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Molecular Genetic Analysis with Microsatellite-like Loci Reveals Specific Dairy-Associated and Environmental Populations of the Yeast Geotrichum candidum.

ABSTRACT: Geotrichum candidum is an environmental yeast, also found as part of the cheese surface microbiota, where it is important in the ripening of many traditional cheeses, such as Camembert. We have previously developed a Multi Locus Sequence Typing (MLST) scheme, which differentiated five clades, of which one contained only environmental isolates, two were composed almost entirely of dairy isolates, and two others contained a mixture of dairy, environmental, and miscellaneous food isolates. In order to provide a simple method to uniquely type G. candidum strains, and in addition to permit investigation of the population structure at a fine level, we describe here a molecular analysis using a set of twelve highly discriminating microsatellite-like markers. The present study consolidates the previously suggested division between dairy and environmental strains, and in addition distinguishes a specifically European group of environmental strains. This analysis permitted the discrimination of 72 genotypes from the collection of 80 isolates, while retaining the underlying meaningful phylogenetic relation between groups of strains.

SUBMITTER: Tinsley CR

PROVIDER: S-EPMC8780849 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The yeast Geotrichum candidum (teleomorph Galactomyces candidus) is inoculated onto mold- and smear-ripened cheeses and plays several roles during cheese ripening. Its ability to metabolize proteins, lipids, and organic acids enables its growth on the cheese surface and promotes the development of organoleptic properties. Recent multilocus sequence typing (MLST) and phylogenetic analyses of G. candidum isolates revealed substantial genetic diversity, which may explain its strain-dependant technological capabilities. Here, we aimed to shed light on the phenotypic and genetic diversity among eight G. candidum and three Galactomyces spp. strains of environmental and dairy origin. Phenotypic tests such as carbon assimilation profiles, the ability to grow at 35°C and morphological traits on agar plates allowed us to discriminate G. candidum from Galactomyces spp. The genomes of these isolates were sequenced and assembled; whole genome comparison clustered the G. candidum strains into three subgroups and provided a reliable reference for MLST scheme optimization. Using the whole genome sequence as a reference, we optimized an MLST scheme using six loci that were proposed in two previous MLST schemes. This new MLST scheme allowed us to identify 15 sequence types (STs) out of 41 strains and revealed three major complexes named GeoA, GeoB, and GeoC. The population structure of these 41 strains was evaluated with STRUCTURE and a NeighborNet analysis of the combined six loci, which revealed recombination events between and within the complexes. These results hint that the allele variation conferring the different STs arose from recombination events. Recombination occurred for the six housekeeping genes studied, but most likely occurred throughout the genome. These recombination events may have induced an adaptive divergence between the wild strains and the cheesemaking strains, as observed for other cheese ripening fungi. Further comparative genomic studies are needed to confirm this phenomenon in G. candidum. In conclusion, the draft assembly of 11 G. candidum/Galactomyces spp. genomes allowed us to optimize a genotyping MLST scheme and, combined with the assessment of their ability to grow under different conditions, provides a reliable tool to cluster and eventually improves the selection of G. candidum strains.

Project description:Enzymes are biocatalysts mainly used for their industrial potential in various applications. The present study aims to understand the enzyme production for biotechnological interest from a local yeast strain. From 100 isolates obtained from various biotopes, 78 strains were selected for their enzymatic heritage. Screening of α-amylase, lipase/esterase, and cellulase activities by rapid plate detection methods was carried out and the PO27 yeast was selected for its high capacity to produce α-amylase. In addition, this yeast strain exhibited good lipolytic and esterolytic activities, as well as low cellulase activity. A sequence analysis of the D1/D2 region of the 26S ribosomal RNA (26S rRNA) and a study of morphological characteristics identified the PO27 strain as Geotrichum candidum. The production of α-amylase has been studied in solid medium fermentation using various natural substrates without any supplementation such as olive pomace, potato peels, leftover bread, and mastic cake. G. candidum PO27 showed an improved production of α-amylase with olive pomace, thus reaching approximately 180.71 U/g. To evaluate the ability of this isolate to produce α-amylase in submerged fermentation, multiple concentrations of olive pomace substrate were tested. The best activity of submerged fermentation was statistically compared to the solid-state fermentation result in order to select the appropriate fermentation type. A high significant difference was found to rank the 6% olive pomace medium as the best substrate concentration with 34.395 U/mL of α-amylase activity. This work showed that the new isolate Geotrichum candidum PO27 has a better potential to produce α-amylase at a low cost in solid-state fermentation compared to submerged fermentation. Optimization conditions for PO27 α-amylase production through solid-state fermentation were achieved ‎ using a one factor at a time (OFAT) approach. The findings revealed that a high temperature (60 °C), an acidic pH, malt extract, and soluble starch were the highly significant medium components for enhancing α-‎amylase production. The use of olive pomace waste by Geotrichum candidum PO27 is expected to be effective in producing an industrially useful α-amylase.

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Molecular Genetic Analysis with Microsatellite-like Loci Reveals Specific Dairy-Associated and Environmental Populations of the Yeast Geotrichum candidum.

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