The Genetic Blueprint of Cycloheximide Resistance: Analysis of 816 Yeast Species

The Genetic Blueprint of Cycloheximide Resistance: Analysis of 816 Yeast Species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Genetic Blueprint of Cycloheximide Resistance: Analysis of 816 Yeast Species Brenda D. Wingfield, Martin P.A. Coetzee, Beverley J. Wingfield, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9313202/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract We examined 816 Saccharomycotina species to identify the link between cycloheximide (CHX) resistance and amino acid changes in the ribosomal proteins eL42 and uL15. This involved determining the gene sequences (genotype) that code for these proteins in the available genomes. The results were compared with the CHX resistance reported from culture-based studies (phenotype) of these species. The correlation between culture-based studies and gene sequences (wild-type or mutant) was strong, with over 90% of the resistance accurately predicted by specific mutations. The P56Q substitution in eL42 was the most common and consistently associated with growth in cultures containing 0.1% CHX. Mutations in uL15, especially Q38L, Q38M, and Q38F, explained tolerance to 0.01% CHX in some species lacking the P56Q mutation. A total of 73 genotype–phenotype mismatches (< 10%) were detected, primarily in CHX-resistant species that lacked typical ribosomal mutations, suggesting the presence of alternative mechanisms of resistance. Eleven of the species with multiple eL42 copies encoding both resistant and sensitive variants were phenotypically CHX sensitive. This supports previous evidence that a single mutant copy of eL42 is insufficient to confer CHX resistance if the wild-type copy is also present. Codon usages at the amino acid position 56 in the putative eL42 protein and position 38 in the putative uL15 proteins were determined. The most common codon in this position for eL42 proline was CCA; a single base change can result in CAA, which codes for glutamine and confers CHX resistance at position 56 in this protein. Overall, mutations in eL42 and uL15 explained most CHX resistance in yeasts, but gene copy number and other pathways affecting uptake of the antibiotic also play roles. The extensive genotype–phenotype framework established in this study provides for accurate, genome-based prediction of CHX resistance across the Saccharomycotina, underscoring the importance of gene dosage and non-ribosomal factors in resistance. Biological sciences/Evolution Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology Figures Figure 1 Figure 2 INTRODUCTION Resistance to Cycloheximide (CHX), an antibiotic with the unique ability to inhibit translation in eukaryotic cells (Schneider-Poetsch et al. 2010 ), is a subject of considerable interest to mycologists. Although it is not commonly used for medical purposes due to its high toxicity, CHX has been extensively used in studies investigating various cellular processes, including protein synthesis (Baliga et al. 1969 ). Fungi exhibit varying degrees of sensitivity to this antibiotic, and some, such as species in the Ophiostomatales , have remarkable resistance to CHX in culture (Wingfield et al. 2022 ). Within Saccharomycotina, a subphylum encompassing a broad range of yeast species, CHX's ability to inhibit the growth of many but not all yeasts has made it an important tool for the identification and classification of Saccharomycetes (Raper, 1966 ). An intriguing aspect of CHX resistance involves the similarities and differences observed across various fungal lineages. Whiffen ( 1948 ) demonstrated that several yeasts, including human fungal pathogens, are resistant to CHX. Fergus ( 1956 ) tested numerous fungi for resistance to CHX, including many species in the Ophiostomatales. While mechanisms underlying resistance in those fungi have recently been elucidated (Wingfield et al. 2022 ), they have been identified for only a limited number of yeast species (Dehoux et al. 1993 ; Sasnauskas et al. 1992 ; Kawai et al. 1992 ; Wingfield et al. 2022 ). Nonetheless, it is well known that yeast species show considerable variation in their sensitivity to CHX in culture, and this trait has been used in taxonomic schemes for these fungi (Kurtzman et al. 2003). Consequently, understanding the molecular basis of CHX resistance in yeasts is important, both to gain fundamental insights into the biology and also due to its practical relevance in yeast taxonomy. Previous research has shown that resistance to CHX in yeasts can result from specific amino acid substitutions in the key ribosomal protein eL42 (Dehoux et al. 1993 ; Sasnauskas et al. 1992 ; Kawai et al. 1992 ). This protein is essential for ribosome function and serves as the target for CHX binding. Notably, replacing a proline (Pro) with a glutamine (Gln) at a particular position in eL42 has been linked to CHX resistance in yeasts. This is similar to the resistance mechanism observed in the Ophiostomatales (Wingfield et al. 2022 ). In addition, Shen et al. ( 2021 ) have reported several amino acid substitutions in ribosomal proteins that confer CHX resistance in Neurospora crassa . Specifically, for eL42, these include P56Q, P56L, and F58L, while for uL15, they identified two substitutions, Q38K and Q38L. The objective of this study was to examine the molecular basis of CHX resistance across the Saccharomycotina, drawing comparisons with the resistance mechanism seen in the Pezizomycotin a and especially the Ophiostomatales . We utilised the genomes published for the Saccharomycotina from the recent publication by Opulente et al. ( 2024 ). The genomic data, specifically the gene sequences of eL42 and uL15 , were then compared with culture-based CHX resistance data for Saccharomycotina available from the Westerdijk Fungal Biodiversity Institute database as well as the published literature. MATERIALS AND METHODS Yeast orders considered The Saccharomycotina consists of twelve orders (Opulente et al. 2024 ; Groenewald et al. 2023 ). These include the Alaninales, Alloascoideales, Ascoideales, Dipodascales, Lipomycetales, Phaffomycetales, Pichiales, Saccharomycetales, Saccharomycodales, Serinales, Sporopachydermiales and Trigonopsidales . For this study, only the orders considered by Opulente et al. ( 2024 ) were studied, and as such, the Alloascoideales was not included. Genomic data A genomic dataset was assembled for all the Saccharomycotina genomes using the phylogenomic framework of Opulente et al. ( 2024 ). The nucleic acid sequences were identified by first extracting the correct amino acid sequences against the locally constructed amino acid database for Saccharomycotina genomes. Amino acid sequences for uL15 from the Saccharomyces cerevisiae strain S288C (GenBank accession: NP_011412) and eL42 from the Kluyveromyces lactis ribosomal protein eL42 (GenBank accession M94988.1) according to Wingfield et al. ( 2022 ), were used as a query against the database. For this purpose, we utilised local BLASTP searches with low stringency (E ≤ 0.05). Amino acid sequences were then filtered for length, removing sequences that were exceptionally short or long. As a second filtering step, sequences were aligned using the default settings in the command-line version of MAFFT v7.520 (Katoh and Standley, 2013 ), and sequences with low similarity were removed from the dataset. The gene identification numbers for each retained amino acid sequence were tabulated and used to search the nucleotide sequence database (CDS files) of the Saccharomycota genomes for their eL42 and uL15 nucleic acid sequences. FASTA files for each predicted ribosomal protein were generated, containing nucleotide sequences from the Saccharomycotina species, and used in downstream analyses to determine the codon usage for each species and gene. To determine whether yeast species in the Saccharomycotina have the genetic potential for resistance or sensitivity to CHX, a tBLASTn (using the amino acid sequences of the protein eL42 provided above as the query sequences against the database ) analysis using NCBI was performed to confirm the presence of Gln or Pro at position 56 in the predicted eL42 sequence. If neither of these amino acids was present, the actual predicted amino acid codon was determined. An additional analysis was conducted on the predicted ribosomal protein uL15 to determine whether it contained a Gln at position 38, a transition to leucine or lysine as reported by Shen et al. ( 2021 ), or another amino acid. Codon usage Codon bias is recognised within the genetic code as being subject to evolutionary selection (Hershberg and Petrov 2008 ). The study by Zavala et al. ( 2024 ) examined codon usage in the Sacchromycotina and found that there was a general preference for A/T-ending codons. For this reason, we examined codon usage at position 56 in the predicted ribosomal protein eL42 and at position 38 in the predicted ribosomal protein uL15 (Supplementary 1). The nucleic acid sequences of these two genes were extracted from the genomes of all genera included in the study of Opulente et al. ( 2024 ). Data from culture-based studies (phenotypic data) Phenotypic