Christiane Haas

Sage in vitro cultures: a promising tool for the production of bioactive terpenes and phenolic substances

Extracts of Salvia species are used in traditional medicine to treat various diseases. The economic importance of this genus has increased in recent years due to evidence that some of its secondary metabolites have valuable pharmaceutical and nutraceutical properties.The bioactivity of sage extracts is mainly due to their content of terpenes and polyphenols. The increasing demand for sage products combined with environmental, ecological and climatic limitations on the production of sage metabolites from field-grown plants have led to extensive investigations into biotechnological approaches for the production of Salvia phytochemicals. The purpose of this review is to evaluate recent progress in investigations of sage in vitro systems as tools for producing important terpenoids and polyphenols and in development of methods for manipulating regulatory processes to enhance secondary metabolite production in such systems.

Growth kinetics of a Helianthus annuus and a Salvia fruticosa suspension cell line: Shake flask cultivations with online monitoring system

Plants produce a variety of secondary metabolites to defend themselves against herbivores or to attract pollinating insects. Plant cell biotechnology offers excellent opportunities in order to use such secondary plant metabolites to produce goods with consistent quality and quantity throughout the year, and therefore to act independently from biotic and abiotic environmental factors. This article presents results of an extensive study of plant cell in vitro cultivation in a modern shake flask system with noninvasive online respiration activity monitoring unit. Comprehensive screening experiments confirm the successful transfer of a model culture (sunflower suspension) into the shake flask monitoring device and the suitability of this respiration activity monitoring unit as qualified tool for screening of plant in vitro cultures (sunflower and sage suspension). The authors demonstrate deviations between online and offline data due to varying water evaporation from different culture flask types. The influence of evaporation on growth‐specific parameters thereby rises with increasing cultivation time. Furthermore, possibilities to minimize the impact of evaporation, either by adjusting the inlet air moisture or by measuring the evaporation in combination with an appropriate correction of the measured growth values are shown.

Ploidy levels in Beta vulgaris (red beet) plant organs and in vitro systems

The ploidy levels of the cells in different organs (leaves, petioles and roots) of red beet (Beta vulgaris L.) plants of different ages, as well as of different in vitro systems (transformed hairy roots, calli derived from leaves and rhizogenic calli), were investigated using flow cytometry. Two callus lines with red and yellow phenotypes, derived by mechanical separation of the morphologically heterogeneous rhizogenic callus, were also examined. All investigated samples experienced several cycles of endoreduplication. The older organs exhibited higher levels of polysomaty than the young ones. The highest degree of endoreduplication was found in old petiole tissue and the lowest in the red callus line (cycle values of 1.81 and 0.55, respectively). Interestingly, the callus derived from leaves did not exhibit a 2Cx peak, but was tetraploid, probably due to genetic instability, which may have been caused by prolonged cultivation under in vitro conditions. Red and yellow calli showed significantly lower polysomaty (cycle values of 0.55 and 0.59, respectively) than the primary rhizogenic callus (cycle value of 1.09). The DNA profiles of the two phenotypes differed, possibly reflecting differences in their metabolism.

Production of Oleanolic and Ursolic Acids by Callus Cultures of Salvia Tomentosa Mill.

ABSTRACT Callus cultures of Salvia tomentosa Mill. were induced and analyzed for their capacity to produce oleanolic and ursolic acids. The obtained callus lines showed high variability in their biosynthetic potentials. Flow cytometric investigations showed that this variability was due to polyploidization of plant cells in calli. Polyploidization of callus cell seems to be promoted by the growth regulators used for callus induction. One octaploid line (consisting of 8C, 16C and 32C cells) was selected as prospective producer of oleanolic (991.57 μg/g DW) and ursolic (641.85 μg/g DW) acids. To our knowledge, this is the first report for obtaining of triterpenes producing callus culture of S. tomentosa Mill.

Flow Cytometry and Phytochemical Analysis of a Sunflower Cell Suspension Culture in a 5-L Bioreactor §

A cell suspension culture of sunflower (Helianthus annuus), a producer of immunologically active polysaccharides, was cultivated in a 5-L stirred tank bioreactor, operated in batch mode. After some changes in the internal bioreactor design a stable growth of Helianthus cells was achieved and the accumulated biomass reached 15.2 g/L (only ~5% lower compared to the accumulated biomass in shake-flasks). Flow cytometry used for measuring the cell cycle parameters of suspended Helianthus cells did not reveal significant differences between shake-flasks and bioreactor cultivation modes. For both cultivation methods significant enhancement of the percentage of S-phase cells was observed at the beginning of the cultivation process. Concerning the metabolite production the maximum in exopolysaccharides was reached at day 9 of the cultivation period (1.9 g/L), while the highest amounts of α-tocopherol were accumulated at the beginning of the cultivation process (day 2 of the cultivation). These finding were related to the respective stress levels caused by the inoculation procedure. The kinetic parameters of growth and polysaccharide production as well as the time course of carbon source utilization were monitored and discussed.

