Defining the role of a caffeic acid 3-O-methyltransferase from Azadirachta indica fruits in the biosynthesis of ferulic acid through heterologous over-expression in Ocimum species and Withania somnifera.

MAIN CONCLUSION: The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and characterized from Neem. The gene is involved in ferulic acid biosynthesis, a key intermediate component of lignin biosynthesis. Azadirachta indica (Neem) is a highly reputed traditional medicinal plant and is phytochemically well-known for its limonoids. Besides limonoids, phenolics are also distinctively present, which add more medicinal attributes to Neem. Caffeic acid is one of such phenolic compound and it can be converted enzymatically into another bioactive phytomolecule, ferulic acid. This conversion requires transfer of a methyl group from a donor to caffeic acid under the catalytic action of an appropriate methyltransferase. In this study, caffeic acid 3-O-methyltransferase gene from Neem (NCOMT) fruits has been isolated and heterologously expressed in E. coli. The recombinant NCOMT enzyme was purified, which exhibited efficient catalytic conversion of caffeic acid into ferulic acid, a highly potential pharmaceutical compound. The purified recombinant enzyme was physico-kinetically characterized for its catalysis. The analysis of tissue-wide expression of NCOMT gene revealed interesting pattern of transcript abundance reflecting its role in the development of fruit tissues. Further, NCOMT was heterologously overexpressed in Withania somnifera and Ocimum species, to analyze its role in ferulic acid biosynthesis in planta. Thus, the study provides insight for the endogenous role of NCOMT in ferulic acid biosynthesis en route to lignin, an important structural component. To the best of our knowledge, NCOMT pertains to be the first enzyme of the secondary metabolism that has been purified and kinetically characterized from Neem. This study may also have important prospects of applications as the observation on heterologous expression of NCOMT showed its involvement in the maintenance of the in vivo pool of ferulic acid in the plants. Thus, the study involving NCOMT opens up new dimensions of metabolic engineering approaches for the biosynthesis of potential therapeutically important phytomolecules in heterologous systems.

WRKY1-mediated regulation of tryptophan decarboxylase in tryptamine generation for withanamide production in Withania somnifera (Ashwagandha).

KEY MESSAGE: WsWRKY1-mediated transcriptional modulation of Withania somnifera tryptophan decarboxylase gene (WsTDC) helps to regulate fruit-specific tryptamine generation for production of withanamides. Withania somnifera is a highly valued medicinal plant. Recent demonstration of novel indolyl metabolites called withanamides in its fruits (berries) prompted us to investigate its tryptophan decarboxylase (TDC), as tryptophan is invariably a precursor for indole moiety. TDC catalyzes conversion of tryptophan into tryptamine, and the catalytic reaction constitutes a committed metabolic step for synthesis of an array of indolyl metabolites. The TDC gene (WsTDC) was cloned from berries of the plant and expressed in E. coli. The recombinant enzyme was purified and characterized for its catalytic attributes. Catalytic and structural aspects of the enzyme indicated its regulatory/rate-limiting significance in generation of the indolyl metabolites. Novel tissue-wise and developmentally differential abundance of WsTDC transcripts reflected its preeminent role in withanamide biogenesis in the fruits. Transgenic lines overexpressing WsTDC gene showed accumulation of tryptamine at significantly higher levels, while lines silenced for WsTDC exhibited considerably depleted levels of tryptamine. Cloning and sequence analysis of promoter of WsTDC revealed the presence of W-box in it. Follow-up studies on isolation of WsWRKY1 transcription factor and its overexpression in W. somnifera revealed that WsTDC expression was substantially induced by WsWRKY1 resulting in overproduction of tryptamine. The study invokes a key role of TDC in regulating the indolyl secondary metabolites through enabling elevated flux/supply of tryptamine at multiple levels from gene expression to catalytic attributes overall coordinated by WsWRKY1. This is the first biochemical, molecular, structural, physiological and regulatory description of a fruit-functional TDC.

Characterization of a novel amylosucrase gene from the metagenome of a thermal aquatic habitat, and its use in turanose production from sucrose biomass.