data for CHX resistance in the Saccharomycotina were obtained from strains maintained in the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, the Netherlands (henceforth Westerdijk). These data were obtained from three sources: the original species description, Westerdijk using the method described by Kurtzman et al. ( 2011 ), or Westerdijk studies using microtiter plate screening. Species for which such phenotypic data were not available were excluded from this part of the study. Comparisons of genomic with culture-based data The results for both the eL42 and uL15 gene data were compared with the phenotypic data from Westerdijk. Where the genetic data from genome analyses did not correlate with the reported phenotypic CHX resistance, we reviewed the published literature for the species to assess the veracity of the Westerdijk data. Original species descriptions were examined, as well as any publications containing data regarding CHX resistance in the Saccharomycotina. RESULTS Codon usage The gene sequences for eL42 and uL15 were extracted from the genomes (supplementary 2 and 3). Not all sequences began with the expected methionine codon, and some were longer than expected, while others were shorter. It was possible to determine codon usage at positions 56 and 38 for all but one of the eL42 gene sequences and in 12 cases for uL15. Rather than utilising the actual position within the predicted gene, we used the motif sequence of the predicted protein. For eL42 and uL15, the sequences were GGQXX and AGG, respectively. Where necessary, codon usage was manually confirmed using the genome sequence in the NCBI database and BLAST analysis. In a single case for eL42, the sequence extracted (genome yHMPu5000035694 Hanseniaspora occidentalis _var_citrica) was found not to make sense. A BLAST analysis of this genome using the eL42 amino acid sequence did not yield a full gene. This sequence was thus removed from further analysis. The codon for Pro at position 56 in the eL42 gene was predominantly CCA, whilst the Gln codon was CAA in most cases (Figure 1). The codon for Gln at position 38 of the uL15 gene was CAA in all but one case (Figure 2). Comparisons of genomic with culture-based data Of the 816 species studied, 57 were predicted to have a Pro at position 56 in eL42 but showed resistance to CHX in culture studies. Twenty two species had at least one wild-type and mutant P56Q eL42 gene, and eleven of these were sensitive to CHX in culture. Eight species were predicted to have P56Q (in eL42) but had been reported as sensitive, while another eight had a mutant uL15 and were CHX sensitive. The results for the amino acid sequences at positions 56 and 38 in the predicted protein sequences of eL42 and uL15 are summarised by yeast order, with details presented in the associated tables (Tables 1-10 and supplementary Tables 1-10). Alaninales There was a perfect correlation between CHX resistance and the presence of Gln at position 56 of eL42 (Table 1). In all cases, Gln was found at position 38 in uL15. Furthermore, both genes are present as a single copy in all genomes. Based on the original description of Nakazawaea siamensis (Kaewwichian and Limtong, 2014), this species grows at 0.1% CHX, albeit only weakly. In addition, Kurtzman et al. (2011) reported that Candida anatomiae (current name Nakazawaea anatomiae ) can grow at 0.1% CHX. In both species, the Westerdijk data list the strains' sensitivity to 0.1% CHX, which could be due to the incubation time not being sufficiently long to observe the very slow or delayed growth. Ascoideales There was a strong correlation between CHX resistance and the predicted presence of the amino acid Gln at position 56 of eL42 (Table 2). In all cases, Gln was found at position 38 in uL15. Additionally, both genes were present as a single copy in all genomes. The only exception to the correlation between culture-based studies and genetic data was for Saccharomycopsis selenospora . While culture-based tests suggest that the sequenced strain of this species is sensitive to 0.1% CHX, Kurtzman et al. (2011) reported that CHX resistance was not tested. There is a second strain in the Westerdijk collection that is recorded to be resistant to 0.1% cycloheximide. Because no additional phenotypic tests have been conducted, there may be another reason why the ex-type strain has been recorded as CHX-sensitive. Dipodascales There was a correlation between CHX resistance and the presence of the amino acid Gln at position 56 of eL42 (Table 3). However, the substitution of Pro with Glu at position 56 in eL42 did not result in CHX resistance in Starmerella davenportii (Table 3). Shen et al. (2021) reported that substitutions at position 38 in uL15, specifically Q38K and Q38L, confer resistance to CHX. This transition was observed in Geotrichum cucujoidarum ; however, a Q38M substitution may also confer resistance, as seen in Blastobotrys americanus , B. muscicola , Candida kazuoi, Crinitomyces ghanaensis , Zygoascus biomembranicola and Zygoascus meyerae (Table 3). Likewise, a Q38F substitution may be associated with resistance, as seen in Spencermartinsiella ligniputridi . This suggests a broader correlation between CHX tolerance and alternative substitutions at position 38 in uL15, such as to leucine, methionine, or phenylalanine. Furthermore, details on other exceptions are provided in the supplementary material (Supplementary 4). Lipomycetales Species in Lipomycetales are typically resistant to CHX, likely due to a P56Q amino acid substitution in the predicted ribosomal protein eL42 (Table 4). There are, however, some inconsistencies in the culture-based studies. The Westerdijk data indicate that both Badjevia anomala (previously Dipodascopsis anomala ) and Myxozyma udenii are CHX-sensitive. In contrast, Müller et al. (2007), who re-examined CHX resistance in the Lipomycetaceae, showed that strain NRRL Y-17387 ( M. udenii ) is not only highly resistant to CHX but also exhibits stimulated growth in its presence. The same study also reported that strain CBS 6740 ( B. anomala ), sequenced by Opulente et al. (2024), is resistant to CHX at concentrations up to 1 g/L. Kurtzman et al. (2011) also identified this species as CHX-tolerant. The predicted amino acid sequence of eL42 and the data from Kurtzman et al. (2011) and Müller et al. (2007) differ from the Westerdijk data, and re-evaluation of this strain is required. Phaffomycetales Only four species in the Phaffomycetales are resistant to CHX, and only these species in the order have the amino acid substitution P56Q in the predicted ribosomal protein eL42 (Table 5). All other species possess a Pro at position 56. Additionally, all species have a Gln predicted at position 38 of uL15. There was consequently a clear correlation between the genotype and phenotype for species in this order, showing that gene sequences accurately predict CHX resistance. Pichiales In the majority of cases examined, CHX resistance in the Pichiales correlated with predicted protein sequences for eL42 and uL15 (Table 6). However, some strains require further investigation (Supplementary 4). Saccharomycetales In the majority of cases, resistance to CHX in Saccharomycetales, as inferred from culture-based studies, correlated with the predicted protein sequences of eL42 and uL15 (Table 7), with Pro for eL42 and Gln for uL15 predicting CHX sensitivity. However, some cases require further investigation (Supplementary 4). Saccharomycodales The genotypes of species in the Saccharomycodales match the phenotypes recorded in the Westerdijk database for all but Hanseniaspora thailandica and Hanseniaspora singularis (Table 8). In both cases, the original description by Jindamorakot et al. (2009) suggests that these species are CHX-sensitive, which is consistent with the Westerdijk database. However, the predicted protein sequence of eL42 suggests CHX resistance. Serinales In most instances, CHX resistance in the Serinales inferred from culture-based assays was consistent with predictions based on the amino acid sequences of eL42 and uL15 (Table 9). Specifically, the presence of Pro in eL42 and Gln in uL15 was associated with CHX sensitivity. Several exceptions were found, and these warrant further investigation (Supplementary 4). Sporopachydermiales and Trigonopsidales There was a strong correlation between the phenotype and the predicted amino acid sequences of eL42 and uL15 in these orders (Table 10). However, there were inconsistencies in some species that merit further study. According to Kurtzman et al. (2011), Sporopachydermia cereana does not grow on media having 0.1% CHX. This is not reflected in the Westerdijk data (where CHX resistance is suggested), and the genotypic data support CHX resistance. The original description (Nadson and Krassil’nikov 1928) of this species was published before yeast taxonomists routinely tested CHX resistance in culture. The original descriptions (Lachance and Kurtzman 2013) of Tortispora caseinolytica (formerly Candida caseinolytica ) and Tortispora mauiana report slow and low growth at 0.01 % CHX and very weak growth of Tortispora agaves and Tortispora sangerardonensis at this concentration. The