Loading...
Please wait, while we are loading the content...
Similar Documents
De novo sequencing and detection of secondary metabolite gene clusters of Penicillium griseofulvum.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Banani, Houda Marcet-Houben, Marina Ballester, Ana-Rosa Abbruscato, Pamela González-Candelas, Luis Gabaldón, Toni Spadaro, Davide |
| Copyright Year | 2016 |
| Abstract | The genus Penicillium comprises different economically important species with the ability to produce a wide array of secondary metabolites. Among different Penicillium species, P. griseofulvum is worldwide distributed and has been associated with blue mold decay. In the present study, the complete genome of P. griseofulvum strain PG3 isolated from rotted apples harvested in Italy was sequenced and some important secondary metabolites clusters present in PG3 were reported. The PG3 estimated genome size was of 29.3 Mb, and the phylogenetic analysis at the wholegenome level revealed that P. griseofulvum is branched off after the divergence of P. oxalicum and before P. chrysogenum. Genome-wide analysis of PG3 genes uncovered a putative gene cluster for patulin biosynthesis. In vitro results clearly confirmed that PG3 is a high patulin producer. In addition to patulin, we detected a functional griseofulvin gene cluster.This study will enable to gain insight into secondary metabolite synthesis in P. griseofulvum and assess its potential applications in biotechnology and threats for food safety. INTRODUCTION P. griseofulvum Dierckx (syn. P. patulum Bain.; P. urticae Bain.) is worldwide distributed and it has been isolated from fruits, decaying plants, cereal grains and animal feed (Shim et al., 2006). P. griseofulvum has been associated with blue mold decay in storage apple fruits, which is considered as one of the most important postharvest diseases of pome fruits worldwide (Pianzzola et al., 2004). Besides the economic losses, P. griseofulvum may represent a potential health risk because of its ability to produce mycotoxins such as patulin. The genes forming the patulin cluster were characterized in P. expansum and Aspergillus clavatus (Artigot et al., 2009; Tannous et al., 2014; Ballester et al., 2015; Li et al., 2015); however no information is yet available about the composition of the patulin cluster in P. griseofulvum. This information is needed to clearly understand the mechanisms leading to patulin production in this fungus and to define strategies for patulin control. P. griseofulvum is also known to produce a wide array of important useful secondary metabolites, including griseofulvin (Samson RA et al., 2004; Shim et al., 2006) which has been in use for many years in medical and veterinary applications (Finkelstein et al., 1996). The griseofulvin biosynthetic gene cluster consists of 13 putative genes and has been reported in P. aethiopicum (Chooi et al., 2010; Cacho et al., 2013, 2015), but it is still not known the genes forming the griseofulvin cluster in P. griseofulvum. To gain insight into secondary metabolite clusters in P. griseofulvum and assess its biotechnological potential and define better the threats for food safety, we have sequenced for the first time its genome. MATERIALS AND METHODS Penicillium griseofulvum Dierckx (syn: P. urticae Bainier) strain PG3 (deposited at Centraalbureau voor Schimmelcultures, with Accession number CBS 140421) was obtained from rotten apples harvested in Piedmont, Northern Italy. Total DNA was extracted from the strain PG3 as previously described by Ballester and collaborators (Ballester et al., 2015) and then DNA concentration and purity were checked by a spectrophotometer (Nanodrop 2000, Thermo Scientific, Wilmington, USA). The genome of P. griseofulvum PG3 was sequenced at the Genomics Platform of the Parco Tecnologico Padano using the Illumina MiSeq technology. SPAdes was used to assemble the P. griseofulvum genome (Bankevich et al., 2012) and the genes encoded in the genome were predicted by Augustus trained with Aspergillus nidulans (Keller et al., 2011). A phylome, designed as the complete collection of phylogenetic trees for each gene encoded in a genome, was reconstructed for P. griseofulvum. Fourteen other species were included in the phylome. These comprised the other sequenced Penicillium genomes (P. chrysogenum, P. oxalicum, P. roqueforti, P. camemberti, P. expansum, P. digitatum and P. italicum) and members of the Aspergillus and Talaromyces clade. Species tree was reconstructed using the method of gene concatenation. RaxML was used to reconstruct the species tree using the PROTGAMMALG model (Stamatakis et al., 2005). A collection of 114 secondary metabolism clusters were used to look for homologous clusters in the P. griseofulvum genome following the method described in Ballester and collaborators (Ballester et al., 2015). PG3 colony diameter (mm) was measured for up to 10 days of growth, and then patulin and griseofulvin production were analyzed by high-performance liquid chromatography (HPLC). RESULTS AND DISCUSSION Genome sequencing and comparative genomics The genome of P. griseofulvum strain PG3 was sequenced. Table 1 shows the final statistics of the genome assembly, which is composed of 363 contigs, 14 of which were larger than 100 kb. The estimated genome size was of 29.3 Mb. Gene annotation showed that 9,631 proteins