Fig 1.
Proposed biosynthetic routes leading from L-phenylalanine to amphetamine-type alkaloids in khat and Ephedra sinica.
A CoA-independent, non-β-oxidative pathway of L-phenylalanine side chain-shortening is shown in blue, whereas a CoA-dependent, β-oxidative route is shown in purple. Benzaldehyde, benzoic acid and/or benzoyl-CoA undergo condensation with pyruvate, a reaction putatively catalyzed by a ThDP-dependent carboligase. 1-Phenylpropane-1,2-dione undergoes transamination to yield (S)-cathinone, which is reduced to cathine and (1R,2S)-norephedrine. N-Methylation is restricted to Ephedra spp. and does not occur in khat. Activity has been detected for enzymes highlighted in yellow, and corresponding genes are available for enzymes highlighted in green. Enzymes highlighted in red have not been isolated, although database mining revealed numerous potential candidates (Tables 2 and 3). Abbreviations: CoA, Coenzyme A; NAD(H), nicotinamide adenine dinucleotide; NADP(H), nicotinamide adenine dinucleotide phosphate. PAL, phenylalanine ammonia lyase; ThDP, thiamine diphosphate.
Table 1.
Summary of the construction and assembly for three Illumina NGS libraries.
Table 2.
Khat unigenes representing enzymes putatively involved in ephedrine alkaloid biosynthesis.
Table 3.
Ephedra sinica unigenes representing enzymes putatively involved in ephedrine alkaloid biosynthesis.
Fig 2.
Functional category analysis based on Gene Ontology (GO) annotations of CED-Trinity (upper panel) and ESI-Trinity (lower panel) unigenes.
Results for ESI-Velvet are found in S2 Fig.
Fig 3.
Phylogenetic analysis of gene candidates.
Abbreviations: benzaldehyde dehydrognase (BDH) (A), thiamin diphosphate-dependent carboligase (ThDPC) (B) and transaminase (TA) (C). Similar analyses for remaining candidates are found in S3 Fig. Sequences were aligned and analyzed for phylogenetic relationships using the neighbor-joining algorithm. Numbers at each node represent bootstrap values calculated using 1000 iterations. Accession numbers are found under Experimental section, and abbreviations are defined in Tables 2 and 3.
Fig 4.
Relative expression of gene candidates identified in khat (CED-Trinity).
FPKM (fragments mapped per kilobase of exon per million reads mapped) is a normalizing statistic measuring gene expression while accounting for variation in gene length [23]. Abbreviations are defined in Tables 2 and 3.
Fig 5.
Relative expression of gene candidates identified in Ephedra sinica (ESI-Trinity).
FPKM (fragments mapped per kilobase of exon per million reads mapped) is a normalizing statistic measuring gene expression while accounting for variation in gene length [23]. Abbreviations are defined in Tables 2 and 3.