slow or weak growth at 0.01% is consistent with the Westerdijk data. Lachance and Kurtzman (2013) also reported that Tortispora ganteri grows slowly at 0.001% CHX, whereas the Westerdijk data suggest no growth. The genotypic data for this species suggest that it should be able to grow in the presence of CHX. DISCUSSION Tolerance to CHX has been well recognised in fungi, for example, in the Ophiostomatales (de Beer et al. 2022 ; Wingfield et al. 2022 ), and this characteristic has been commonly used in taxonomic studies of yeasts. To gain an improved understanding of the conserved nature of these CHX resistance mechanisms, we analysed 816 yeast species across the Sachromycotina. This revealed a strong correlation between CHX resistance and specific amino acid substitutions in predicted proteins of genes eL42 and uL15 . Of these the P56Q substitution in eL42 was the most consistent marker for resistance, typically conferring growth at 0.1% CHX, whereas a P56E substitution in Starmerella davenportii resulted in a sensitive phenotype. In uL15, the Q38L, Q38M, and Q38F substitutions generally corresponded to 0.01% CHX tolerance, with occasional tolerance at 0.1%. It was notable that the Q32K transition reported by Shen et al. ( 2021 ) was absent from our dataset. Very few mismatches were found between the CHX resistance phenotype and the mutations found in the genomes of the studied species. A small number (eleven) of mismatches involved species carrying both resistant and sensitive eL42 alleles. Consistent with earlier research (Bae et al. 2003 ; Kawai et al. 1992 ), resistance is suppressed when a wild-type eL42 allele is present unless multiple mutant copies are expressed. This explains why species with two eL42 alleles—one resistant (P56Q) and one sensitive—are often recorded as CHX-sensitive in culture. Interestingly, eleven species having two eL42 alleles—one resistant (P56Q) and one sensitive—were resistant to CHX in culture. In three of these species, more than one copy of the resistance gene was present in the genome, which could explain why they have been recorded as CHX-resistant in culture. Discrepancies between genotypic and phenotypic data were cross-checked against peer-reviewed original literature. In some cases, the species descriptions did not mention CHX resistance, or the data in the various papers were conflicting. The comprehensive treatise by Kurtzman et al. ( 2011 ) was particularly useful in determining CHX resistance. It is important to consider that data suggesting slow, low, or delayed growth patterns can lead to variation across studies for the same species, as only small differences in incubation conditions, such as incubation time and growth medium, and the age of cultures could yield different outcomes. In addition, tests for CHX resistance in culture have typically been conducted on one or only a few isolates of a species and consequently fail to take population genetic differences into consideration. In a few instances, the Westerdijk strains representing these species need to be re-evaluated for growth in 0.01% and/or 0.1% CHX, as indicated in the tables (Table 1 – 10 and supplementary Tables – 15 cases). The majority of species resistant to CHX had the P56Q predicted mutation in the eL42 gene; however, some species were resistant due to a mutation in the uL15 gene. Substitutions Q38L and Q38K in the uL15 gene were reported by Shen et al. ( 2021 ) to confer CHX resistance. The Q38L mutation is thus supported by our results. In contrast, we did not observe the Q38K mutation in the uL15 amino acid sequence. We also found that the Q38M and Q38F substitutions likely confer CHX resistance. In some cases, this resistance is evident only at lower CHX concentrations. Some CHX-resistant species lacked ribosomal gene changes altogether, suggesting alternative mechanisms of CHX resistance. One example among yeasts is the Agp2 permease pathway (Mohanty et al. 2023 ), in which deletion of AGP2 confers resistance by preventing CHX uptake. Agp2 is rapidly degraded upon CHX exposure in a Brp1-dependent manner, providing a plausible model for non-ribosomal resistance in a small number of taxa. We extracted the codons from all the genomes described by Opulente et al. ( 2024 ) at amino acid positions 56 and 38 in eL42 and uL15. For the majority of the species with a Pro in position 56 in eL42, the most common codon is CCA (548 species). What makes this particularly interesting is that a single base mutation at this codon (CCA to CAA) can result in the substitution of Gln at this position and the potential to confer resistance to CHX. More than 50% of the species with a Gln at this position have the codon CAA. Over evolutionary time, switching from CHX resistance to CHX sensitivity has occurred, thereby maintaining the use of these codons in these species. In the interesting case of the Ophiostomatales , CHX tolerance can be fully explained by a single, conserved P56Q substitution in ribosomal protein eL42 (Wingfield et al. 2022 ). No evidence was found for uL15 changes or alternative resistance mechanisms in that Order of fungi. This is in contrast to the Saccharomycotina considered in this study, where CHX resistance in some species can be explained by a mutation in the uL15 gene. In other cases, species that display resistance to CHX in culture lack ribosomal resistance markers. They most likely rely on other mechanisms, such as altered antibiotic uptake and protein turnover, or Agp2-mediated regulation of uptake (Gerlinger et al. 1997 , Culakova et al. 2013 , Harris et al. 2021 , Mohanty et al. 2023 ). Taken collectively, the results of this study demonstrate that cycloheximide resistance across the Saccharomycotina is largely predictable from specific mutations in ribosomal proteins, particularly the P56Q substitution in eL42 and, in some taxa, substitutions at position 38 of uL15. The strong concordance between genotype and phenotype across 816 species indicates that CHX resistance is primarily governed by conserved molecular mechanisms affecting the ribosome. The few exceptions observed highlight the biological complexity of antifungal tolerance, revealing that additional mechanisms, such as altered antibiotic uptake, gene copy number variation, or permease-mediated regulation, can contribute to resistance in a minority of taxa. The codon-level patterns observed in eL42 further suggest that simple single-nucleotide changes may facilitate rapid evolutionary transitions between sensitivity and resistance, providing a plausible explanation for the widespread yet uneven distribution of CHX tolerance across the yeasts. These findings not only clarify the molecular basis of a trait widely used in yeast taxonomy but also illustrate how ribosomal protein evolution, gene dosage, and cellular transport pathways interact to shape antifungal tolerance across large evolutionary scales. Declarations Ethics, Consent to Participate, and Consent to Publish declarations: not applicable. Funding for this study was provided by the DSTI/NRF SARChI chair in Fungal Genomics. Author Contribution BDW led the study, writing the main manuscript and conducting the literature review. MPAC extracted gene sequences from the genomes using a bioinformatic pipeline. BJW prepared Figures 1–2 and performed the codon usage analyses in Excel. CP and MG contributed critical expertise in taxonomy and a broader understanding of Saccharomycotina. MJW provided substantial input on the structure and refinement of the manuscript, contributing to multiple rounds of revision. All authors reviewed and approved the final version of the manuscript. Data Availability We utilised the genomes published for the Saccharomycotina from the recent publication by Opulente et al. (2024). The genome sequence data is freely available on in GenBank. References Bae, J.H., Sohn, J.H., Park, C.S., Rhee, J.S. and Choi, E.S., 2003. Integrative transformation system for the metabolic engineering of the sphingoid base-producing yeast Pichia ciferrii . Applied and Environmental Microbiology , 69 (2), pp.812-819. Baliga, B.S., Pronczuk, A.W. and Munro, H.N., 1969. Mechanism of cycloheximide inhibition of protein synthesis in a cell-free system prepared from rat liver. Journal of Biological Chemistry , 244 (16), pp.4480-4489. 