Heike Dörnenburg

Formation of cyclotides and variations in cyclotide expression in Oldenlandia affinis suspension cultures.

Cyclotides, a family of disulfide-rich mini-proteins, show a wide range of biological activities, making them interesting targets for pharmaceutical and agrochemical applications, but little is known about their natural function and the events that trigger their expression. An investigation of nutritional variations and irradiation during a batch process involving plant cell cultures has been performed, using the native African medical herb, Oldenlandia affinis, as a model plant. The results demonstrated the biosynthesis of kalata B1, the main cyclotide in O. affinis, in a combined growth/nongrowth-associated pattern. The highest concentration, 0.37xa0mg g−1 dry weight, was accumulated in irradiated cells at 35xa0μmol m−2 s−1. Furthermore, 12 novel cyclotides were identified and the expression of various cyclotides compared in irradiated vs non-irradiated cultures. The results indicate that cyclotide expression varies greatly depending on physiological conditions and environmental stress. Kalata B1 is the most abundant cyclotide in plant suspension cultures, which underlies its importance as a natural defense molecule. The identification of novel cyclotides in suspension cultures, compared to whole plants, indicates that there may be more novel cyclotides to be discovered and that the genetic network regulating cyclotide expression is a very sensitive system, ready to adapt to the current environmental growth condition.

Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides

In vitro cultured plants from Oldenlandia affinis were established from seeds and grown on a hormone-free medium. In vitro plants produced the cyclic peptide kalata B1 in concentrations of 0.67xa0mgxa0g−1xa0dry weight after growth of 30xa0days. This was approximately 50% of the concentration analysed in green house plants (shoot tips), where different concentrations have been determined in leaves (1.82xa0mgxa0g−1), shoot tips (1.36xa0mgxa0g−1), stems (0.36xa0mgxa0g−1), and in flowers (0.16xa0mgxa0g−1). Callus and cell suspension cultures could be initiated from aseptic root, stem and leaf explants of O. affinis seedlings and plants. Different light intensities were shown to affect culture growth as well as chlorophyll synthesis. The friable callus was then used for the establishment of a cell suspension culture. Fresh and dry weight measurements showed that growth was optimal on MS medium supplemented with 0.4xa0mgxa0l−1 2,4-dichlorophenoxyacetic acid (2,4-d). Leaf suspensions cultured on this medium showed a 4-fold increase of biomass by the first week of incubation. No quantifiable amounts of kalata B1 were produced under these conditions. Morphological differentiation seems to be essential for cyclic peptide production. Therefore, several undifferentiated as well as organised cell lines of O. affinis have been developed. These cell lines will constitute a worthwhile starting point for the optimisation of kalata B1 synthesis in liquid media to the objective of producing cyclic peptides under controlled and defined conditions in bioreactors.

Plant cell culture technology–harnessing a biological approach for competitive cyclotides production

Cyclotides are naturally occurring mini-proteins that have a cyclic backbone and a knotted arrangement of three disulfide bonds. They are remarkably stable and have a diverse range of therapeutically useful biological activities, including antimicrobial and anti-HIV activity, although their natural function appears to be plant defence agents. Cyclotides are amenable to chemical synthesis; however currently most bioactivity studies have involved the use of peptides extracted from plants. Plant cell culture technology shows promise towards the goal of producing therapeutically active cyclotides in qualities and quantities required for drug development.

Cyclotide synthesis and supply: From plant to bioprocess

Cyclotides are disulfide‐rich miniproteins with a circular backbone and a knotted arrangement of disulfide bonds. Because these plant‐derived peptides are resistant to degradation and exhibit a diverse range of bioactivities, they have become important agronomic and industrial objectives. They belong to a group of compounds with low market volume and high price that are poorly processed by microorganisms, are too complex for economic chemical synthesis, and thus are valuable candidates for the synthesis in plant cell bioprocesses. This review highlights current research aimed at production routes of cyclotides in Oldenlandia affinis plantlets and cell cultures, and summarizes recent advances in bioprocessing aspects, with particular emphasis on the development of suitable bioreactor configurations for plant cell culture‐based processes, the optimization of culture environments as a powerful means to improve yields, bioreactor operational modes, and trends in protein recovery.

