Lessard et al. BMC Genomics 2014, 15:235 http://www.biomedcentral.com/1471-2164/15/235 RESEARCH ARTICLE Open Access Metatranscriptome analysis of fungal strains Penicillium camemberti and Geotrichum candidum reveal cheese matrix breakdown and potential development of sensory properties of ripened Camembert-type cheese Marie-Hélène Lessard1, Catherine Viel1, Brian Boyle2, Daniel St-Gelais1,3 and Steve Labrie1* Abstract Background: Camembert-type cheese ripening is driven mainly by fungal microflora including Geotrichum candidum and Penicillium camemberti. These species are major contributors to the texture and flavour of typical bloomy rind cheeses. Biochemical studies showed that G. candidum reduces bitterness, enhances sulphur flavors through amino acid catabolism and has an impact on rind texture, firmness and thickness, while P. camemberti is responsible for the white and bloomy aspect of the rind, and produces enzymes involved in proteolysis and lipolysis activities. However, very little is known about the genetic determinants that code for these activities and their expression profile over time during the ripening process. Results: The metatranscriptome of an industrial Canadian Camembert-type cheese was studied at seven different sampling days over 77 days of ripening. A database called CamemBank01was generated, containing a total of 1,060,019 sequence tags (reads) assembled in 7916 contigs. Sequence analysis revealed that 57% of the contigs could be affiliated to molds, 16% originated from yeasts, and 27% could not be identified. According to the functional annotation performed, the predominant processes during Camembert ripening include gene expression, energy-, carbohydrate-, organic acid-, lipid- and protein- metabolic processes, cell growth, and response to different stresses. Relative expression data showed that these functions occurred mostly in the first two weeks of the ripening period. Conclusions: These data provide further advances in our knowledge about the biological activities of the dominant ripening microflora of Camembert cheese and will help select biological markers to improve cheese quality assessment. Background culture, on culture media or on model cheese medium [3], Camembert cheese is a soft, mold-ripened cheese. The biochemical pathways potentially involved in the deve- mold Penicillium camemberti and the yeast Geotrichum lopment of sensory properties and even microbiological candidum are the two major Fungi that give the white succession during Camembert ripening [4-10]. Surprisingly, coated characteristic of this cheese variety. Their asso- only limited genetic information is available for these Fungi, ciation is crucial not only for appearance, but also for typ- since fewer than 30 different genes of each organism have ical sensory characteristics of Camembert cheese [1,2]. been deposited in public databases. Previous studies considered aroma production in pure Molecular biology techniques were recently used to evaluate several aspects of sensory characteristics of cheese. * Correspondence: [email protected] For example, multispecies DNA microarrays combined 1Department of Food Sciences and Nutrition, Institute of Nutrition and with biochemical analysis (HPLC and SPME-GCMS) Functional Foods (INAF), STELA Dairy Research Centre, Université Laval, 2425 has been a useful tool to evaluate L-methionine catabo- rue de l′Agriculture, G1V 0A6 Québec City, QC, Canada Full list of author information is available at the end of the article lism, production of volatile sulfur compounds (VSC) and © 2014 Lessard et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lessard et al. BMC Genomics 2014, 15:235 Page 2 of 13 http://www.biomedcentral.com/1471-2164/15/235 lactose/lactate consumption during yeast growth [11,12]. quantified using a TaqMan-based qPCR method [5,41], Even though microarrays provide information about gene G. candidum and P. camemberti had similar growth pro- expression under various conditions, their utility is limited files with an active phase in the first 5 days of ripening. to organisms for which genetic information is available Their maximum cell density was 6.45 × 109 and 4.69 × [13]. Next-Generation sequencing (NGS) methods are now 1010 gene copies/cm2, respectively, at the end of ripening widely used for de novo- and re- sequencing of genomes, (Figure 1). transcriptomes, epigenomes and metagenomes [14-19]. The first metagenomic analysis using 454 pyrosequenc- Sequencing and assembly of the Camembert ing was performed on bacterial communities in