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The yeast genus Tortispora gen. nov., description of Tortispora ganteri sp. nov., Tortispora mauiana f.a., sp. nov., Tortispora agaves f.a., sp. nov., Tortispora sangerardonensis f.a., sp. nov., Tortispora cuajiniquilana f.a., sp. nov., Tortispora starmeri f.a., sp. nov. and Tortispora phaffii f.a., sp. nov., reassignment of Candida caseinolytica to Tortispora caseinolytica f.a., comb. nov., emendation of Botryozyma , and assignment of Botryozyma, Tortispora gen. nov. and Trigonopsis to the family Trigonopsidaceae fam. nov. International Journal of Systematic and Evolutionary Microbiology , 63 (Pt_8), pp.3104-3114. Limtong, S., Kaewwichian, R., Am-In, S., Nakase, T., Lee, C.F. and Yongmanitchai, W., 2010. Candida asiatica sp. nov., an anamorphic ascomycetous yeast species isolated from natural samples from Thailand, Taiwan, and Japan. Antonie van Leeuwenhoek , 98 (4), pp.475-481. Limtong, S., Nitiyon, S., Kaewwichian, R., Jindamorakot, S., Am-In, S. and Yongmanitchai, W., 2012. Wickerhamomyces xylosica sp. nov. and Candida phayaonensis sp. nov., two xylose-assimilating yeast species from soil. International Journal of Systematic and Evolutionary Microbiology , 62 (Pt_11), pp.2786-2792. Mohanty, A., Alhaj Sulaiman, A., Moovarkumudalvan, B., Ali, R., Aouida, M. and Ramotar, D., 2023. The yeast permease Agp2 senses cycloheximide and undergoes degradation that requires the small protein brp1-cellular fate of Agp2 in response to cycloheximide. International Journal of Molecular Sciences , 24 (8), p.6975. Müller, W.J., Albertyn, J. and Smit, M.S., 2007. Cycloheximide resistance in the Lipomycetaceae revisited. Canadian Journal of Microbiology , 53 (4), pp. 509-513. Nadson, G. and Krassil’inkov, N., 1928.Un nouveau genre d'Endomycétacées: Guilliermondella , nov. gen. Comptes Rendus Hebdomadaires des Séances de l'Académie , 187 , pp.307-309. Naganishi, H., 1917. Three new species of yeasts. Botanical Magazine (Tokyo), 31 , pp.107-115. Nagatsuka, Y., Saito, S. and Sugiyama, J., 2008. Ogataea neopini sp. nov. and O. corticis sp. nov., with the emendation of the ascomycete yeast genus Ogataea, and transfer of Pichia zsoltii, P. dorogensis , and P. trehaloabstinens to it. The Journal of General and Applied Microbiology , 54 (6), pp.353-365 Nakase, T., Jindamorakot, S., Am-In, S., Lee, C.F. and Limtong, S., 2011. Four novel species of the anamorphic yeast genus Candida found in Thailand and Taiwan. The Journal of General and Applied Microbiology , 57 (4), pp.231-242. Nouri, H., Moghimi, H., Geranpayeh Vaghei, M. and Nasr, S., 2018. Blastobotrys persicus sp. nov., an ascomycetous yeast species isolated from cave soil. Antonie van Leeuwenhoek , 111 (4), pp.517-524. Opulente, D.A., LaBella, A.L., Harrison, M.C., Wolters, J.F., Liu, C., Li, Y., Kominek, J., Steenwyk, J.L., Stoneman, H.R., VanDenAvond, J. and Miller, C.R., 2024. Genomic factors shape carbon and nitrogen metabolic niche breadth across Saccharomycotina yeasts. Science , 384 (6694), p.eadj4503. Pereira, L.F., Costa Jr, C.R.L., Brasileiro, B.T.R.V. and de Morais Jr, M.A., 2011. Lachancea mirantina sp. nov., an ascomycetous yeast isolated from the cachaca fermentation process. International Journal of Systematic and Evolutionary Microbiology , 61 (4), pp.989-992. Raper, K.B., 1966. Antagonistic action of cycloheximide on filamentous fungi and yeast. Bacteriological Reviews , 30(1), pp1-17. Ruivo, C.C., Lachance, M.A., Rosa, C.A., Bacci Jr, M. and Pagnocca, F.C., 2006. Candida heliconiae sp. nov., Candida picinguabensis sp. nov. and Candida saopaulonensis sp. nov., three ascomycetous yeasts from Heliconia velloziana (Heliconiaceae). International Journal of Systematic and Evolutionary Microbiology , 56 (5), pp.1147-1151. Sasnauskas, K., Jomantienė, R., Lebedienė, E., Lebedys, J., Janusˇka, A. and Janulaitis, A., 1992. Cloning and sequence analysis of a Candida maltosa gene which confers resistance to cycloheximide. Gene , 116 (1), pp.105-108. Schneider-Poetsch, T., Ju, J., Eyler, D.E., Dang, Y., Bhat, S., Merrick, W.C., Green, R., Shen, B. and Liu, J.O., 2010. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nature Chemical Biology , 6 (3), pp.209-217. Shen, L., Su, Z., Yang, K., Wu, C., Becker, T., Bell-Pedersen, D., Zhang, J. and Sachs, M.S., 2021. Structure of the translating Neurospora ribosome arrested by cycloheximide. Proceedings of the National Academy of Sciences , 118 (48), p.e2111862118. Steensels, J., Daenen, L., Malcorps, P., Derdelinckx, G., Verachtert, H. and Verstrepen, K.J., 2015. Brettanomyces yeasts—From spoilage organisms to valuable contributors to industrial fermentations. International Journal of Food Microbiology , 206 , pp.24-38. Suh, S.O. and Blackwell, M., 2005. Four new yeasts in the Candida mesenterica clade associated with basidiocarp-feeding beetles. Mycologia , 97 (1), pp.167-177. Van der Walt, J.P., Johannsen, E., Opperman, A. and Halland, L., 1986. Kluyveromyces yarrowii sp. nov., a haploid, heterothallic, arboreal species. Systematic and Applied Microbiology , 8 (3), pp.208-212. Vidal-Leiria, M., 1966. Torulopsis vanderwaltii sp. n. Antonie van Leeuwenhoek , 32 (1), pp.447-449. Whiffen, A.J., 1948. The production, assay, and antibiotic activity of actidione, an antibiotic from Streptomyces griseus . Journal of Bacteriology , 56 (3), pp.283-291. Wickerham, L.J., 1969. New homothallic taxa of Hansenula . Mycopathologia et Mycologia Applicata , 37 (1), pp.15-32. Wingfield, B.D., Wingfield, M.J. and Duong, T.A., 2022. Molecular basis of cycloheximide resistance in the Ophiostomatales revealed. Current Genetics , 68 (3), pp.505-514. Zavala, B., Dineen, L., Fisher, K.J., Opulente, D.A., Harrison, M.C., Wolters, J.F., Shen, X.X., Zhou, X., Groenewald, M., Hittinger, C.T. and Rokas, A., 2024. Genomic factors shaping codon usage across the Saccharomycotina subphylum. G3: Genes, Genomes, Genetics , 14(11), p.jkae207. Tables Tables 1 to 10 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files summarisedtablesrevised.docx Supplementaryfile1.docx Supplementary2nucleicacidsequencesEL42.xlsx Supplementary3uL15L28sequences.xlsx Supplementary4resultsyeasts.docx Supplementarytablesyeastms.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 12 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers invited by journal 20 Apr, 2026 Editor invited by journal 09 Apr, 2026 Editor assigned by journal 04 Apr, 2026 Submission checks completed at journal 04 Apr, 2026 First submitted to journal 03 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Wingfield","email":"data:image/png;base64,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","orcid":"","institution":"University of Pretoria","correspondingAuthor":true,"prefix":"","firstName":"Brenda","middleName":"D.","lastName":"Wingfield","suffix":""},{"id":628639632,"identity":"45d125fa-50aa-4f12-939d-f09b145e4484","order_by":1,"name":"Martin P.A. 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Wingfield","email":"","orcid":"","institution":"University of Pretoria","correspondingAuthor":false,"prefix":"","firstName":"Beverley","middleName":"J.","lastName":"Wingfield","suffix":""},{"id":628639634,"identity":"2ea43701-1999-4bda-83ee-8863f8982732","order_by":3,"name":"Marizeth Groenewald","email":"","orcid":"","institution":"Westerdijk Fungal Biodiversity Institute","correspondingAuthor":false,"prefix":"","firstName":"Marizeth","middleName":"","lastName":"Groenewald","suffix":""},{"id":628639635,"identity":"7d6242ea-c43d-47b0-9822-16681d613af6","order_by":4,"name":"Carolina Pohl","email":"","orcid":"","institution":"University of the Free State","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Pohl","suffix":""},{"id":628639636,"identity":"5905f5ad-9c50-4ecc-94b4-c461af93020f","order_by":5,"name":"Michael J. 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Although it is not commonly used for medical purposes due to its high toxicity, CHX has been extensively used in studies investigating various cellular processes, including protein synthesis (Baliga et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1969\u003c/span\u003e). Fungi exhibit varying degrees of sensitivity to this antibiotic, and some, such as species in the \u003cem\u003eOphiostomatales\u003c/em\u003e, have remarkable resistance to CHX in culture (Wingfield et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Within Saccharomycotina, a subphylum encompassing a broad range of yeast species, CHX's ability to inhibit the growth of many but not all yeasts has made it an important tool for the identification and classification of Saccharomycetes (Raper, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1966\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn intriguing aspect of CHX resistance involves the similarities and differences observed across various fungal lineages. Whiffen (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1948\u003c/span\u003e) demonstrated that several yeasts, including human fungal pathogens, are resistant to CHX. Fergus (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1956\u003c/span\u003e) tested numerous fungi for resistance to CHX, including many species in the Ophiostomatales. While mechanisms underlying resistance in those fungi have recently been elucidated (Wingfield et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), they have been identified for only a limited number of yeast species (Dehoux et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Sasnauskas et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Kawai et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Wingfield et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nonetheless, it is well known that yeast species show considerable variation in their sensitivity to CHX in culture, and this trait has been used in taxonomic schemes for these fungi (Kurtzman et al. 2003). Consequently, understanding the molecular basis of CHX resistance in yeasts is important, both to gain fundamental insights into the biology and also due to its practical relevance in yeast taxonomy.