Combination of Plant Metabolic Modules Yields Synthetic Synergies

Abstract

The great potential of pharmacologically active secondary plant metabolites is often limited by low yield and availability of the producing plant. Chemical synthesis of these complex compounds is often too expensive. Plant cell fermentation offers an alternative strategy to overcome these limitations. However, production in batch cell cultures remains often inefficient. One reason might be the fact that different cell types have to interact for metabolite maturation, which is poorly mimicked in suspension cell lines. Using alkaloid metabolism of tobacco, we explore an alternative strategy, where the metabolic interactions of different cell types in a plant tissue are technically mimicked based on different plant-cell based metabolic modules. In this study, we simulate the interaction found between the nicotine secreting cells of the root and the nicotine-converting cells of the senescent leaf, generating the target compound nornicotine in the model cell line tobacco BY-2. When the nicotine demethylase NtomCYP82E4 was overexpressed in tobacco BY-2 cells, nornicotine synthesis was triggered, but only to a minor extent. However, we show here that we can improve the production of nornicotine in this cell line by feeding the precursor, nicotine. Engineering of another cell line overexpressing the key enzyme NtabMPO1 allows to stimulate accumulation and secretion of this precursor. We show that the nornicotine production of NtomCYP82E4 cells can be significantly stimulated by feeding conditioned medium from NtabMPO1 overexpressors without any negative effect on the physiology of the cells. Co-cultivation of NtomCYP82E4 with NtabMPO1 stimulated nornicotine accumulation even further, demonstrating that the physical presence of cells was superior to just feeding the conditioned medium collected from the same cells. These results provide a proof of concept that combination of different metabolic modules can improve the productivity for target compounds in plant cell fermentation.

Grapevine fatty acid hydroperoxide lyase generates actin-disrupting volatiles and promotes defence-related cell death

“…JA is known to activate basal immunity, and we asked whether activation of JA signalling can mitigate the harpin-triggered cell death response observed in the HPL1ox line. Previous experiments had shown that the JA sensitivity of BY-2 is low, such that 100 µM racemic JA is required to activate JA-dependent gene expression ( Rajabi et al , 2017 ). When this concentration of JA was administered prior to harpin treatment, the otherwise strong activation of cell death by harpin could be suppressed ( Fig.…”

“…It should be noted that the relatively high concentration (100 µM) of JA required for this phenomenon is due to the low sensitivity of BY-2 cells to JA. For instance, the alkaloid pathway as metabolic readout for stress responses requires 100 µM JA in order to be efficiently activated, while lower concentrations (10 µM) induce this only very poorly ( Rajabi et al , 2017 ).…”

“…Cells of the strain Nicotiana tabacum L. cv. ‘Bright Yellow 2’ (BY-2; Nagata et al , 1992 ) were cultivated as described in Rajabi et al (2017) . To visualize actin filaments in vivo , a transgenic tobacco BY-2 line expressing the actin-binding domain of plant fimbrin (FABD2) in fusion with GFP was used ( Sano et al , 2005 ).…”

Nanosecond pulsed electrical fields enhance product recovery in plant cell fermentation

“…In our previous work (Rajabi et al 2017), we had observed that cells overexpressing NtomCYP82E4 were able to accumulate high amounts of nornicotine, if they were confronted with the culture filtrate from cells overexpressing NtabMPO1. This could, alternatively, also be achieved by simply co-cultivating the two cell lines, which was the strategy chosen for nsPEF treatment in the current work.…”

Nicotine Biosynthesis inNicotiana: A Metabolic Overview

“…Previously, MPO1 was described as acting in the peroxisome, the same subcellular localization of DAO1 (54). This added a new cellular compartment to the nicotine biosynthesis pathway, contributing to a better understanding of the route (58). The product derived from the MPO1 reaction, 4-methyl-aminobutanal, spontaneously undergoes intramolecular cyclization to form the N-methylpyrrolinium cation (36).…”