Transcriptome analysis based on next-generation sequencing of non-model plants producing specialized metabolites of biotechnological interest

Abstract

Plants produce a vast array of specialized metabolites, many of which are used as pharmaceuticals, flavors, fragrances, and other high-value fine chemicals. However, most of these compounds occur in non-model plants for which genomic sequence information is not yet available. The production of a large amount of nucleotide sequence data using next-generation technologies is now relatively fast and cost-effective, especially when using the latest Roche-454 and Illumina sequencers with enhanced base-calling accuracy. To investigate specialized metabolite biosynthesis in non-model plants we have established a data-mining framework, employing next-generation sequencing and computational algorithms, to construct and analyze the transcriptomes of 75 non-model plants that produce compounds of interest for biotechnological applications. After sequence assembly an extensive annotation approach was applied to assign functional information to over 800,000 putative transcripts. The annotation is based on direct searches against public databases, including RefSeq and InterPro. Gene Ontology (GO), Enzyme Commission (EC) annotations and associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps are also collected. As a proof-of-concept, the selection of biosynthetic gene candidates associated with six specialized metabolic pathways is described. A web-based BLAST server has been established to allow public access to assembled transcriptome databases for all 75 plant species of the PhytoMetaSyn Project (www.phytometasyn.ca).

Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae

“…Next generation DNA sequencing technology has provided rapid access to the genetic diversity underpinning the immense biosynthetic capacity of plants and microbes 5,6 . Although Saccharomyces cerevisiae has traditionally been used for the biosynthesis of simple molecules derived from central metabolism 7 , advances in recombinant DNA technology streamlining the cloning of large DNA sequences make this microbe an attractive platform for functional characterization of enzymes as well as the reconstitution of complex metabolic pathways 8,9 .…”

A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids

“…were processed for combined 454 sequencing and annotation (www.phytometasyn.ca; Facchini et al, 2012;Xiao et al, 2013). Mining of each annotated database for g-TLMTs related to CrDhtNMT produced nine candidate genes that could be investigated for their possible roles as MIA N-methyltransferases.…”

“…A novel class of g-TLMTs involved in the third-to-last step in vindoline biosynthesis was recently discovered (Liscombe et al, 2010;Liscombe and O'Connor, 2011), and its sequence was used in this study for large-scale transcriptome analysis of several MIA-producing plant species (www.phytometasyn.ca; Facchini et al, 2012;Xiao et al, 2013). This study identifies nine new g-TLMTs (Supplemental Fig.…”

“…The database assembly and transcript annotations from A. hubrichtii, C. roseus, R. serpentina, Tabernaemontana elegans, V. minor, and Lonicera japonica transcriptomes (Facchini et al, 2012;Xiao et al, 2013), found at www. phytometasyn.ca, were used to search for g-TLMTs.…”

“…Candidate g-TLMT genes were identified by searching annotated EST databases (www. phytometasyn.ca; Facchini et al, 2012;Xiao et al, 2013) of C. roseus, V. minor, and R. serpentina for a number of cDNAs that encode putative g-TLMTs related to CrDhtNMT. We also report the molecular cloning and functional characterization of two novel candidate g-TLMTs isolated from R. serpentina (RsPiNMT) and V. minor VmPiNMT), respectively, that N-methylate picrinine to generate ervincine (Fig.…”