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RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice

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Abstract

Background

Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice.

Methods and results

We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method’s capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains.

Conclusions

The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

This study was carried out within the framework of a master thesis conducted by Hacettepe University, Graduate School of Science and Engineering.

Funding

This work was supported by Hacettepe University, Department of Food Engineering FoodOmics Laboratory.

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Authors and Affiliations

  1. FoodOmics Laboratory, Department of Food Engineering, Hacettepe University, Beytepe Campus, 06800, Ankara, Turkey

    Elif Bircan Muyanlı & Remziye Yılmaz

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  1. Elif Bircan Muyanlı
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Contributions

EBM and RY designed the study. Material preparation, data collection, and analysis were performed by EBM under the supervision of RY. The manuscript was written by EBM and RY.

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Correspondence to Remziye Yılmaz.

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Muyanlı, E.B., Yılmaz, R. RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice. Mol Biol Rep 51, 547 (2024). https://doi.org/10.1007/s11033-024-09444-2

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