\u003c/p\u003e \u003cp\u003ePrevious research has shown that resistance to CHX in yeasts can result from specific amino acid substitutions in the key ribosomal protein eL42 (Dehoux et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Sasnauskas et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Kawai et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). This protein is essential for ribosome function and serves as the target for CHX binding. Notably, replacing a proline (Pro) with a glutamine (Gln) at a particular position in eL42 has been linked to CHX resistance in yeasts. This is similar to the resistance mechanism observed in the \u003cem\u003eOphiostomatales\u003c/em\u003e (Wingfield et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, Shen et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have reported several amino acid substitutions in ribosomal proteins that confer CHX resistance in \u003cem\u003eNeurospora crassa\u003c/em\u003e. Specifically, for eL42, these include P56Q, P56L, and F58L, while for uL15, they identified two substitutions, Q38K and Q38L.\u003c/p\u003e \u003cp\u003eThe objective of this study was to examine the molecular basis of CHX resistance across the Saccharomycotina, drawing comparisons with the resistance mechanism seen in the Pezizomycotin\u003cem\u003ea\u003c/em\u003e and especially the \u003cem\u003eOphiostomatales\u003c/em\u003e. We utilised the genomes published for the Saccharomycotina from the recent publication by Opulente et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The genomic data, specifically the gene sequences of \u003cem\u003eeL42\u003c/em\u003e and \u003cem\u003euL15\u003c/em\u003e, were then compared with culture-based CHX resistance data for Saccharomycotina available from the Westerdijk Fungal Biodiversity Institute database as well as the published literature.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eYeast orders considered\u003c/h2\u003e \u003cp\u003eThe Saccharomycotina consists of twelve orders (Opulente et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Groenewald et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These include the \u003cem\u003eAlaninales, Alloascoideales, Ascoideales, Dipodascales, Lipomycetales, Phaffomycetales, Pichiales, Saccharomycetales, Saccharomycodales, Serinales, Sporopachydermiales\u003c/em\u003e and \u003cem\u003eTrigonopsidales\u003c/em\u003e. For this study, only the orders considered by Opulente et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) were studied, and as such, the \u003cem\u003eAlloascoideales\u003c/em\u003e was not included.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenomic data\u003c/h3\u003e\n\u003cp\u003eA genomic dataset was assembled for all the Saccharomycotina genomes using the phylogenomic framework of Opulente et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The nucleic acid sequences were identified by first extracting the correct amino acid sequences against the locally constructed amino acid database for Saccharomycotina genomes. Amino acid sequences for uL15 from the \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain S288C (GenBank accession: NP_011412) and eL42 from the \u003cem\u003eKluyveromyces lactis\u003c/em\u003e ribosomal protein eL42 (GenBank accession M94988.1) according to Wingfield et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), were used as a query against the database. For this purpose, we utilised local BLASTP searches with low stringency (E \u0026le; 0.05). Amino acid sequences were then filtered for length, removing sequences that were exceptionally short or long. As a second filtering step, sequences were aligned using the default settings in the command-line version of MAFFT v7.520 (Katoh and Standley, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and sequences with low similarity were removed from the dataset. The gene identification numbers for each retained amino acid sequence were tabulated and used to search the nucleotide sequence database (CDS files) of the Saccharomycota genomes for their eL42 and uL15 nucleic acid sequences. FASTA files for each predicted ribosomal protein were generated, containing nucleotide sequences from the Saccharomycotina species, and used in downstream analyses to determine the codon usage for each species and gene.\u003c/p\u003e \u003cp\u003eTo determine whether yeast species in the Saccharomycotina have the genetic potential for resistance or sensitivity to CHX, a tBLASTn (using the amino acid sequences of the protein eL42 provided above as the query sequences against the database ) analysis using NCBI was performed to confirm the presence of Gln or Pro at position 56 in the predicted eL42 sequence. If neither of these amino acids was present, the actual predicted amino acid codon was determined. An additional analysis was conducted on the predicted ribosomal protein uL15 to determine whether it contained a Gln at position 38, a transition to leucine or lysine as reported by Shen et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), or another amino acid.\u003c/p\u003e\n\u003ch3\u003eCodon usage\u003c/h3\u003e\n\u003cp\u003eCodon bias is recognised within the genetic code as being subject to evolutionary selection (Hershberg and Petrov \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The study by Zavala et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) examined codon usage in the Sacchromycotina and found that there was a general preference for A/T-ending codons. For this reason, we examined codon usage at position 56 in the predicted ribosomal protein eL42 and at position 38 in the predicted ribosomal protein uL15 (Supplementary 1). The nucleic acid sequences of these two genes were extracted from the genomes of all genera included in the study of Opulente et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eData from culture-based studies (phenotypic data)\u003c/h3\u003e\n\u003cp\u003ePhenotypic data for CHX resistance in the Saccharomycotina were obtained from strains maintained in the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, the Netherlands (henceforth Westerdijk). These data were obtained from three sources: the original species description, Westerdijk using the method described by Kurtzman et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), or Westerdijk studies using microtiter plate screening. Species for which such phenotypic data were not available were excluded from this part of the study.\u003c/p\u003e\n\u003ch3\u003eComparisons of genomic with culture-based data \u003c/h3\u003e\n\u003cp\u003eThe results for both the \u003cem\u003eeL42\u003c/em\u003e and \u003cem\u003euL15\u003c/em\u003e gene data were compared with the phenotypic data from Westerdijk. Where the genetic data from genome analyses did not correlate with the reported phenotypic CHX resistance, we reviewed the published literature for the species to assess the veracity of the Westerdijk data. Original species descriptions were examined, as well as any publications containing data regarding CHX resistance in the Saccharomycotina.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eCodon usage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene sequences for \u003cem\u003eeL42\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;uL15\u003c/em\u003e were extracted from the genomes (supplementary 2 and 3). Not all sequences began with the expected methionine codon, and some were longer than expected, while others were shorter. It was possible to determine codon usage at positions 56 and 38 for all but one of the eL42 gene sequences and in 12 cases for uL15. Rather than utilising the actual position within the predicted gene, we used the motif sequence of the predicted protein. For eL42 and uL15, the sequences were GGQXX and AGG, respectively. Where necessary, codon usage was manually confirmed using the genome sequence in the NCBI database and BLAST analysis.\u003c/p\u003e\n\u003cp\u003eIn a single case for eL42, the sequence extracted (genome yHMPu5000035694 \u003cem\u003eHanseniaspora occidentalis\u003c/em\u003e_var_citrica) was found not to make sense. A BLAST analysis of this genome using the eL42 amino acid sequence did not yield a full gene. This sequence was thus removed from further analysis.\u003c/p\u003e\n\u003cp\u003eThe codon for Pro at position 56 in the \u003cem\u003eeL42\u003c/em\u003e gene was predominantly CCA, whilst the Gln codon was CAA in most cases (Figure 1). The codon for Gln at position 38 of the \u003cem\u003euL15\u003c/em\u003e gene was CAA in all but one case (Figure 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparisons of genomic with culture-based data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOf the 816 species studied, 57 were predicted to have a Pro at position 56 in eL42 but showed resistance to CHX in culture studies. Twenty two species had at least one wild-type and mutant P56Q \u003cem\u003eeL42\u003c/em\u003e gene, and eleven of these were sensitive to CHX in culture. Eight species were predicted to have P56Q (in eL42) but had been reported as sensitive, while another eight had a mutant uL15 and were CHX sensitive.