Turanose is a natural isomer of sucrose. It is an emerging functional sweetener of the next generation. Turanose is catalytically synthesized from the sucrose biomass by employing amylosucrase enzyme. In this study, a novel gene encoding amylosucrase (Asmet) has been identified from the metagenome of a thermal aquatic habitat. Asmet exhibits 37-55% identity at the protein level with the known amylosucrases characterized till date. Asmet was cloned and expressed in Escherichia coli, followed by protein purification, and characterization. Asmet protein exhibited the maximum total activity at 9.0 pH and 60 °C temperature, whereas, 8.0 pH and 50 °C temperature were found optimum for transglycosylation activity. Asmet showed fairly high thermal tolerance at 50 °C. The conjugation of Asmet protein with functionalized iron nanoparticles significantly improved its thermal tolerance, showing hardly any loss in the enzyme's activity even after 72 h of heat (50 °C) exposure. The turanose yield of about 47% was achieved from 1.5 M sucrose, containing 0.5 M fructose in the reaction. Turanose was purified (˜95%) via a bio-physical process, and characterized by TLC, HPLC, and NMR. The novel amylosucrase gene was demonstrated to be a potential candidate for turanose production, utilizing various sucrose containing feedstocks.

Kinetic characterization of laccase from Bacillus atrophaeus, and its potential in juice clarification in free and immobilized forms.

In the present study, a laccase gene (BaLc) from a lignin degrading bacterium, Bacillus atrophaeus, has been cloned and expressed in Escherichia coli. The optimal catalytic activity of the protein was achieved at 5.5 pH and 35°C temperature, measured by oxidation of ABTS. The Km and Vmax values were determined as 1.42 mM and 4.16 µmole/min, respectively. To achieve the enzyme recovery, the biocatalyst (BaLc) was covalently attached onto the functionalized iron magnetic-nanoparticles. The nanoparticles were characterized by zeta-potential and FTIR analyses. The immobilized BaLc enzyme was physico-kinetically characterized, exhibiting retention of 60% of the residual activity after ten reaction cycles of ABTS oxidation. The immobilized biocatalyst system was tested for its biotechnological exploitability in plant juice processing, achieving 41-58% of phenol reduction, 41-58% decolorization, 50-59% turbidity reduction in the extracts of banana pseudo-stem and sweet sorghum stalk, and apple fruit juice. This is the first study to demonstrate the use of nanoparticle-laccase conjugate in juice clarification. The findings suggest that B. atrophaus laccase is a potential catalytic tool for plant juice bioprocessing activities.

Long noncoding RNAs and miRNAs regulating terpene and tartaric acid biosynthesis in rose-scented geranium.

This study aimed to explore the noncoding RNAs, which have emerged as key regulatory molecules in biological processes, in rose-scented geranium. We analyzed RNA-seq data revealing 26 784 long noncoding RNAs (lncRNAs) and 871 miRNAs in rose-scented geranium. A total of 466 lncRNAs were annotated using different plant lncRNA public databases. Furthermore, 372 lncRNAs and 99 miRNAs were detected that target terpene and tartarate biosynthetic pathways. An interactome, comprising of lncRNAs, miRNAs, and mRNAs, was constructed that represents a noncoding RNA regulatory network of the target mRNAs. Real-time quantitative PCR expression validation was done for selected lncRNAs involved in the regulation of terpene and tartaric acid pathways. This study provides the first insights into the regulatory functioning of noncoding RNAs in rose-scented geranium.

Catalytic biosynthesis of levan and short-chain fructooligosaccharides from sucrose-containing feedstocks by employing the levansucrase from Leuconostoc mesenteroides MTCC10508.

Levansucrase gene (LmLEVS) was cloned from Leuconostoc mesenteroides MTCC 10508. The heterologous expression and purification of the truncated (TrLmLEVS) gene, lacking the N-terminal signal peptide, was performed in Escherichia coli. The recombinant enzyme (TrLmLEVS) was physico-kinetically characterized using sucrose as substrate. TrLmLEVS exhibited the maximum activity at pH 6 and temperature 30 °C. Thin layer chromatography and high performance liquid chromatography analyses unveiled the biosynthesis of fructooligosaccharides and levan by TrLmLEVS using sucrose as substrate. The catalytically synthesized polymer was characterized by Fourier-Transform Infrared Spectroscopy and Nuclear Magnetic Resonance analyses, confirming it as levan. TrLmLEVS was capable of catalyzing the transformation of raffinose-derived molecules, besides sucrose, into fructans. Further, TrLmLEVS was employed for the genesis of non-digestible fructans from sucrose-containing feedstocks like table sugar, jaggery, cane molasses, and sweet sorghum juice. The results suggest that Leu. mesenteroides MTCC 10508 levansucrase is a potential candidate for the production of levan-type biomolecules in plant-based food products.

HMG-CoA reductase from Camphor Tulsi (Ocimum kilimandscharicum) regulated MVA dependent biosynthesis of diverse terpenoids in homologous and heterologous plant systems.

