The complete mitochondrial genome of huperzine A-producing endophytic fungus Penicillium polonicum.

Huperzine A-producing fungus Penicillium polonicum Hy4 (CCTCC No.M2010086) was isolated from Huperzia serrata (Thunb) Trev. The complete mitochondrial genome of P. polonicum is 28 192 bp in length, containing 15 protein-encoding genes, 27 tRNA genes and two rRNA genes. The whole mitogenome is high in AT content (74.40%) and low in GC content (25.60%). The mitochondrial gene order and arrangement of P. polonicum are identical to those of other Penicillium. Phylogenetic analysis based on 14 concatenated protein-encoding genes showed that P. polonicum was close to P. solitum. This study reports the complete mitogenome of P. polonicum for the first time and provides valuable information for further exploration of mitochondrial evolution.

Penicillium polonicum strain Hy4 (CCTCC No.M2010086) , isolated from Huperzia serrata in Shaoyang, Hunan, China (27.22 North, 111.50 East) was found to produce Huperzine A (Hup A) and was deposited in China Center for Type Culture Collection (CCTCC). Hup A is used to treat dementia as a reversible and selective acetylcholinesterase (AChE) inhibitor with low toxicity (Zhang & Tang 2006). Up to now, Hup A is mainly isolated from H. serrata which have an extremely long vegetative growth cycle and low Hup A content (Ma et al. 2005). Endophytic fungi are one of the most creative groups of high-value secondary metabolite producers (Venugopalan & Srivastava 2015).

Initial taxonomic evaluation of P. polonicum Hy4 was made based on ITS sequence (Makimura et al. 2001). The mitogenome was sequenced by Illumina Hiseq 2000 and assembled by Allpaths-LG (Gnerre et al. 2011), with two gaps filled by PCR amplification. It was a circular-mapping DNA molecule of 28 192 bp with a low GC content of 25.60% (Genbank accession number: KU530219). The mitogenome contained 15 conserved protein-encoding genes (13 728 bp, 48.69% of the mitogenome) that were discovered by searching NCBI NR database using Exonerate (Slater & Birney 2005; Lin et al. 2015). Those genes encoded ATP synthase subunits (atp6, atp8 and atp9), cytochrome oxidase subunits (cox1, cox2 and cox3), apocytochrome b (cob), NADH dehydrogenase subunits (nad1, nad2 and nad3, nad4, nad4L, nad5 and nad6) and ribosomal protein (rps3). All genes were located on one strand and apparently transcribed in one direction. Additionally, 27 tRNA genes were predicted by tRNAscan-SE (Lowe & Eddy 1997) and the large/small subunits ribosomal RNA genes (rns, rnl) were identified by searching against Rfam database (Burge et al. 2013). Similar to many filamentous fungi, P. polonicum mitochondrial tRNA genes were organized into two dense gene clusters (KGGDSWISP, TEVMMLAFLQM), which were flanked by cox3, rnl and cox1 (Sun et al. 2011; Eldarov et al. 2012; Joardar et al. 2012). The comparison of trn distribution in the mitogenomes showed that the trn order in Penicillium was relatively conserved in contrast to that in Aspergillus.

The codon frequency analysis showed that a total of 56 codons were used for transcription, with the absence of ACC, CCC, CGA, CGC, CGG, CTC, GGC and TCG. All protein-encoding sequences started with the typical ATG codon and ended with TAA as the stop codons except for nad6 and cox3 (TAG). AT-rich codons were abundant, up to 82.36%, reflecting the high AT content of the P. polonicum mitogenome. The most used codon is TTA for Leu and followed by ATA for Ile. The ratio of codons encoding hydrophobic amino acids (Met, Trp, Phe, Val, Leu, Ile, Pro, Ala) was 54.74%, reflecting the hydrophobic nature of respiratory membrane complexes. A maximum likelihood tree generated by 14 protein-coding genes from 17 fungal strains (Figure 1) suggested the closest relationship between P. polonicum and P. solitum. Moreover, Penicillium was closely related to Aspergillus, except Penicillium marneffei, which was positioned on a distinct branch. This observation was identical with the earlier study based on nuclear genome (van den Berg et al. 2008).

Figure 1.

Phylogenetic relationship between P. polonicum and other 16 fungal mitogenomes. The maximum likelihood tree was generated by MEGA v6.06 (Tamura et al. 2013) based on 14 concatenated protein-coding genes (nad1-6, nad4L, atp6, atp8-9, cob and cox1-3). Verticillium dahliae was used as outgroup. The model for phylogenetic analysis was GTR + G+I with bootstrap value of 1000. The sequence data for these 17 fungi with complete mitochondrial genomes were used: Aspergillus clavatus (JQ354999), Aspergillus fumigatus (JQ346807), Aspergillus terreus (JQ355001), Aspergillus niger (NC_007445), Aspergillus tubingensis (NC_007597), Penicillium chrysogenum (JQ354996), Penicillium digitatum (NC_015080), Penicillium polonicum (KU530219), Penicillium solitum (NC_016187), Talaromyces stipitatus (JQ354994), Penicillium marneffei (JQ354997), Talaromyces marneffei (NC_005256), Arthroderma uncinatum (NC_012828), Arthroderma obtusum (NC_012830), Trichophyton mentagrophytes (NC_012826), Trichophyton rubrum (NC_012824), Verticillium dahliae (DQ351941).