Glycyrrhiza glabra cambial meristematic cells as a production platform for high value chemicals

Abstract

Plant secondary metabolites represent both a current and future source of diverse molecules with desirable pharmaceutical characteristics. Exploration and utilisation of these highly chiral Plant Natural Products (PNPs) has been historically limited by both low or trace concentrations in whole plants and the lack of a suitable production platform. The isolation of Cambial Meristematic Cells (CMCs) in 2010 showed many advantages over traditional dedifferentiated plant cell culture (DDCs): namely reduced aggregation, improved growth rates and vastly improved metabolite concentrations. It remains to be seen if these desirable characteristics are present in species additional to the small subset already generated. The primary aim of this project was to explore the suitability of Glycyrrhiza glabra CMCs as a production platform for natural products. CMC and DDC lines were generated and characterised at the physical, chemical and transcript level for the first time. The generated CMCs were found to possess the key markers for classification as CMC cells, namely higher levels of the marker genes PXY and WOL, preservation of genomic integrity, presence of numerous small vacuoles and retention of the ability to differentiate into tracheary elements. A de-novo transcriptome was successfully assembled from RNA sequencing reads and assessed for differential gene expression, identifying a number of potential genes of interest and providing a valuable resource for future studies. Metabolic analysis of the CMC cells using HPLC/MS revealed a major peak, the isoflavone formononetin. Upon elicitation with a Methyl jasmonate/Coronatine combination this peak was eliminated and pinocembrin dominance expressed as a percentage of total peak area increased by 54-77 fold. This pattern was mirrored in the DDC samples to a lesser degree with the formononetin peak not being completely eliminated and pinocembrin increasing by just 3.5-8 fold. Other potential future industrial considerations in the screening of high performing lines, scalability, the implications of different elicitation regimes, amenability to cryopreservation and metabolic engineering were also explored.