylceramide (IPC) synthase, a key enzyme in sphingolipid biosynthesis, making it an attractive candidate for dominant selection. In this study, we evaluated the sensitivity to AbA across phylogenetically diverse yeast species and developed a resistance marker based on a codon-optimised variant of the
Saccharomyces cerevisiae AUR1
gene, designed to ensure correct translation in CTG-clade yeasts. Sensitivity assays confirmed that AbA efficiently inhibited the growth of multiple yeast species at low concentrations, supporting its use as a selective agent. The native
C. albicans AUR1
gene did not confer functional resistance in
C. famata
despite successful transformation, highlighting limitations imposed by host-dependent gene expression. In contrast, the codon-optimized
S. cerevisiae AUR1
construct restored robust resistance across the tested yeast species. Transformation yielded stable mutants with reproducible resistance phenotypes, confirmed by molecular validation and maintained through serial passaging. Importantly, the construct remained functional not only in CTG-clade yeasts but also in species using the standard genetic code. Codon usage analysis showed that replacement of CTG codons eliminated ambiguous decoding while introducing leucine codons broadly preferred among yeast species, providing a plausible explanation for the observed cross-species functionality of the
AUR1
marker. Fusion with
GFP
confirmed correct expression and intracellular localisation without detectable impact on host physiology, while fermentation experiments demonstrated that the system did not significantly affect riboflavin production under the tested conditions. Collectively, these results establish AbA as a highly effective selective agent and demonstrate that rational recoding of
AUR1*
enables the development of an efficient dominant selection marker across the yeast species evaluated in this study. This platform expands the genetic toolkit available for non-conventional yeasts and provides a versatile solution for strain engineering across multiple yeast hosts.
Dominik Wojdyla, J. Ruchała· Journal of Biological Engine...· 0 citations
In this work, selective pressure was applied in E. coli to enrich the population expressing recombinant proteins and suppress the emergence of low-expression phenotypes. To this end, plasmids were constructed containing two reporter genes, a degradable green fluorescent protein (GFP) and a stable red fluorescent protein (RFP), positioned upstream of either a gentamicin acetyltransferase gene or a d-serine deaminase gene. Flow cytometry analysis revealed that gentamicin selection prevented the formation of bimodal populations and maintained predominantly homogeneous expression profiles, accompanied by up to 12-fold and 10-fold increases in RFP and GFP fluorescence, respectively. These results suggest that selective pressure favoured the enrichment and maintenance of highly expressing phenotypes while preserving operon functionality. Transcriptomic analysis indicated extensive physiological adaptation under gentamicin selection, including changes in translation-related functions and increased transcript abundance of the genes of interest. To establish an antibiotic-free strategy, d-serine was employed as an alternative selective agent. Detoxification of d-serine by d-serine deaminase similarly reduced population heterogeneity and resulted in predominantly single-expression populations with up to 4-fold and 6-fold higher RFP and GFP fluorescence, respectively. Furthermore, d-serine was evaluated as the sole nitrogen source, combining selective pressure with an auxotrophic strategy and yielding up to 6-fold and 15-fold increases in RFP and GFP fluorescence, respectively. These findings support the use of selective pressure to reduce recombinant expression heterogeneity and suppress low-expression subpopulations. Additionally, we highlighted the potential use of d-serine and similar toxic substrates to simultaneously function as selective agents, inducers, and sources of essential metabolites through detoxification.
L. Gelain, Jing Wui Yeoh, Benjamin Shang Yong Au-Yeung et al.· Synthetic and Systems Biotec...· 0 citations
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
