RESUMO
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.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Kluyveromyces , Regulação da Expressão Gênica , Kluyveromyces/genética , ÉsteresRESUMO
Antibiotic resistance in foodborne pathogens is an increasing threat to global human health. Among the most prevalent antibiotic-resistant bacteria are Salmonella enterica serovar Typhimurium, Campylobacter jejuni and E. coli 0157:H7. Control of these and other pathogens requires innovative approaches, i.e., discovering new molecules that will inactivate them, or render them less virulent without inducing resistance. Recently, several polyphenol molecules have been shown to possess such characteristics. Also, the use of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) approaches has recently been proposed for such purpose. This review summarizes the main findings regarding the application of both approaches to control the above-mentioned foodborne pathogens by relying on Quorum Sensing interference (Quorum Quenching) mechanisms and highlights the avenues needed for further research.
RESUMO
Fructans are the main sugar in agave pine used by yeasts during mezcal fermentation processes, from which Candida apicola NRRL Y-50540 and Torulaspora delbrueckii NRRL Y-50541 were isolated. De novo transcriptome analysis was carried out to identify genes involved in the hydrolysis and assimilation of Agave fructans (AF). We identified a transcript annotated as SUC2, which is related to ß-fructofuranosidase activity, and several differential expressed genes involved in the transcriptional regulation of SUC2 such as: MIG1, MTH1, SNF1, SNF5, REG1, SSN6, SIP1, SIP2, SIP5, GPR1, RAS2, and PKA. Some of these genes were specifically expressed in some of the yeasts according to their fructans assimilation metabolism. Different hexose transporters that could be related to the assimilation of fructose and glucose were found in both the transcriptomes. Our findings provide a better understanding of AF assimilation in these yeasts and provide resources for further metabolic engineering and biotechnology applications.
