Enhancing the Accumulation of Rosavins in Rhodiola rosea L. Plants Grown In Vitro by Precursor Feeding

Rhodiola rosea produces nearly 150 bioactive compounds. Cinnamyl alcohol glycosides (CAGs) are among the most important secondary metabolites which are specific to this plant species, exhibiting adaptogenic properties along with salidroside. However, raw material supplies for the pharmaceutical industry are hindered by limited access to the plant material. The species is endangered and protected in many areas: cultivation is long and ineffective. Precursor feeding has been found to be an effective strategy for improving the production of secondary metabolites in various plant tissues cultures, including in Rhodiola species. In this study, whole R. rosea plants grown in vitro were subjected to three different precursor treatments, including with trans-cinnamic acid, cinnamaldehyde and cinnamyl alcohol at 2 mM concentrations. The different treatments affected the secondary metabolite production differently. Trans-cinnamic acid did not affect the synthesis significantly, which contradicts earlier studies with cell suspensions. On the other hand, cinnamyl alcohol and cinnamaldehyde were beneficial, improving the production rate of rosin and rosavin by 13.8- and 6.9-fold, and 92.7- and 8.0-fold, respectively. The significant improvement in CAG accumulation due to cinnamaldehyde treatment was unexpected based on previous studies. In addition, cinnamaldehyde triggered the production of rosarin, which the other two treatments failed to do. The study presents the beneficial application of precursors to whole plants grown in vitro.

Cell Type Matters: Competence For Alkaloid Metabolism Differs In Two Seed-Derived Cell Strains of Catharanthus Roseus.

Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question, how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seeds of Catharanthus roseus were found not only to differ morphologically, but also in their metabolic competence. This differential competence became manifest under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not just “de-differentiated”, but can differ in metabolic competence. In addition to elicitation, and precursor feeding, the cellular properties of the “biomatter” are highly relevant for the success of plant cell fermentation.

Precursor-Boosted Production of Metabolites in Nasturtium officinale Microshoots Grown in Plantform Bioreactors, and Antioxidant and Antimicrobial Activities of Biomass Extracts

The study demonstrated the effects of precursor feeding on the production of glucosinolates (GSLs), flavonoids, polyphenols, saccharides, and photosynthetic pigments in Nasturtium officinale microshoot cultures grown in Plantform bioreactors. It also evaluated the antioxidant and antimicrobial activities of extracts. L-phenylalanine (Phe) and L-tryptophan (Trp) as precursors were tested at 0.05, 0.1, 0.5, 1.0, and 3.0 mM. They were added at the beginning (day 0) or on day 10 of the culture. Microshoots were harvested after 20 days. Microshoots treated with 3.0 mM Phe (day 0) had the highest total GSL content (269.20 mg/100 g DW). The qualitative and quantitative profiles of the GSLs (UHPLC-DAD-MS/MS) were influenced by precursor feeding. Phe at 3.0 mM stimulated the best production of 4-methoxyglucobrassicin (149.99 mg/100 g DW) and gluconasturtiin (36.17 mg/100 g DW). Total flavonoids increased to a maximum of 1364.38 mg/100 g DW with 3.0 mM Phe (day 0), and polyphenols to a maximum of 1062.76 mg/100 g DW with 3.0 mM Trp (day 0). The precursors also increased the amounts of p-coumaric and ferulic acids, and rutoside, and generally increased the production of active photosynthetic pigments. Antioxidant potential increased the most with 0.1 mM Phe (day 0) (CUPRAC, FRAP), and with 0.5 mM Trp (day 10) (DPPH). The extracts of microshoots treated with 3.0 mM Phe (day 0) showed the most promising bacteriostatic activity against microaerobic Gram-positive acne strains (MIC 250–500 µg/mL, 20–21 mm inhibition zones). No extract was cytotoxic to normal human fibroblasts over the tested concentration range (up to 250 μg/mL).

High performance liquid chromatography based quantification of reserpine in Rauwolfia tetraphylla L. and enhanced production through precursor feeding

Rauwolfia tetraphylla L., is an important medicinal plant in Apocynaceae family and is recognized as an alternative source to Rauwolfia serpentina L., in terms of anti-hypertensive alkaloid production i.e. reserpine. In view of this, the present study is conducted to estimate the reserpine content in different parts (leaf, stem and root) of field grown plants (2 years old), tissue cultured plantlets (R1) (two months old) and cell suspensions cultures (two months old with and without precursor feeding) of R. tetraphylla by using high performance liquid chromatography (HPLC) technique. Overall maximum content of reserpine (in %) was estimated from the root samples. Roots of field grown plants has recorded high percent of reserpine (0.39%) followed by roots of tissue cultured plantlets (0.35%) and root callus based cell suspension cultures (0.38 %) which was fed with precursor amino acid (100 mg/L of tryptophan). In control type of root callus based cell suspension cultures, reserpine content was quantified as 0.14%; by precursor feeding (100 mg/L of tryptophan) it was enhanced to 0.38%. In conclusion, the reserpine content (0.35 and 0.38%) produced by the roots of tissue cultured plantlets (R1) and 100 mg/L tryptophan fed root callus based cell suspensions was comparable to that of the reserpine content (0.39%) of root parts of field grown plants. The present study demonstrates the reserpine production by in vitro cell suspension cultures throughout the year without sacrificing the medicinal plants.

Biosynthesis of Chlorinated Lactylates in Sphaerospermopsis sp. LEGE 00249

Lactylates are an important group of molecules in the food and cosmetic industries. A series of natural halogenated 1-lactylates – chlorosphaerolactyaltes (<b>1</b>-<b>4</b>) – were recently reported from <i>Sphaerospermopsis</i> sp. LEGE 00249. Here, we identify the <i>cly</i> biosynthetic gene cluster, containing all the necessary functionalities to generate and release the natural lactylates. Using a combination of stable isotope-labeled precursor feeding and bioinformatic analysis, we propose that dodecanoic acid and pyruvate are the key building blocks in the biosynthesis of <b>1</b>-<b>4</b>. We additionally report minor analogues of these molecules<b> </b>with varying alkyl chains. The discovery of the <i>cly</i> gene cluster paves the way to accessing industrially-relevant lactylates through pathway engineering.

Biosynthesis of Chlorinated Lactylates in Sphaerospermopsis sp. LEGE 00249

Lactylates are an important group of molecules in the food and cosmetic industries. A series of natural halogenated 1-lactylates – chlorosphaerolactyaltes (<b>1</b>-<b>4</b>) – were recently reported from <i>Sphaerospermopsis</i> sp. LEGE 00249. Here, we identify the <i>cly</i> biosynthetic gene cluster, containing all the necessary functionalities to generate and release the natural lactylates. Using a combination of stable isotope-labeled precursor feeding and bioinformatic analysis, we propose that dodecanoic acid and pyruvate are the key building blocks in the biosynthesis of <b>1</b>-<b>4</b>. We additionally report minor analogues of these molecules<b> </b>with varying alkyl chains. The discovery of the <i>cly</i> gene cluster paves the way to accessing industrially-relevant lactylates through pathway engineering.