A new synthetic biology approach for the production of curcumin and its glucoside in Atropa belladonna hairy roots.

Curcumin has ignited global interest as an elite drugable molecule, owing to its time-honoured pharmacological activities against diverse human ailments. Limited natural accessibility and poor oral bioavailability caused major hurdles in the curcumin-based drug development process. We report the first successful testimony of curcumin and its glucoside synthesis in Atropa belladonna hairy roots (HR) through metabolic engineering. Re-routing the inherent biosynthetic precursors of the phenylpropanoid pathway of A. belladonna by heterologous expression of key curcumin biosynthetic pathway genes (i.e., Diketide-CoA synthase-DCS and Curcumin synthase-CURS3) and glucosyltransferase gene (CaUGT2) resulted in the production of curcumin and its glucoside in HR clones. Under shake-flask cultivation, the PGD2-HR1clone bearing DCS/ CURS3 genes showed the maximum curcumin yield (180.62 ± 4.7 µg/g DW), while the highest content of curcumin monoglucoside (32.63 ± 2.27 µg/g DW) along with curcumin (67.89 ± 2.56 µg/g DW) were noted in the PGD3-HR3 clone co-expressing DCS/CURS3 and CaUGT2 genes. Bioreactor up-scaling showed yield improvements in the PGD2-HR1 (2.3 fold curcumin) and the PGD3-HR3 clone (0.9 and 1.65 folds of curcumin-monoglucoside and curcumin respectively). These findings proved the advantageous use of HR cultures as the production source for curcumin and its glucoside, which remained unexplored so far.

Anti-cancer labdane diterpenoids from adventitious roots of Andrographis paniculata: augmentation of production prospect endowed with pathway gene expression.

Andrographolide (AD) is the time-honoured pharmacologically active constituent of the traditionally renowned medicinal plant-Andrographis paniculata. Advancements in the target-oriented drug discovery process have further unravelled the immense therapeutic credibility of another unique molecule-neoandrographolide (NAD). The escalated market demand of these anti-cancer diterpenes is increasingly facing unrelenting hurdles of demand and supply disparity, attributable to their limited yield. Callus and adventitious root cultures were generated to explore their biosynthetic potentials which first time revealed NAD production along with AD. Optimization of the types and concentrations of auxins along with media form and cultivation time led to the successful tuning towards establishing adventitious roots as a superior production alternative for both AD/NAD. Supplementation of IBA to the NAA + Kn-containing MS medium boosted the overall growth and AD/NAD synthesis in the adventitious roots. Compared to control leaves, the adventitious root exhibited about 2.61- and 8.8-fold higher contents of AD and NAD, respectively. The qRT-PCR involving nine key pathway genes was studied, which revealed upregulation of GGPS1 and HMGR1/2 genes and downregulation of DXS1/2 and HDR1/2 genes in the adventitious root as compared to that in the control leaves. Such observations highlight that in vitro cultures can serve as efficient production alternatives for AD/NAD as the cytosolic genes (HMGR1/2 of MVA pathway) are competent enough to take over from the plastidial genes (DXS1/2 and HDR1/2 of MEP pathway), provided the accredited first branch-point regulatory gene (GGPS) expression and the culture requirements are optimally fulfilled.

Advances in Boerhaavia diffusa hairy root technology: a valuable pursuit for identifying strain sensitivity and up-scaling factors to refine metabolite yield and bioactivity potentials.

The present study reports the Agrobacterium rhizogenes-mediated hairy root induction in of an ethno-medicinally significant herb-Boerhaavia diffusa L., for elucidating the underlying competence regarding its biosynthetic (i.e. boeravinone B and eupalitin) and bioactivity (antibacterial, antioxidant and anti-inflammatory) potentials. Host plant-specific receptiveness towards A. rhizogenes strains and disparity in compatibility threshold of leaf and nodal explants were evident. Only leaf explants responded, attaining hairy root induction with the ATCC 15834 followed by A4 and SA79 strains in reducing order of transformation efficiency. The growth behaviours differed amongst independent rhizoclones, and two clones of A4 (RBH) and ATCC 15834 (RBT8) origin demonstrated higher growth potentials. Polymerase chain reaction amplification of rol genes confirmed their transformed nature. Optimization of the appropriate solvent and reverse phase high-performance liquid chromatography parameters relating to the targeted metabolite production in the selected RBH and RBT8 clones revealed higher accumulation of eupalitin with the RBH clone having the best result of 1.44 times greater yield over the control root. Compared to the selected rhizoclones, the control roots however showed higher boeravinone B content. Devising a modified "stirred-tank" reactor through equipping with marine impellers and ring spargers facilitated high-density RBH root biomass yield with 6.1-fold and 1.15-fold yield increment of the boeravinone B and eupalitin respectively compared to shake-flask cultures. Considering the control roots, the RBH clone revealed analogous antioxidant/antibacterial activities with improved anti-inflammatory potential. The hairy root mediated higher production of boeravinone B and eupalitin could be achieved for the first time in bioreactor.

Production of anti-cancer triterpene (betulinic acid) from callus cultures of different Ocimum species and its elicitation.

