Cloning and characterization of terpenoid biosynthesis genes of Antrodia cinnamomea

Antrodia cinnamomea (Neu-Chang-Tsu), a potent medicinal resupinate mushroom, was documented with the capacity to produce numerous metabolites with biological activity, notably the terpenoids, and merit for study further. Attempt at molecular level to reveal the activity of these secondary metabolites of A. cinnamomea, we conducted cloning and characterization of the terpenoid biosynthesis genes. The genomic DNA gene fragments of farnesyl diphosphatesynthase (FDS) (ca. 300 bp ), geranylgeranyltransferase (GT) (ca. 700 bp), sesquiterpene cyclase (SC) (ca. 1 kb), and squalene monooxygenase (SM) (ca. 700 bp) were obtained by using specific primers derived from previously constructed cDNA EST library by polymerase chain reaction (PCR). The unique genomic DNA fragments were labeled to probe the constructed Fosmid library. Four Fosmid clones, bdf01008-B24 (37,783 bp), bdf01020-E06 (34,826 bp), bdf01010-N11 (37,395 bp), and bdf01002-P14 (26,567 bp) which exhibited positive signal towards FDS, GT, SC, and SM specific probes, respectively, were accessed by Fosmid colony hybridization. Of the four Fosmid clones selected and sequenced, in addition to FDS, GT, SC, and SM, 22 putative genes, including monooxygenase, heat shock protein, expressed protein, glutamate dehydrogenase/leucine dehydrogenas, phosphoethanolamine N-methyltransferase, ppg3, Pol-like protein Pol-2, pol protein, H25N7.04, nuclear mRNA splicing protein, vicilin storage protein, and 10 hypothetical proteins, were annotated after BlastX with the NCBI database. Furthermore, the full-length cDNA of FDS, GT, SC, and SM were achieved by rapid amplification of cDNA ends (RACE), of them, FDS cDNA was 1359 bp , with 1092 bp open reading frame (ORF), encoding translated 364 amino acids; GT 1181 bp, ORF 1026 bp, translated 342 amino acids; SC 1402 bp, ORF 831 bp, translated 277 amino acids; SM 1546 bp, ORF 1443 bp, translated 481 amino acids, respectively. In order to verify the function of the four FDS, GT, SC, and SM genes by gene disruption, four specific replacement vectors were constructed by insertion the pAN7-1 vector with the hygromycin resistance cassette (hph cassette) flanked with the terminal specific homologus sequences of FDS, GT, SC, and SM genes. Although the four constructed replacement vectors have been tried to transform the monokaryotic strain AC T1 MT14 of A. cinnamomea by biolistic gun, no positive transformants have been secured yet, and not available for functional analysis. To overcome the obstacles, refine the transformation protocol and expression of these genes in yeast host will be continued later on.