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results for the amino acid sequences at positions 56 and 38 in the predicted protein sequences of eL42 and uL15 are summarised by yeast order, with details presented in the associated tables (Tables 1-10 and supplementary Tables 1-10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAlaninales\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a perfect correlation between CHX resistance and the presence of Gln at position 56 of eL42 (Table 1). In all cases, Gln was found at position 38 in uL15. Furthermore, both genes are present as a single copy in all genomes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the original description of \u003cem\u003eNakazawaea siamensis\u0026nbsp;\u003c/em\u003e(Kaewwichian and Limtong, 2014), this species grows at 0.1% CHX, albeit only weakly. In addition, Kurtzman et al. (2011) reported that \u003cem\u003eCandida anatomiae\u0026nbsp;\u003c/em\u003e(current name \u003cem\u003eNakazawaea anatomiae\u003c/em\u003e) can grow at 0.1% CHX. In both species, the Westerdijk data list the strains\u0026apos; sensitivity to 0.1% CHX, which could be due to the incubation time not being sufficiently long to observe the very slow or delayed growth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAscoideales\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a strong correlation between CHX resistance and the predicted presence of the amino acid Gln at position 56 of eL42 (Table 2). In all cases, Gln was found at position 38 in uL15. Additionally, both genes were present as a single copy in all genomes. The only exception to the correlation between culture-based studies and genetic data was for \u003cem\u003eSaccharomycopsis selenospora\u003c/em\u003e. While culture-based tests suggest that the sequenced strain of this species is sensitive to 0.1% CHX, Kurtzman et al. (2011) reported that CHX resistance was not tested. There is a second strain in the Westerdijk collection that is recorded to be resistant to 0.1% cycloheximide. Because no additional phenotypic tests have been conducted, there may be another reason why the ex-type strain has been recorded as CHX-sensitive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDipodascales\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a correlation between CHX resistance and the presence of the amino acid Gln at position 56 of eL42 (Table 3). However, the substitution of Pro with Glu at position 56 in eL42 did not result in CHX resistance in \u003cem\u003eStarmerella davenportii\u003c/em\u003e (Table 3). Shen et al. (2021) reported that substitutions at position 38 in uL15, specifically Q38K and Q38L, confer resistance to CHX. This transition was observed in \u003cem\u003eGeotrichum cucujoidarum\u003c/em\u003e\u003cem\u003e;\u0026nbsp;\u003c/em\u003ehowever, a Q38M substitution may also confer resistance, as seen in \u003cem\u003eBlastobotrys americanus\u003c/em\u003e, \u003cem\u003eB. muscicola\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Candida kazuoi,\u003c/em\u003e \u003cem\u003eCrinitomyces ghanaensis\u003c/em\u003e, \u003cem\u003eZygoascus biomembranicola\u003c/em\u003e and \u003cem\u003eZygoascus meyerae\u003c/em\u003e (Table 3). Likewise, a Q38F substitution may be associated with resistance, as seen in \u003cem\u003eSpencermartinsiella ligniputridi\u003c/em\u003e. This suggests a broader correlation between CHX tolerance and alternative substitutions at position 38 in uL15, such as to leucine, methionine, or phenylalanine. Furthermore, details on other exceptions are provided in the supplementary material (Supplementary 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLipomycetales\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecies in \u003cem\u003eLipomycetales\u003c/em\u003e are typically resistant to CHX, likely due to a P56Q amino acid substitution in the predicted ribosomal protein eL42 (Table 4). There are, however, some inconsistencies in the culture-based studies. The Westerdijk data indicate that both \u003cem\u003eBadjevia anomala\u0026nbsp;\u003c/em\u003e(previously\u003cem\u003e\u0026nbsp;Dipodascopsis anomala\u003c/em\u003e) and\u003cem\u003e\u0026nbsp;Myxozyma\u003c/em\u003e \u003cem\u003eudenii\u003c/em\u003e are CHX-sensitive. In contrast, M\u0026uuml;ller et al. (2007), who re-examined CHX resistance in the \u003cem\u003eLipomycetaceae,\u0026nbsp;\u003c/em\u003eshowed that strain NRRL Y-17387 (\u003cem\u003eM.\u003c/em\u003e \u003cem\u003eudenii\u003c/em\u003e) is not only highly resistant to CHX but also exhibits stimulated growth in its presence. The same study also reported that strain CBS 6740 (\u003cem\u003eB. anomala\u003c/em\u003e), sequenced by Opulente et al. (2024), is resistant to CHX at concentrations up to 1 g/L. Kurtzman et al. (2011) also identified this species as CHX-tolerant. The predicted amino acid sequence of eL42 and the data from Kurtzman et al. (2011) and M\u0026uuml;ller et al. (2007) differ from the Westerdijk data, and re-evaluation of this strain is required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhaffomycetales\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnly four species in the \u003cem\u003ePhaffomycetales\u003c/em\u003e are resistant to CHX, and only these species in the order have the amino acid substitution P56Q in the predicted ribosomal protein eL42 (Table 5). All other species possess a Pro at position 56. Additionally, all species have a Gln predicted at position 38 of uL15. There was consequently a clear correlation between the genotype and phenotype for species in this order, showing that gene sequences accurately predict CHX resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePichiales\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the majority of cases examined, CHX resistance in the \u003cem\u003ePichiales\u003c/em\u003e correlated with predicted protein sequences for eL42 and uL15 (Table 6). However, some strains require further investigation (Supplementary 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSaccharomycetales\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the majority of cases, resistance to CHX in \u003cem\u003eSaccharomycetales,\u003c/em\u003e as inferred from culture-based studies, correlated with the predicted protein sequences of eL42 and uL15 (Table 7), with Pro for eL42 and Gln for uL15 predicting CHX sensitivity. However, some cases require further investigation (Supplementary 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSaccharomycodales\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genotypes of species in the \u003cem\u003eSaccharomycodales\u003c/em\u003e match the phenotypes recorded in the Westerdijk database for all but \u003cem\u003eHanseniaspora thailandica\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Hanseniaspora singularis\u0026nbsp;\u003c/em\u003e(Table 8). In both cases, the original description by\u003cem\u003e\u0026nbsp;\u003c/em\u003eJindamorakot et al. (2009) suggests that these species are CHX-sensitive, which is consistent with the Westerdijk database. However, the predicted protein sequence of eL42 suggests CHX resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSerinales\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn most instances, CHX resistance in the \u003cem\u003eSerinales\u003c/em\u003e inferred from culture-based assays was consistent with predictions based on the amino acid sequences of eL42 and uL15 (Table 9). Specifically, the presence of Pro in eL42 and Gln in uL15 was associated with CHX sensitivity. Several exceptions were found, and these warrant further investigation (Supplementary 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSporopachydermiales\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and \u003cem\u003eTrigonopsidales\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a strong correlation between the phenotype and the predicted amino acid sequences of eL42 and uL15 in these orders (Table 10). However, there were inconsistencies in some species that merit further study. According to Kurtzman et al. (2011),\u0026nbsp;\u003cem\u003eSporopachydermia\u003c/em\u003e \u003cem\u003ecereana\u003c/em\u003e does not grow on media having 0.1% CHX. This is not reflected in the Westerdijk data (where CHX resistance is suggested), and the genotypic data support CHX resistance. The original description (Nadson and Krassil\u0026rsquo;nikov 1928) of this species was published before yeast taxonomists routinely tested CHX resistance in culture. The original descriptions (Lachance and Kurtzman 2013) of\u0026nbsp;\u003cem\u003eTortispora caseinolytica\u0026nbsp;\u003c/em\u003e(formerly\u003cem\u003e\u0026nbsp;Candida caseinolytica\u003c/em\u003e)\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Tortispora mauiana\u003c/em\u003e report slow and low growth at 0.01 % CHX and very weak growth of \u003cem\u003eTortispora agaves\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Tortispora sangerardonensis\u003c/em\u003e at this concentration. The slow or weak growth at 0.01% is consistent with the Westerdijk data.