Plant cell-based processes for cyclotides production

Cyclotides are naturally occurring mini-proteins that have a diverse range of therapeutically useful biological activities. Although a choice of approaches is available for cyclotides synthesis; most studies have involved the use of peptides extracted from plants. In order to facilitate the screening for structure-activity studies or to exploit them in drug development, a convenient and reliable route for the biosynthesis of cyclotides is of vital importance. Callus, suspension cultures and hydroponic plants of Oldenlandia affinis were established and have been evaluated for effective cyclotides production processes. The specific accumulation of kalata B1 was affected by cell differentiation as well as agitation; highest accumulation of 2.7 mgg(-1) dry weight was detected in agitated hydroponic plant cultures resulting in a productivity of 1.4 mg kalata B1l(-1)day(-1).

Effect of irradiation intensity on cell growth and kalata B1 accumulation in Oldenlandia affinis cultures

Light irradiation had remarkable effects on callus growth of Oldenlandia affinis with an optimum intensity of 35xa0μmol m−2xa0s−1. Biosynthesis of kalata B1, the main cyclic peptide in O. affinis, was induced and triggered with rising irradiation intensities. The highest concentration of kalata B1, 0.49xa0mgxa0g−1 DW characterised by the maximum productivity of 3.88xa0μg per litre and day was analysed at 120xa0μmolxa0m−2xa0s−1, although callus growth was repressed. The light saturation point was established to be 35xa0μmolxa0m−2xa0s−1, where kalata B1 productivity was in a similar order (3.41xa0μg per day) due to the higher growth index. O. affinis suspension cultures were shown to accumulate comparable specific kalata B1 concentrations in a delayed growth associated production pattern. These were dependent on irradiation intensity (0.16xa0mgxa0g−1 at 2xa0μmolxa0m−2xa0s−1; 0.28xa0mgxa0g−1 at 35xa0μmolxa0m−2xa0s−1). The batch cultivation process resulted in a maximum productivity of 27.30xa0μg per litre and day with culture doubling times of 1.16xa0d−1. Submers operation represented a 8-fold product enhancement compared to callus cultivation.

Scale‐up of Oldenlandia affinis suspension cultures in photobioreactors for cyclotide production

Cyclotides are a family of backbone‐cyclized cystine‐knot‐containing macrocyclic peptides from plants that possess extremely interesting biological activities. Suspension cultures of Oldenlandia affinis, a model plant containing cyclotides, were scaled‐up from shake flask to photobioreactor operation in order to produce these plant peptides under controlled conditions. Cell growth was highly dependent on inoculation culture; cell density as well as culture age had an effect on the growth rates and thus affected the kalata B1 productivity of the bioprocess.

Progress in kalata peptide production via plant cell bioprocessing

Cyclotides are disulfide‐rich mini‐proteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. They typically comprise 28–37 amino acids and are produced from linear precursors, and translational modification via oxidative folding, proteolytic processing and N‐C cyclization. Because these plant‐derived peptides are resistant to degradation and do exhibit a diverse range of biological activities, they have become important agronomic and industrial objectives. Due to its tolerance to sequence variation, the cyclotide backbone is also potentially useful as a molecular scaffold for protein‐engineering applications. Several production options are available for bioactive plant metabolites including natural harvesting, total chemical synthesis, and expression of plant pathways in microbial systems. For the cyclotides with low yields in nature, chemical complexity and lack of knowledge of the complete biosynthetic pathway, however, many of these options are precluded. Plant cell‐culture technology shows promise towards the goal of producing therapeutically active cyclotides in quality and quantities required for drug development as they are amenable to process optimization, scale‐up, and metabolic engineering. It is conceivable that plant‐based production systems may ultimately prove to be the preferred route for the production of native or designed cyclotides, and will contribute towards the development of target‐specific drugs.