were encoded in the genome. We compared the genome of P. griseofulvum with the genomes of 14 other fully-sequenced Penicillium and Aspergillus species. To determine the phylogenetic position of P. griseofulvum in relation with other sequenced species, we reconstructed a species tree based on the concatenation of 2,134 genes that were found to be single copy in all considered species. Our results show that P. griseofulvum is branched off between P. chrysogenum and P. oxalicum (Figure 1A). Genome-wide analysis of P. griseofulvum PG3 genes revealed two putative gene clusters for patulin and griseofulvin biosynthesis The presence of secondary metabolites clusters in PG3 was analyzed by searching for homologs of about 114 gene clusters present in the database. A patulin gene cluster is identified for the first time in P. griseofulvum, PG3 strain, containing 15 genes gathered together ordered similarly to the patulin cluster of P. expansum strain PEXP (Ballester et al., 2015) (Figure 1B). This result confirms that the changes in gene order observed between the cluster in A. clavatus and the cluster in P. griseofulvum and P. expansum happened before the two Penicillium species diverged. Besides the patulin gene cluster, we found a griseofulvin gene cluster (Figure 1C). This gene cluster was originally described in P. aethiopicum and consists of 13 genes (Chooi et al., 2010). When we compared the griseofulvin gene cluster of P. aethiopicum with the one found in PG3, we found that three genes (gsfR2, gsfK and gsfH) were not located within the PG3 griseofulvin gene cluster: gsfR2 codes for a putative transcription factor, gsfK, encodes a putative NAD(P)-dependent oxidoreductase, and gsfH codes for a isochorismatase-like protein. However these proteins have homologs in a different position of the genome and therefore we could not discard the option that they are still playing a function in the synthesis of griseofulvin. Patulin and griseofulvin production in vitro by PG3 Patulin and griseofulvin production by P. griseofulvum PG3 was quantified in vitro for up to 10 days. After 3 days of incubation in vitro, PG3 produces considerable amount of patulin (about 446.86 μg/plate), then increased significantly to reach 3498.71 μg/plate at day 10 (figure 2B). Interestingly, these concentrations are in the same range as the patulin production by P. expansum strain PEXP which has similar genes number and order as the putative PG3 patulin cluster (Ballester et al., 2015), although PG3 exhibited distinct differences in colony morphology and slower growth kinetics compared with PEXP (figure 2A/B). As mentioned before, P. griseofulvum is known to produce thesecondary metabolite griseofulvin that has been used for many years in medical and veterinary applications.In the present work, griseofulvin production by PG3 was investigated, and the results clearly confirm that PG3 produced significant levels of griseofulvin in vitro, which increased over the time to reach at the day 10 about 215 μg/plate (Figure 2C). The production of griseofulvin by the newly sequenced P. griseofulvum PG3 strain indicates that the lack of three genes within the gene cluster is apparently not affecting the synthesis of griseofulvin. However we do not know whether or not these genes are nevertheless involved in the synthesis of this compound. CONCLUSIONS In this study, we sequenced and annotated for the first time the genome of the postharvest pathogen P. griseofulvum PG3 isolated from rotted apples in Italy. Our data suggest that PG3 genome size is 29.3 Mb, and the phylogenetic analysis at the wholegenome level revealed that P. griseofulvum is branched off after the divergence of P. oxalixum and before P. chrysogenum. Then our analyses uncovered two important secondary metabolite gene clusters in PG3: a griseofulvin cluster and a patulin gene clusterthat is similar to the cluster identified in P. expansum. Finally, in vitro analysis conducted in the present study revealed that PG3 produces a considerable amount of patulin and griseofulvin. Our data give insight into secondary metabolites synthesis in P. griseofulvum PG3 and assess its potentiality in term of threats for food safety, but also pave the way for its future biotechnological applications such as using PG3-knocked out of PKS of the patulin cluster for producing griseofulvin. Literature cited Artigot, M.P., Loiseau, N., Laffitte, J., Mas-Reguieg, L., Tadrist, S., Oswald, I.P. and Puel, O. 2009. Molecular cloning and functional characterization of two CYP619 cytochrome P450s involved in biosynthesis of patulin in Aspergillus clavatus. Microbiology 155:1738–1747. Ballester, A., Marcet-Houben, M., Levin, E., Sela, N., Selma-Lázaro, C., Carmona, L., Wisniewski, M., Droby, S., González-Candelas, L. and Gabaldón, T. 2015. Genome , Transcriptome , and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity. Mol. Plant-Microbe Interact. 28:232–248. Bankevich, A., Nurk, S., |
| Starting Page | 157 |
| Ending Page | 162 |
| Page Count | 6 |
| File Format | PDF HTM / HTML |
| DOI | 10.17660/ActaHortic.2016.1144.22 |
| Volume Number | 1144 |
| Alternate Webpage(s) | https://iris.unito.it/retrieve/handle/2318/1620756/448637/Banani%20et%20al%20POSTPRINT.pdf |
| Alternate Webpage(s) | https://doi.org/10.17660/ActaHortic.2016.1144.22 |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