\u0026nbsp;Lachance and Kurtzman (2013)\u0026nbsp;also reported that \u003cem\u003eTortispora ganteri\u003c/em\u003e grows slowly at 0.001% CHX, whereas the Westerdijk data suggest no growth. The genotypic data for this species suggest that it should be able to grow in the presence of CHX.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eTolerance to CHX has been well recognised in fungi, for example, in the Ophiostomatales (de Beer et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wingfield et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and this characteristic has been commonly used in taxonomic studies of yeasts. To gain an improved understanding of the conserved nature of these CHX resistance mechanisms, we analysed 816 yeast species across the Sachromycotina. This revealed a strong correlation between CHX resistance and specific amino acid substitutions in predicted proteins of genes \u003cem\u003eeL42\u003c/em\u003e and \u003cem\u003euL15\u003c/em\u003e. Of these the P56Q substitution in eL42 was the most consistent marker for resistance, typically conferring growth at 0.1% CHX, whereas a P56E substitution in \u003cem\u003eStarmerella davenportii\u003c/em\u003e resulted in a sensitive phenotype. In uL15, the Q38L, Q38M, and Q38F substitutions generally corresponded to 0.01% CHX tolerance, with occasional tolerance at 0.1%. It was notable that the Q32K transition reported by Shen et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was absent from our dataset.\u003c/p\u003e \u003cp\u003eVery few mismatches were found between the CHX resistance phenotype and the mutations found in the genomes of the studied species. A small number (eleven) of mismatches involved species carrying both resistant and sensitive eL42 alleles. Consistent with earlier research (Bae et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Kawai et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), resistance is suppressed when a wild-type eL42 allele is present unless multiple mutant copies are expressed. This explains why species with two eL42 alleles\u0026mdash;one resistant (P56Q) and one sensitive\u0026mdash;are often recorded as CHX-sensitive in culture. Interestingly, eleven species having two eL42 alleles\u0026mdash;one resistant (P56Q) and one sensitive\u0026mdash;were resistant to CHX in culture. In three of these species, more than one copy of the resistance gene was present in the genome, which could explain why they have been recorded as CHX-resistant in culture.\u003c/p\u003e \u003cp\u003eDiscrepancies between genotypic and phenotypic data were cross-checked against peer-reviewed original literature. In some cases, the species descriptions did not mention CHX resistance, or the data in the various papers were conflicting. The comprehensive treatise by Kurtzman et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) was particularly useful in determining CHX resistance. It is important to consider that data suggesting slow, low, or delayed growth patterns can lead to variation across studies for the same species, as only small differences in incubation conditions, such as incubation time and growth medium, and the age of cultures could yield different outcomes. In addition, tests for CHX resistance in culture have typically been conducted on one or only a few isolates of a species and consequently fail to take population genetic differences into consideration. In a few instances, the Westerdijk strains representing these species need to be re-evaluated for growth in 0.01% and/or 0.1% CHX, as indicated in the tables (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e10\u003c/span\u003e and supplementary Tables \u0026ndash; 15 cases).\u003c/p\u003e \u003cp\u003eThe majority of species resistant to CHX had the P56Q predicted mutation in the \u003cem\u003eeL42\u003c/em\u003e gene; however, some species were resistant due to a mutation in the \u003cem\u003euL15\u003c/em\u003e gene. Substitutions Q38L and Q38K in the uL15 gene were reported by Shen et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to confer CHX resistance. The Q38L mutation is thus supported by our results. In contrast, we did not observe the Q38K mutation in the uL15 amino acid sequence. We also found that the Q38M and Q38F substitutions likely confer CHX resistance. In some cases, this resistance is evident only at lower CHX concentrations.\u003c/p\u003e \u003cp\u003eSome CHX-resistant species lacked ribosomal gene changes altogether, suggesting alternative mechanisms of CHX resistance. One example among yeasts is the Agp2 permease pathway (Mohanty et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), in which deletion of \u003cem\u003eAGP2\u003c/em\u003e confers resistance by preventing CHX uptake. Agp2 is rapidly degraded upon CHX exposure in a Brp1-dependent manner, providing a plausible model for non-ribosomal resistance in a small number of taxa.\u003c/p\u003e \u003cp\u003eWe extracted the codons from all the genomes described by Opulente et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) at amino acid positions 56 and 38 in eL42 and uL15. For the majority of the species with a Pro in position 56 in eL42, the most common codon is CCA (548 species). What makes this particularly interesting is that a single base mutation at this codon (CCA to CAA) can result in the substitution of Gln at this position and the potential to confer resistance to CHX. More than 50% of the species with a Gln at this position have the codon CAA. Over evolutionary time, switching from CHX resistance to CHX sensitivity has occurred, thereby maintaining the use of these codons in these species.\u003c/p\u003e \u003cp\u003eIn the interesting case of the \u003cem\u003eOphiostomatales\u003c/em\u003e, CHX tolerance can be fully explained by a single, conserved P56Q substitution in ribosomal protein eL42 (Wingfield et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). No evidence was found for uL15 changes or alternative resistance mechanisms in that Order of fungi. This is in contrast to the Saccharomycotina considered in this study, where CHX resistance in some species can be explained by a mutation in the \u003cem\u003euL15\u003c/em\u003e gene. In other cases, species that display resistance to CHX in culture lack ribosomal resistance markers. They most likely rely on other mechanisms, such as altered antibiotic uptake and protein turnover, or Agp2-mediated regulation of uptake (Gerlinger et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Culakova et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Harris et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Mohanty et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken collectively, the results of this study demonstrate that cycloheximide resistance across the Saccharomycotina is largely predictable from specific mutations in ribosomal proteins, particularly the P56Q substitution in \u003cem\u003eeL42\u003c/em\u003e and, in some taxa, substitutions at position 38 of uL15. The strong concordance between genotype and phenotype across 816 species indicates that CHX resistance is primarily governed by conserved molecular mechanisms affecting the ribosome. The few exceptions observed highlight the biological complexity of antifungal tolerance, revealing that additional mechanisms, such as altered antibiotic uptake, gene copy number variation, or permease-mediated regulation, can contribute to resistance in a minority of taxa. The codon-level patterns observed in \u003cem\u003eeL42\u003c/em\u003e further suggest that simple single-nucleotide changes may facilitate rapid evolutionary transitions between sensitivity and resistance, providing a plausible explanation for the widespread yet uneven distribution of CHX tolerance across the yeasts. These findings not only clarify the molecular basis of a trait widely used in yeast taxonomy but also illustrate how ribosomal protein evolution, gene dosage, and cellular transport pathways interact to shape antifungal tolerance across large evolutionary scales.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics, Consent to Participate, and Consent to Publish declarations: not applicable.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003efor this study was provided by the DSTI/NRF SARChI chair in Fungal Genomics.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBDW led the study, writing the main manuscript and conducting the literature review. MPAC extracted gene sequences from the genomes using a bioinformatic pipeline. BJW prepared Figures 1\u0026ndash;2 and performed the codon usage analyses in Excel. CP and MG contributed critical expertise in taxonomy and a broader understanding of Saccharomycotina. MJW provided substantial input on the structure and refinement of the manuscript, contributing to multiple rounds of revision. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eWe utilised the genomes published for the Saccharomycotina from the recent publication by Opulente et al. (2024). The genome sequence data is freely available on in GenBank.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBae, J.H., Sohn, J.H., Park, C.S., Rhee, J.S. and Choi, E.S., 2003. Integrative transformation system for the metabolic engineering of the sphingoid base-producing yeast \u003cem\u003ePichia ciferrii\u003c/em\u003e. \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e, \u003cem\u003e69\u003c/em\u003e(2), pp.812-819.