The Epigenetic Regulation in Plant Specialized Metabolism: DNA Methylation Limits Paclitaxel in vitro Biotechnological Production

Abstract

Environmental conditions are key factors in the modulation of the epigenetic mechanisms regulating gene expression in plants. Specifically, the maintenance of cell cultures in optimal in vitro conditions alters methylation patterns and, consequently, their genetic transcription and metabolism. Paclitaxel production in Taxus x media cell cultures is reduced during its maintenance in in vitro conditions, compromising the biotechnological production of this valuable anticancer agent. To understand how DNA methylation influences taxane production, the promoters of three genes (GGPPS, TXS, and DBTNBT) involved in taxane biosynthesis have been studied, comparing the methylation patterns between a new line and one of ~14 years old. Our work revealed that while the central promoter of the GGPPS gene is protected from cytosine methylation accumulation, TXS and DBTNBT promoters accumulate methylation at different levels. The DBTNBT promoter of the old line is the most affected, showing a 200 bp regulatory region where all the cytosines were methylated. This evidence the existence of specific epigenetic regulatory mechanisms affecting the last steps of the pathway, such as the DBTNBT promoter. Interestingly, the GGPPS promoter, a regulatory sequence of a non-specific taxane biosynthetic gene, was not affected by this mechanism. In addition, the relationship between the detected methylation points and the predicted transcription factor binding sites (TFBS) showed that the action of TFs would be compromised in the old line, giving a further explanation for the production reduction in in vitro cell cultures. This knowledge could help in designing novel strategies to enhance the biotechnological production of taxanes over time.

Light-Dependent Expression and Promoter Methylation of the Genes Encoding Succinate Dehydrogenase, Fumarase, and NAD-Malate Dehydrogenase in Maize (Zea mays L.) Leaves

“…The regulation of plant metabolism by light has been studied for many years [ 1 ]; however, it has only been assessed recently in terms of distinguishing the mechanisms involved, including the rearrangement of transcriptional networks that mediate photoreceptor signals [ 30 , 31 , 32 ]. Epigenetic mechanisms are directly involved in the control of light-responsive genes via modulation of their binding to transcription factors [ 33 , 34 ]. Analysis of the nucleotide sequences of promoters of the genes encoding respiratory enzymes indicated the presence of specific binding sites in their structures for transcription factors of the basic helix-loop-helix (bHLH) family, among them the phytochrome-interacting factors (PIFs) that mediate the action of phytochrome.…”

Suspension cell cultures of Panax vietnamensis as a biotechnological source of ginsenosides: growth, cytology, and ginsenoside profile assessment

“…DNA methylation, which may be accumulated over time in cell culture due to multiple subcultures and constant proliferation, is also believed to be one of the factors responsible for alterations in the biosynthesis of secondary metabolites ( Dubrovina and Kiselev, 2016 ; Sanchez-Muñoz et al., 2019 ). Such effects have been demonstrated for cell cultures of P. ginseng and Taxus media ( Kiselev et al., 2013 ; Escrich et al., 2022 ). Some authors stressed that auxins in the nutrient medium might increase DNA methylation frequency in plant cells ( Gao et al., 2014 ; Grzybkowska et al., 2020 ).…”

Integrated mass spectrometry imaging and single‐cell transcriptome atlas strategies provide novel insights into taxoid biosynthesis and transport in Taxus mairei stems

“…Also, a new MYB TF gene, T. mairei MYB47, and another TF-target pair, MYB47-T5OH, were identified. In woody trees, NAC family TFs are reportedly involved in secondary cell wall formation (Chen et al, 2022). NAC154 overexpression modulated phenolic glycoside levels in poplar stem wood (Jervis et al, 2015).…”

“…Metabolic engineering relies on the selection of key rate‐limiting enzymes; thus, multiple encoding genes can provide several outcomes. TS is the rate‐limiting enzyme in the taxol biosynthesis pathway and was encoded by three genes (Escrich et al., 2022). Among these genes, ctg5306_gene.4 and ctg7747_gene.1 showed extremely low cell coverage (<0.1%), suggesting silenced expression in most stem cells.…”