Betulinic acid (BA), a pentacyclic triterpenoid, is gaining unmatched attention owing to its unique anti-cancer activity with selective melanoma growth inhibition without damaging normal cells. It is also well-known for its multifaceted pharmacokinetics, entailing antibacterial, antimalarial, anti-HIV and antioxidant merits. Considering the escalating demand with diminishing bioresource of this molecule, the present study was undertaken that revealed the untapped potentials of Ocimum calli, contrasting to that in the in vitro derived leaves, as effective production alternative of BA in three out of four tested species (i.e. Ocimum basilicum, Ocimum kilimandscharicum, Ocimum sanctum excluding Ocimum grattisimum). Callus inductions were obtained in all the four species with different 2,4-dichlorophenoxyacetic acid (2,4-D)/α-naphthaleneacetic acid (NAA) concentrations with kinetin. Notably, 2,4-D favoured maximum callus growth in all whereas NAA proved beneficial for the highest metabolite yield in the calli of each BA-producing species. The O. basilicum calli demonstrated the maximum growth (growth index (GI) 678.7 ± 24.47) and BA yield (2.59 ± 0.55 % dry weight [DW]), whereas those in O. kilimandscharicum (GI 533.33 ± 15.87; BA 1.87 ± 0.6 % DW) and O. sanctum (GI 448 ± 16.07; BA 0.39 ± 0.12 % DW) followed a descending order. The O. gratissimum calli revealed minimum growth (GI 159 ± 13.25) with no BA accumulation. Elicitation with methyl jasmonate at 200-µM concentration after 48-h exposure doubled the BA yield (5.10 ± 0.18 % DW) in NAA-grown O. basilicum calli compared to that in the untreated counterpart (2.61 ± 0.19 % DW), which further enthused its future application.

Long-term stability in biomass and production of terpene indole alkaloids by hairy root culture of Rauvolfia serpentina and cost approximation to endorse commercial realism.

The effect of 6 years of cultivation and use of table-sugar (TS) on the biomass/terpene alkaloid productivities and rol gene expression were studied in a hairy root (HR) clone of Rauvolfia serpentina. The media cost could be reduced >94 % by replacing sucrose (SUC) with TS­an unexplored avenue for HR cultivation. The overall productivities increased over long-term cultivation with sugar proving superior to SUC for biomass (24.4 ± 2.11 g/l DW after 40 days to 17.31 % higher) and reserpine (0.094 ± 0.008 % DW after 60 days to 193.8 % more) production. The latter however revealed comparatively better yields concerning ajmaline (0.507 ± 0.048 % DW after 60 days to 61.98 % higher) and yohimbine (0.628 ± 0.062 % DW after 60 days to 38.32 % higher), respectively. PCR amplification of rol genes confirmed long-term expression stability.

Isolation, structure determination, and antiaging effects of 2,3-pentanediol from endophytic fungus of Curcuma amada and docking studies.

An endophytic fungus was isolated from the rhizomes of Curcuma amada (Zingiberaceae), which was identified as Fusarium oxysporum on the basis of its morphological and molecular characters. Chromatographic separation and spectroscopic analysis of the fungal metabolite (chloroform extract) led to the identification of one pure compound having molecular formula C5H12O2, i.e., 2,3-pentanediol (1). Activity analysis of compound 1 demonstrated improved antiaging (antioxidant, thermotolerance) properties against Caenorhabditis elegans, in comparison to a similar, commercially available molecule i.e., 1,5-pentanediol (2). The effective (lower) concentration of 1 significantly showed (28.6%) higher survival percentage of the worms under thermal stress (37 ºC) compared to its higher concentration (25.3%), while similar trends were followed in oxidative stress where (22.2%) higher survival percentage was recorded in comparison to untreated control. The compound 1, however, lacked potential antimicrobial activity, indicating the plausible ramification of the position of OH group in such bioactive molecules. In silico evaluation of these molecules against common as well as unique targets corroborated better antiaging potential of 1 in comparison to that of 2. The results for the first time indicated that the utilization of the endophytic fungi of C. amada could, thus, be a possible source for obtaining non-plant-based bioactive compounds having broader therapeutic applications pertaining to age-related progressions.

Biotransformation studies using hairy root cultures - A review.

Agrobacterium rhizogenes induced hairy root cultures are entering into a new juncture of functional research in generating pharmaceutical lead compounds by bringing about chemical transformations aided through its inherent enzyme resources. Rational utilization of hairy root cultures as highly effective biotransformation systems has come into existence in the last twenty years involving a wide range of plant systems as well as exogenous substrates and diverse chemical reactions. To date, hairy root cultures are preferred over plant cell/callus and suspension cultures as biocatalyst due to their genetic/biochemical stability, hormone-autotrophy, multi-enzyme biosynthetic potential mimicking that of the parent plants and relatively low-cost cultural requirements. The resultant biotransformed molecules, that are difficult to make by synthetic organic chemistry, can unearth notable practical efficacies by acquiring improved physico-chemical properties, bioavailability, lower toxicity and broader therapeutic properties. The present review summarizes the overall reported advances made in the area of hairy root mediated biotransformation of exogenous substrates with regard to their reaction types, plant systems associated, bacterial strains/molecules involved and final product recovery.

A bioactive labdane diterpenoid from Curcuma amada and its semisynthetic analogues as antitubercular agents.

A labdane diterpene dialdehyde was first time isolated from the chloroform extract of rhizomes of Curcuma amada. This compound exhibited antitubercular activity (MIC=500 microg/mL) against Mycobacterium tuberculosis H(37)Rv strain in BACTEC-460 assay. Two of its semisynthetic analogues also exhibited antitubercular activity at 250-500 microg/mL. It is the first report on isolation and antimycobacterial activity of this dialdehyde from C. amada.