\u003c/li\u003e\n\u003cli\u003eBaliga, B.S., Pronczuk, A.W. and Munro, H.N., 1969. 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Genomic factors shape carbon and nitrogen metabolic niche breadth across Saccharomycotina yeasts. \u003cem\u003eScience\u003c/em\u003e, \u003cem\u003e384\u003c/em\u003e(6694), p.eadj4503.\u003c/li\u003e\n\u003cli\u003ePereira, L.F., Costa Jr, C.R.L., Brasileiro, B.T.R.V. and de Morais Jr, M.A., 2011. \u003cem\u003eLachancea mirantina\u003c/em\u003e sp. nov., an ascomycetous yeast isolated from the cachaca fermentation process. \u003cem\u003eInternational Journal of Systematic and Evolutionary Microbiology\u003c/em\u003e, \u003cem\u003e61\u003c/em\u003e(4), pp.989-992.\u003c/li\u003e\n\u003cli\u003eRaper, K.B., 1966. Antagonistic action of cycloheximide on filamentous fungi and yeast. \u003cem\u003eBacteriological Reviews\u003c/em\u003e, 30(1), pp1-17.\u003c/li\u003e\n\u003cli\u003eRuivo, C.C., Lachance, M.A., Rosa, C.A., Bacci Jr, M. and Pagnocca, F.C., 2006. \u003cem\u003eCandida heliconiae\u003c/em\u003e sp. nov., \u003cem\u003eCandida picinguabensis\u003c/em\u003e sp. nov. and \u003cem\u003eCandida saopaulonensis\u003c/em\u003e sp. nov., three ascomycetous yeasts from \u003cem\u003eHeliconia velloziana\u003c/em\u003e (Heliconiaceae). \u003cem\u003eInternational Journal of Systematic and Evolutionary Microbiology\u003c/em\u003e, \u003cem\u003e56\u003c/em\u003e(5), pp.1147-1151.\u003c/li\u003e\n\u003cli\u003eSasnauskas, K., Jomantienė, R., Lebedienė, E., Lebedys, J., Janusˇka, A. and Janulaitis, A., 1992. Cloning and sequence analysis of a \u003cem\u003eCandida maltosa\u003c/em\u003e gene which confers resistance to cycloheximide. \u003cem\u003eGene\u003c/em\u003e, \u003cem\u003e116\u003c/em\u003e(1), pp.105-108.\u003c/li\u003e\n\u003cli\u003eSchneider-Poetsch, T., Ju, J., Eyler, D.E., Dang, Y., Bhat, S., Merrick, W.C., Green, R., Shen, B. and Liu, J.O., 2010. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. \u003cem\u003eNature Chemical Biology\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(3), pp.209-217.\u003c/li\u003e\n\u003cli\u003eShen, L., Su, Z., Yang, K., Wu, C., Becker, T., Bell-Pedersen, D., Zhang, J. and Sachs, M.S., 2021. Structure of the translating \u003cem\u003eNeurospora\u003c/em\u003e ribosome arrested by cycloheximide. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e, \u003cem\u003e118\u003c/em\u003e(48), p.e2111862118.\u003c/li\u003e\n\u003cli\u003eSteensels, J., Daenen, L., Malcorps, P., Derdelinckx, G., Verachtert, H. and Verstrepen, K.J., 2015. \u003cem\u003eBrettanomyces\u003c/em\u003e yeasts\u0026mdash;From spoilage organisms to valuable contributors to industrial fermentations. \u003cem\u003eInternational Journal of Food Microbiology\u003c/em\u003e, \u003cem\u003e206\u003c/em\u003e, pp.24-38.\u003c/li\u003e\n\u003cli\u003eSuh, S.O. and Blackwell, M., 2005. Four new yeasts in the \u003cem\u003eCandida mesenterica\u003c/em\u003e clade associated with basidiocarp-feeding beetles. \u003cem\u003eMycologia\u003c/em\u003e, \u003cem\u003e97\u003c/em\u003e(1), pp.167-177.\u003c/li\u003e\n\u003cli\u003eVan der Walt, J.P., Johannsen, E., Opperman, A. and Halland, L., 1986. \u003cem\u003eKluyveromyces yarrowii\u003c/em\u003e sp. nov., a haploid, heterothallic, arboreal species. \u003cem\u003eSystematic and Applied Microbiology\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(3), pp.208-212.\u003c/li\u003e\n\u003cli\u003eVidal-Leiria, M., 1966. \u003cem\u003eTorulopsis vanderwaltii\u003c/em\u003e sp. n. \u003cem\u003eAntonie van Leeuwenhoek\u003c/em\u003e, \u003cem\u003e32\u003c/em\u003e(1), pp.447-449.\u003c/li\u003e\n\u003cli\u003eWhiffen, A.J., 1948. The production, assay, and antibiotic activity of actidione, an antibiotic from \u003cem\u003eStreptomyces griseus\u003c/em\u003e. \u003cem\u003eJournal of Bacteriology\u003c/em\u003e, \u003cem\u003e56\u003c/em\u003e(3), pp.283-291.\u003c/li\u003e\n\u003cli\u003eWickerham, L.J., 1969. New homothallic taxa of \u003cem\u003eHansenula\u003c/em\u003e. \u003cem\u003eMycopathologia et Mycologia Applicata\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e(1), pp.15-32.\u003c/li\u003e\n\u003cli\u003eWingfield, B.D., Wingfield, M.J. and Duong, T.A., 2022. Molecular basis of cycloheximide resistance in the Ophiostomatales revealed. \u003cem\u003eCurrent Genetics\u003c/em\u003e, \u003cem\u003e68\u003c/em\u003e(3), pp.505-514.\u003c/li\u003e\n\u003cli\u003eZavala, B., Dineen, L., Fisher, K.J., Opulente, D.A., Harrison, M.C., Wolters, J.F., Shen, X.X., Zhou, X., Groenewald, M., Hittinger, C.T. and Rokas, A., 2024. Genomic factors shaping codon usage across the Saccharomycotina subphylum. \u003cem\u003eG3: Genes, Genomes, Genetics\u003c/em\u003e, 14(11), p.jkae207.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 10 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9313202/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9313202/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe examined 816 Saccharomycotina species to identify the link between cycloheximide (CHX) resistance and amino acid changes in the ribosomal proteins eL42 and uL15. This involved determining the gene sequences (genotype) that code for these proteins in the available genomes. The results were compared with the CHX resistance reported from culture-based studies (phenotype) of these species. The correlation between culture-based studies and gene sequences (wild-type or mutant) was strong, with over 90% of the resistance accurately predicted by specific mutations. The P56Q substitution in eL42 was the most common and consistently associated with growth in cultures containing 0.1% CHX. Mutations in uL15, especially Q38L, Q38M, and Q38F, explained tolerance to 0.01% CHX in some species lacking the P56Q mutation. A total of 73 genotype\u0026ndash;phenotype mismatches (\u0026lt;\u0026thinsp;10%) were detected, primarily in CHX-resistant species that lacked typical ribosomal mutations, suggesting the presence of alternative mechanisms of resistance. Eleven of the species with multiple eL42 copies encoding both resistant and sensitive variants were phenotypically CHX sensitive. This supports previous evidence that a single mutant copy of eL42 is insufficient to confer CHX resistance if the wild-type copy is also present. Codon usages at the amino acid position 56 in the putative eL42 protein and position 38 in the putative uL15 proteins were determined. The most common codon in this position for eL42 proline was CCA; a single base change can result in CAA, which codes for glutamine and confers CHX resistance at position 56 in this protein. Overall, mutations in \u003cem\u003eeL42\u003c/em\u003e and \u003cem\u003euL15\u003c/em\u003e explained most CHX resistance in yeasts, but gene copy number and other pathways affecting uptake of the antibiotic also play roles. The extensive genotype\u0026ndash;phenotype framework established in this study provides for accurate, genome-based prediction of CHX resistance across the Saccharomycotina, underscoring the importance of gene dosage and non-ribosomal factors in resistance.\u003c/p\u003e","manuscriptTitle":"The Genetic Blueprint of Cycloheximide Resistance: Analysis of 816 Yeast Species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 16:59:44","doi":"10.21203/rs.3.rs-9313202/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"178048486115284458815933091867470230888","date":"2026-05-12T08:36:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88432144239394917883769343073533690150","date":"2026-05-06T16:39:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89863899943558039091842263942778692080","date":"2026-05-06T13:15:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T00:28:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-09T11:17:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-04T04:47:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-04T04:46:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-03T12:51:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"312d52ed-4fe5-4a11-9b44-c6dff279dd6c","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"178048486115284458815933091867470230888","date":"2026-05-12T08:36:58+00:00","index":79,"fulltext":""},{"type":"reviewerAgreed","content":"88432144239394917883769343073533690150","date":"2026-05-06T16:39:02+00:00","index":76,"fulltext":""},{"type":"reviewerAgreed","content":"89863899943558039091842263942778692080","date":"2026-05-06T13:15:32+00:00","index":75,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66911047,"name":"Biological sciences/Evolution"},{"id":66911048,"name":"Biological sciences/Genetics"},{"id":66911049,"name":"Biological sciences/Microbiology"},{"id":66911050,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-04-29T16:59:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 16:59:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9313202","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9313202","identity":"rs-9313202","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}