Ocimum kilimandscharicum is unique in possessing terpenoids whereas other Ocimum species are renowned for phenylpropanoids as major constituents of essential oil. The key enzyme of MVA/terpenoid metabolic pathway viz 3-hydroxy-3-methylglutaryl Co-A reductase (OkHMGR) of 1.7-Kb ORF encoding ~60-kDa protein was cloned from O. kilimandscharicum and its kinetic characteristics revealed the availability of HMG-CoA as a control point of MVA-pathway. Transcript profiling of the OkHMGR elucidated tissue-specific functions of the gene in flower and leaf tissues in accumulation of terpenoidal essential oil. OkHMGR was differentially regulated in response to exposure to methyl-jasmonate, salicylic-acid, and stress conditions such-as salt and temperature stress, demonstrating its key role in managing signaling and stress-responses. To elucidate its functional role, OkHMGR was transiently over-expressed in homologous and heterologous plants such as O. sanctum, O. basilicum, O. gratissimum, Withania somnifera and Artemisia annua. The over-expression and inhibition dual strategy revealed that the additional OkHMGR in-planta could afford endogenous flow of isoprenoid units towards synthesis of terpenoids. The present study provides in-depth insight of OkHMGR in regulation of biosynthesis of non-plastidal isoprenoids. This is first report on any gene of MVA/isoprenoid pathway from under-explored Camphor Tulsi belonging to genus Ocimum. Studies also suggested that OkHMGR could be a potential tool for attempting metabolic engineering for enhancing medicinally important terpenoidal metabolites in plants.

Transcription factor repertoire in Ashwagandha (Withania somnifera) through analytics of transcriptomic resources: Insights into regulation of development and withanolide metabolism.

Transcription factors (TFs) are important regulators of cellular and metabolic functions including secondary metabolism. Deep and intensive RNA-seq analysis of Withania somnifera using transcriptomic databases provided 3532 annotated transcripts of transcription factors in leaf and root tissues, belonging to 90 different families with major abundance for WD-repeat (174 and 165 transcripts) and WRKY (93 and 80 transcripts) in root and leaf tissues respectively, followed by that of MYB, BHLH and AP2-ERF. Their detailed comparative analysis with Arabidopsis thaliana, Capsicum annum, Nicotiana tabacum and Solanum lycopersicum counterparts together gave interesting patterns. However, no homologs for WsWDR representatives, LWD1 and WUSCHEL, were observed in other Solanaceae species. The data extracted from the sequence read archives (SRA) in public domain databases were subjected to re-annotation, re-mining, re-analysis and validation for dominant occurrence of WRKY and WD-repeat (WDR) gene families. Expression of recombinant LWD1 and WUSCHEL proteins in homologous system led to enhancements in withanolide content indicating their regulatory role in planta in the biosynthesis. Contrasting expression profiles of WsLWD1 and WsWUSCHEL provided tissue-specific insights for their participation in the regulation of developmental processes. The in-depth analysis provided first full-spectrum and comparative characteristics of TF-transcripts across plant species, in the perspective of integrated tissue-specific regulation of metabolic processes including specialized metabolism.

Withania coagulans tryptophan decarboxylase gene cloning, heterologous expression, and catalytic characteristics of the recombinant enzyme.

Tryptophan decarboxylase (EC 4.1.1.28) catalyzes pyridoxal 5'-phosphate (PLP)-dependent decarboxylation of tryptophan to produce tryptamine for recruitment in a myriad of biosynthetic pathways of metabolites possessing indolyl moiety. A recent report of certain indolyl metabolites in Withania species calls for a possible predominant functional role of tryptophan decarboxylase (TDC) in the genome of Withania species to facilitate production of the indolyl progenitor molecule, tryptamine. Therefore, with this metabolic prospection, we have identified and cloned a full-length cDNA sequence of TDC from aerial tissues of Withania coagulans. The functional WcTDC gene comprises of 1506 bp open reading frame (ORF) encoding a 502 amino acid protein with calculated molecular mass and pI value of 56.38 kDa and 8.35, respectively. The gene was expressed in Escherichia coli, and the recombinant enzyme was affinity-purified to homogeneity to discern its kinetics of catalysis. The enzyme (WcTDC) exhibited much higher Km value for tryptophan than for pyridoxal 5'-phosphate and was dedicated to catalyze decarboxylation of only tryptophan or, to a limited extent, of its analogue (like 5-hydroxy tryptophan). The observed optimal catalytic functionality of the enzyme on the slightly basic side of the pH scale and at slightly higher temperatures reflected adaptability of the plant to hot and arid regions, the predominant natural habitat of the herb. This pertains to be the first report on cloning and characterization of heterologously expressed recombinant enzyme from W. coagulans and forms a starting point to further understanding of withanamide biosynthesis.