Milen I. Georgiev

Hairy root type plant in vitro systems as sources of bioactive substances.

Abstract“Hairy root” systems, obtained by transforming plant tissues with the “natural genetic engineer” Agrobacterium rhizogenes, have been known for more than three decades. To date, hairy root cultures have been obtained from more than 100 plant species, including several endangered medicinal plants, affording opportunities to produce important phytochemicals and proteins in eco-friendly conditions. Diverse strategies can be applied to improve the yields of desired metabolites and to produce recombinant proteins. Furthermore, recent advances in bioreactor design and construction allow hairy root-based technologies to be scaled up while maintaining their biosynthetic potential. This review highlights recent progress in the field and outlines future prospects for exploiting the potential utility of hairy root cultures as “chemical factories” for producing bioactive substances.

Bioprocessing of plant cell cultures for mass production of targeted compounds

More than a century has passed since the first attempt to cultivate plant cells in vitro. During this time, plant cell cultures have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. Furthermore, plant cell culture is the only economically feasible way of producing some high-value metabolites (e.g., paclitaxel) from rare and/or threatened plants. This review summarizes recent advances in bioprocessing aspects of plant cell cultures, from callus culture to product formation, with particular emphasis on the development of suitable bioreactor configurations (e.g., disposable reactors) for plant cell culture-based processes; the optimization of bioreactor culture environments as a powerful means to improve yields; bioreactor operational modes (fed-batch, continuous, and perfusion); and biomonitoring approaches. Recent trends in downstream processing are also considered.

Advances in the biotechnological glycosylation of valuable flavonoids.

The natural flavonoids, especially their glycosides, are the most abundant polyphenols in foods and have diverse bioactivities. The biotransformation of flavonoid aglycones into their glycosides is vital in flavonoid biosynthesis. The main biological strategies that have been used to achieve flavonoid glycosylation in the laboratory involve metabolic pathway engineering and microbial biotransformation. In this review, we summarize the existing knowledge on the production and biotransformation of flavonoid glycosides using biotechnology, as well as the impact of glycosylation on flavonoid bioactivity. Uridine diphosphate glycosyltransferases play key roles in decorating flavonoids with sugars. Modern metabolic engineering and proteomic tools have been used in an integrated fashion to generate numerous structurally diverse flavonoid glycosides. In vitro, enzymatic glycosylation tends to preferentially generate flavonoid 3- and 7-O-glucosides; microorganisms typically convert flavonoids into their 7-O-glycosides and will produce 3-O-glycosides if supplied with flavonoid substrates having a hydroxyl group at the C-3 position. In general, O-glycosylation reduces flavonoid bioactivity. However, C-glycosylation can enhance some of the benefits of flavonoids on human health, including their antioxidant and anti-diabetic potential.

Genetically transformed roots: from plant disease to biotechnological resource

Hairy root syndrome is a disease that is induced by Agrobacterium rhizogenes infection and characterized by a proliferation of excessively branching roots. However, in the past 30 years A. rhizogenes-mediated transformation has also provided a valuable platform for studying biosynthesis pathways in plants. Furthermore, the genetically transformed root cultures are becoming increasingly attractive, cost-effective options for mass-producing desired plant metabolites and expressing foreign proteins. Numerous proof-of-concept studies have demonstrated the feasibility of scaling up hairy-root-based processes while maintaining their biosynthetic potential. Recently, hairy roots have also shown immense potential for applications in phytoremediation, that is, plant-based decontamination of polluted environments. This review highlights recent progress and limitations in the field, and outlines future perspectives for the industrial exploitation of hairy roots.

Hosting the plant cells in vitro: recent trends in bioreactors

Biotechnological production of high-value metabolites and therapeutic proteins by plant in vitro systems has been considered as an attractive alternative of classical technologies. Numerous proof-of-concept studies have illustrated the feasibility of scaling up plant in vitro system-based processes while keeping their biosynthetic potential. Moreover, several commercial processes have been established so far. Though the progress on the field is still limited, in the recent years several bioreactor configurations has been developed (e.g., so-called single-use bioreactors) and successfully adapted for growing plant cells in vitro. This review highlights recent progress and limitations in the bioreactors for plant cells and outlines future perspectives for wider industrialization of plant in vitro systems as “green cell factories” for sustainable production of value-added molecules.

