Abstract
Mezcal is a traditional Mexican distilled beverage, known for its marked organoleptic profile, which is influenced by several factors, such as the fermentation process, where a wide variety of microorganisms are present. Kluyveromyces marxianus is one of the main yeasts isolated from mezcal fermentations and has been associated with ester synthesis, contributing to the flavors and aromas of the beverage. In this study, we employed CRISPR interference (CRISPRi) technology, using dCas9 fused to the Mxi1 repressor factor domain, to down-regulate the expression of the IAH1 gene, encoding for an isoamyl acetate-hydrolyzing esterase, in K. marxianus strain DU3. The constructed CRISPRi plasmid successfully targeted the IAH1 gene, allowing for specific gene expression modulation. Through gene expression analysis, we assessed the impact of IAH1 down-regulation on the metabolic profile of volatile compounds. We also measured the expression of other genes involved in volatile compound biosynthesis, including ATF1, EAT1, ADH1, and ZWF1 by RT-qPCR. Results demonstrated successful down-regulation of IAH1 expression in K. marxianus strain DU3 using the CRISPRi system. The modulation of IAH1 gene expression resulted in alterations in the production of volatile compounds, specifically ethyl acetate, which are important contributors to the beverage's aroma. Changes in the expression levels of other genes involved in ester biosynthesis, suggesting that the knockdown of IAH1 may generate intracellular alterations in the balance of these metabolites, triggering a regulatory response. The application of CRISPRi technology in K. marxianus opens the possibility of targeted modulation of gene expression, metabolic engineering strategies, and synthetic biology in this yeast strain.
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Data availability
The complete KmDU3 genome sequence has been deposited in the National Center of Biotechnology Information (NCBI) database with the accession number JAQIJY000000000. The raw reads obtained from the Oxford Nanopore Sequence and Illumina Sequences used to assemble the KmDU3 genome were deposited in the NCBI—Sequence Read Archive (SRA) under the BioProject ID PRJNA909832. RNA-Seq data supporting the genomic annotation of KmDU3 are not openly available for reasons of sensitivity of another publication and can be requested from the corresponding author.
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Acknowledgements
The authors thank Dr. Cesar Arturo Nava Valdivia and M. en C. Martin Zermeño Ruíz (Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara) for the space and technical assistance during the RT-qPCR experiments
Funding
The present work was financed by the CONAHCYT Basic Science fund with project number 252465, with the title "Study of ester production by non-Saccharomyces yeasts with a multidisciplinary approach".
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Conceptualization: [LJFY], Methodology: [LAMM, ACZP, ACGM, AMZ, APS, LCG, ICBM], Formal analysis [APS, LAMM, AMZ, LJFY], Data curation [APS, LAMM], Investigation: [LAMM, ACZP, ACGM, AMZ], Funding acquisition: [ACGM], Resources: [ACGM, LJFY, LCG, ICBM, LAMM, APS], Supervision: [ACGM, LJFY], Software [APS, LAMM], Validation [LAMM, LJFY], Visualization [LAMM], Project administration [LJFY, ACGM], Writing—original draft preparation: [LAMM], Writing—review and editing: [LAMM, ACZP, ACGM, AMZ, APS, LCG, ICBM, LCRZ].
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Muñoz-Miranda, L.A., Zepeda-Peña, A.C., Casas-Godoy, L. et al. CRISPRi-induced transcriptional regulation of IAH1 gene and its influence on volatile compounds profile in Kluyveromyces marxianus DU3. World J Microbiol Biotechnol 40, 121 (2024). https://doi.org/10.1007/s11274-023-03811-0
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DOI: https://doi.org/10.1007/s11274-023-03811-0