De novo transcriptome analysis of Dysoxylum binectariferum to unravel the biosynthesis of pharmaceutically relevant specialized metabolites.

The tropical tree, D. binectariferum, is a prominent source of chromone alkaloid rohitukine, which is used in the semi-syntheses of anticancer molecules such as flavopiridol and P-276-00. The biosynthetic pathway of rohitukine or its derivatives is currently unknown in plants. Here, we explored chromone alkaloid biosynthesis in D. binectariferum through targeted transcriptome sequencing. Illumina sequencing of leaves and roots of a year-old D. binectariferum seedling generated, 42.43 and 38.74 million paired-end short reads, respectively. Quality filtering and de novo assembly of the transcriptome generated 274,970 contigs and 126,788 unigenes with an N50 contig length of 1560 bp. The assembly generated 117,619 translated unigene protein sequences and 51,598 non-redundant sequences. Nearly 80% of these non-redundant sequences were annotated to publicly available protein and nucleotide databases, suggesting the completeness and effectiveness of the transcriptome assembly. Using the assembly, we identified a chalcone synthase (CHS) and three type III polyketide synthases (PKS-III; non-CHS type) that are likely to be involved in the biosynthesis of chromone ring/noreugenin moiety of rohitukine. We also identified key enzymes like lysine decarboxylase in the piperidine pathway that make the piperidine moiety of rohitukine. Besides these, the upstream enzymes in flavonoid biosynthesis like phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H),4-coumarate-CoA ligase (4CL), and chalcone isomerase (CHI) have also been identified. Also, terpene synthases that are likely to be involved in the biosynthesis of various terpenoid scaffolds have been identified. Together, the D. binectariferum transcriptome resource forms a basis for further exploration of biosynthetic pathways of these valuable compounds through functional validation of the candidate genes and metabolic engineering in heterologous hosts. Additionally, the transcriptome dataset generated will serve as an important resource for research on functional genomics and enzyme discovery in D. binectariferum and comparative analysis with other Meliaceae family members.

An endophytic fungus, Gibberella moniliformis from Lawsonia inermis L. produces lawsone, an orange-red pigment.

Lawsone (2-hydroxy-1, 4-napthoquinone), also known as hennotannic acid, is an orange red dye used as a popular skin and hair colorant. The dye is produced in the leaves of Lawsonia inermis L, often referred to as the "henna" tree. In this study, we report the production of lawsone by an endophytic fungus, Gibberella moniliformis isolated from the leaf tissues of Lawsonia inermis. The fungus produced the orange-red dye in potato dextrose agar and broth, independent of the host tissue. Presence of lawsone was confirmed spectrometrically using HPLC and ESI-MS/MS analysis. The fragmentation pattern of lawsone was identical to both standard lawsone and that extracted from plant tissue. This is a first report of lawsone being produced by an endophytic fungus, independent of the host tissue. The study opens up interesting questions on the possible biosynthetic pathway through which lawsone is produced by the fungus.

Pyrenacantha volubilis Wight, (Icacinaceae) a rich source of camptothecine and its derivatives, from the Coromandel Coast forests of India.

Camptothecine, a potent eukaryotic topoisomerase inhibitor, is an important anticancer compound. The global demand for this compound was estimated to be $1 billion in 2003 and is only further expected to increase. Partly to meet the expected increase in demand, in the recent past, several efforts have been made to discover newer and alternative plant and fungal sources of camptothecine. In this study we report a rich source of camptothecine and its natural derivatives, Pyrenacantha volubilis (Icacinaceae) from the eastern coast of peninsular India. Camptothecine and its derivatives were analyzed using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in all plant parts such as twigs, leaves, roots, seedling, ripened whole fruit, fruit coat, seed coat and cotyledons. Cotyledons and ripened whole fruits contained the highest amount of camptothecine (1.35% and 0.60% dry weight respectively). LC-MS and ESI-MS/MS analyses revealed besides camptothecine, other derivatives and precursors such as 10-hydroxycamptothecine, 9-methoxycamptothecine, 20-deoxycamptothecine, deoxypumiloside, strictosidine and strictosamide. Pure camptothecine was isolated from fruits and structurally confirmed using NMR. Seed extracts were found to be effective against breast cancer, ovarian, colon and carcinoma cell lines (with IC50 values of 4.0 µg/mL, 6.5 µg/mL, 25.0 µg/mL and 25.0 µg/mL respectively). We discuss the results in the context of exploring alternative sources of camptothecine.