mines cheese transcriptome [20] and since then, high quality information is available Since only scarce genetic information is available for G. about ecosystems from soil [21,22], sea water [23,24], candidum and P. camemberti, the metatranscriptomic humans [25,26], and even cheese [27], most of them iden- approach using massive parallel sequencing had the ad- tifying microorganisms and establishing their phylogenetic vantage of simultaneously identifying new genes and de- relationships [28]. Genome and metagenome sequencing termining their expression profile during cheese ripening. are powerful tools, but massive transcriptome sequencing A de novo assembly performed using all 1,019,060 reads using NGS provides a more dynamic and functional view generated 8,909 contigs (length > 99 nt, average length of microbial activity under particular conditions by accu- of 916 nt). After sorting data for a minimum contig mulating data on RNA and its expression profile. length of 200 nt and a minimum of 6 assembled reads, Several studies used NGS technologies to compare the 8,318 contigs were conserved in the original cheese transcriptomic response of a single organism exposed to database. Reads were mapped back to the de novo as- different conditions [29-35]. In multiple-organism envi- sembly to enable semi-quantitative analysis and quality ronments, establishing the metatranscriptome reveals control of the assembly. De novo assembly and mapping the activity of a community, but only rare and very re- data were compared to remove artefacts, such as dupli- cent papers selected this approach [36-39]. This study is cated transcript models, resulting in the exclusion of the first comprehensive metatranscriptome analysis of 402/8,318 contigs. The assembly contigs were free of the Camembert cheese complex fungal ripening ecosys- fungal rDNA and mt-rDNA contamination as revealed tem. Here, the fungal metatranscriptome was sequenced by local BLAST search. This high quality dataset of 7,916 using a Roche 454 pyrosequencing NGS strategy, without contigs (average length of 988 nt; Table 1) represents the prior knowledge of the Penicillium camemberti and Geo- fungal metatranscriptome of the Canadian Camembert- trichum candidum genome sequences. The longer reads type cheese selected and was called CamemBank01, hence- produced by the 454 instruments enabled the discovery forth compensating the absence of available sequenced and characterization of new genetic information for these genomes for ripening species Penicillium camemberti Fungi and simultaneously established their activity profile. and Geotrichum candidum. Many fungal activities were identified using this strategy, including the central metabolism and the response to en- Identification and functional annotation of contigs found vironmental stresses and nutrient availability in the cheese in CamemBank01 matrix. This semi-quantitative gene expression profiling All 7,916 contigs were analyzed using the Blast2GO plat- revealed the adaptation of G. candidum and P. camem- form [42]. Because no genome of the yeast G. candidum berti during the 77-day ripening period of a commercial and the mold P. camemberti are currently available in Canadian Camembert-type cheese. public databases, sequence analysis was performed with caution. Therefore, contigs were assigned according to Results and discussion their similarity to mold or yeast relatives if sequences Cheese characteristics and fungal growth had a >70% identity with known proteins in GenBank. Commercial Camembert-type cheeses made from pas- Globally, 56,7% contigs originated from molds (M, n = teurized milk were obtained from a processing plant lo- 4,491 contigs) and 16,4% from yeasts (Y, n = 1,299 contigs). cated in Canada. Cheeses used in the present study The other 26,9% was defined as of uncharacterized origin developed no obvious defects during the ripening period (U), either because the Blastx protein similarity was under and met the high quality criteria of the company who 70% or because they had no significant homology. Over the provided the cheeses for the characteristics of cheese 563,733 reads assembled, 275,586 reads (48.89%) were con- texture, fat matter, salt and water content (confidential fidently assigned to molds and 105,017 reads (18.63%) to data, not presented). Also, the measured pH increase fit yeasts, while 183,130 reads are still unassigned. The aver- the normal alkalinisation of the rind over time observed age expression was 71 reads/contig, or 71 transcripts/ for similar Canadian mold ripened