Metabolic differentiations and classification of Verbascum species by NMR-based metabolomics

The genus Verbascum L. (mulleins) comprises of about 360 species of flowering plants in the Scrophulariaceae family. Mulleins have been used in the traditional folk medicine for centuries, for treatment of a wide range of human ailments, inter alia bronchitis, tuberculosis, asthma, and different inflammations. Despite all applications the knowledge of the metabolites, accumulated in different mullein species, is still limited and based mainly on determination of the major compounds. Here we report the application of 1H NMR metabolic fingerprinting in combination with principal component analyses (PCA) in five different Verbascum species. Based on the obtained results mulleins were divided in two groups: group A (Verbascum phlomoides and Verbascum densiflorum) and group B (Verbascum xanthophoeniceum, Verbascum nigrum and Verbascum phoeniceum). Further it was found that the plants in group B accumulate higher amounts of bioactive iridoid and phenylethanoid glycosides. V. xanthophoeniceum and V. nigrum accumulate higher amounts of the pharmaceutically-important harpagoside (∼0.5% on dry weight basis) and verbascoside, forsythoside B and leucosceptoside B (in total 5.6-5.8% on dry weight basis), which underlines the possibility for their application in pharmaceutical industry. To the best of our knowledge this is the first report on the analyses of Verbascum sp. leaf metabolome.

Modifications of Dietary Flavonoids towards Improved Bioactivity: An Update on Structure-activity Relationship

ABSTRACT Over the past two decades, extensive studies have revealed that inflammation represents a major risk factor for various human diseases. Chronic inflammatory responses predispose to pathological progression of chronic illnesses featured with penetration of inflammatory cells, dysregulation of cellular signaling, excessive generation of cytokines, and loss of barrier function. Hence, the suppression of inflammation has the potential to delay, prevent, and to treat chronic diseases. Flavonoids, which are widely distributed in humans daily diet, such as vegetables, fruits, tea and cocoa, among others, are considered as bioactive compounds with anti-inflammatory potential. Modification of flavonoids including hydroxylation, o-methylation, and glycosylation, can alter their metabolic features and affect mechanisms of inflammation. Structure–activity relationships among naturally occurred flavonoids hence provide us with a preliminary insight into their anti-inflammatory potential, not only attributing to the antioxidant capacity, but also to modulate inflammatory mediators. The present review summarizes current knowledge and underlies mechanisms of anti-inflammatory activities of dietary flavonoids and their influences involved in the development of various inflammatory-related chronic diseases. In addition, the established structure–activity relationships of phenolic compounds in this review may give an insight for the screening of new anti-inflammatory agents from dietary materials.

Ginsenosides: prospective for sustainable biotechnological production

Panax ginseng C.A. Meyer (ginseng) is a well-known medicinal plant that has been traditionally used in the oriental countries for centuries. Wild ginseng is a scarce and rare commodity. Field cultivation of the ginseng plant is a time-consuming and labor-intensive process. Ginsenosides, a group of glycosylated triterpenes, also known as saponins, are the principal bioactive constituents of ginseng. The use of cell and organ culture processes has been sought as a potential alternative for the efficient mass production of ginseng raw material. Various bioprocessing strategies have been developed to date. Cells and adventitious roots have been cultured in large-scale bioreactors and various strategies have been developed accordingly for the enhancement of biomass and ginsenoside accumulation. This review highlights the recent progress in the cultivation of ginseng cell and organ cultures for the production of ginsenosides from bioreactor cultures. In addition, the metabolism and biochemistry of ginsenoside biosynthesis, genomic and proteomic studies in ginseng, metabolic engineering, biosafety, toxicological evaluation, and efficacy assessment of ginseng raw material are also summarized and thoroughly discussed.

Optimization of Rosmarinic Acid Production by Lavandula vera MM Plant Cell Suspension in a Laboratory Bioreactor

The all‐round effect of dissolved oxygen concentration, agitation speed, and temperature on the rosmarinic acid production by Lavandula veraMM cell suspension was studied in a 3‐L laboratory bioreactor by means of the modified Simplex method. Polynomial regression models were elaborated for description of the process of rosmarinic acid production (Y) in the bioreactor as a consequence of the variation of the dissolved oxygen (X1) concentration between 10% and 50%; agitation (X2) between 100 and 400 rpm; and temperature (X3) between 22 and 30 °C. The optimization made it possible to establish the optimal conditions for the biosynthesis of rosmarinic acid by L. veraMM: dissolved oxygen (X1*), 50% of air saturation; agitation (X2*), 400 rpm; and temperature (X3*), 29.9 °C, where maximal yield (Ymax) of 3489.4 mg/L of rosmarinic acid was achieved (2 times higher compared with the shake‐flasks cultivation).

Harpagoside: from Kalahari Desert to pharmacy shelf.

Harpagoside is an iridoid glycoside that was first isolated from Harpagophytum procumbens (devils claw, Pedaliaceae), a medicinal plant in which it is the major constituent of the iridoid pool. Both the pure compound and devils claw extracts have potent anti-rheumatic, anti-inflammatory and analgesic effects. According to the European Pharmacopoeia commercial devils claw products should contain at least 1.2% harpagoside. However, the compound has also been isolated from several other plant species and in vitro plant culture systems. Recent advances in knowledge of harpagoside distribution, biosynthesis/accumulation and pharmacology are summarized in this review. We also discuss the possible synergism and/or antagonism between major constituents in harpagoside-containing phytopharmaceutical products. Finally, future perspectives for its potential application are highlighted.