Phototrophic growth of Arthrospira platensis in a respiration activity monitoring system for shake flasks (RAMOS

Optimizing illumination is essential for optimizing the growth of phototrophic cells and their production of desired metabolites and/or biomass. This requires appropriate modulation of light and other key inputs and continuous online monitoring of their metabolic activities. Powerful noninvasive systems for cultivating heterotrophic organisms include shake flasks in online monitoring units, but they are rarely used for phototrophs because they lack the appropriate illumination design and necessary illuminatory power. This study presents the design and characterization of a photosynthetic shake flask unit, illuminated from below by warm white light‐emitting diodes with variable light intensities up to 2300 μmol m−2 s−1. The photosynthetic unit was successfully used, in combination with online monitoring of oxygen production, to cultivate Arthrospira platensis. In phototrophic growth under continuous light and a 16 h light/8 h dark cycle (light intensity: 180 μmol m−2 s−1), the oxygen transfer rate and biomass‐related oxygen production were −1.5 mmol L−1 h−1 and 0.18 mmol O2 gx−1 h−1, respectively. The maximum specific growth rate was 0.058 h−1, during the exponential growth phase, after which the biomass concentration reached 0.75 g L−1.

Hairy Roots of Salvia Species for Bioactive Substances Production

The biosynthesis of biologically active secondary metabolites in plant tissues is frequently related to their differentiation. From this point of view, the hairy roots are a suitable plant in vitro system for producing bioactive substances due to their degree of differentiation and metabolic profiles identical to or similar to that of the root systems of the intact plants from which they are derived. In addition, they are grown in growth regulators-free nutrient media, which is essential for the subsequent application of their metabolites in variety of nutritional and pharmaceutical systems. They are characterized by high genetic and biochemical stability, making possible the scale-up of the cultivation processes in bioreactors. This provides complete control and management of the biosynthetic process and results in high yields of target metabolites for a short period of time, regardless of seasons. Salvia plant species are widely used in traditional medicine. The major biological activities they possessed are antioxidant, anti-inflammatory, anti-tumor activities and etc. These activities are consequence of biosynthesized biologically active substances: sterols, various polyphenol structures, di- and triterpenes, etc. Data about Salvia hairy roots in the scientific literature are limited. Nevertheless, it is clear that they biosynthesized mainly diterpenes and phenolic compounds. Current progress of investigation of Salvia hairy roots is limited to the phytochemical profiling and assessment of the influence of different independent variables (mainly elicitors) on the yields of the target bioactive substances and the development of commercial production process is not visible at the time. Therefore in the near future it is necessary an integrated approach for optimizing production processes to be developed. In our opinion, this is the basis for successful achievement of the commercially important yields of bioactive substances produced by hairy roots of Salvia species.

Two parametric cell cycle analyses of plant cell suspension cultures with fragile, isolated nuclei to investigate heterogeneity in growth of batch cultivations.

Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU‐antibodies or 5‐ethynyl‐2′‐deoxyuridine (EdU) click‐it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU‐Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72 h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells’ fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures. Biotechnol. Bioeng. 2016;113: 1244–1250.

Whole-cell biotransformation of oleanolic acid by free and immobilized cells of Nocardia iowensis: Characterization of new metabolites

In this study, Nocardia iowensis was used to transform oleanolic acid (OA) into oleanane derivatives. The first derivative, which was found after 24 h of cultivation, was the known and already described OA methyl ester. After 1 week, two other derivatives (oleanonic acid methyl ester and an unknown metabolite) were identified as new products of a biotransformation by N. iowensis. These oleanane metabolites were characterized by HPLC, HPLC‐ESI‐MS, and HPLC‐1H NMR spectroscopy. The biotransformation was performed by suspended and immobilized cells (ICs) of N. iowensis. Cells immobilized in alginate beads were used in order to prepare a continuous process. The substrate uptake of free and ICs was similar, whereas the peak area of OA methyl ester of the ICs was only about 10% of the native cells. However, the final product (oleanonic acid methyl ester) concentrations were similar in both approaches, whereas the unknown metabolite 3 was only detected transiently in the medium of ICs. Based on these results, a new biosynthetic pathway for the biotechnological production of oleanonic acid methyl ester is proposed.

Uptake of iron by Kluyveromyces marxianus DSM 5422 cultivated in a whey-based medium

The ability of Kluyveromyces marxianus for converting lactose into ethyl acetate offers a chance for the economical reuse of whey. Iron plays a significant role in this process as ester synthesis requires a low intracellular iron content, xFe. The iron content in turn is decreased by growth due to cell expansion and increased by iron uptake. Thus, the iron‐uptake rate, ψ, is important for the considered process. Iron uptake by K. marxianus DSM 5422 was studied in aerobic cultivation on a whey‐borne medium with varied initial iron content, in part combined with a feed of iron under intensive growth conditions. A possible precipitation of iron that would pretend iron uptake was verified not to have occurred. Regularly measured dissolved iron concentrations, CFe,L, allowed the xFe and ψ parameters to be obtained by model‐based iron balancing. The achieved data were used for establishing a ψ(CFe,L, xFe) model. Mathematical simulations based on this iron‐uptake model reproduced the performed cultivation processes. The created iron‐uptake model allows for a future predictive system to be developed for the optimization of biotechnological ester production.