Biology subjects

Knoch, E.

Publications and source records attributed to Knoch, E..

4 recordsLinked to original sources

bioRxiv2024

Elucidation of gene clusters underlying withanolide biosynthesis in ashwagandha through yeast metabolic engineering

Withanolides are medicinally relevant steroidal lactones produced by Withania somnifera (ashwagandha) amongst other Solanaceae family plants. However, the biosynthetic pathway to withanolides is largely unknown, preventing scale-up and hindering pharmaceutical applications. We sequenced the genome of W. somnifera and identified two biosynthetic gene clusters exhibiting a segmented tissue-specific expression pattern. We characterized the cluster enzymes through stepwise pathway reconstitution in yeast and transient expression in Nicotiana benthamiana, leading to the identification of three cytochrome P450s (CYP87G1, CYP88C7, and CYP749B2) and a short-chain dehydrogenase that produce a lactone ring- containing intermediate when co-expressed. A fourth cytochrome P450 (CYP88C10) and a sulfotransferase convert this into an intermediate with the characteristic withanolide A-ring structure, featuring a C1 ketone and C2-C3 unsaturation. The discovery of the sulfotransferase as a core pathway enzyme challenges the conventional paradigm of sulfotransferases as tailoring enzymes. These insights pave the way for an efficient biomanufacturing process for withanolides and future development of withanolide-derived drugs.

biochemistry↗

bioRxiv2024

Sub-functionalization and epigenetic regulation of a biosynthetic gene cluster in Solanaceae

Biosynthetic gene clusters (BGCs) are sets of often heterologous genes that are genetically and functionally linked. Among eukaryotes, BGCs are most common in plants and fungi, where they ensure the co-expression of the different enzymes coordinating the biosynthesis of specialised metabolites. Here, we report on a newly identified withanolide BGC in Physalis grisea (ground-cherry), a member of the nightshade family (Solanaceae). A combination of transcriptomic, epigenomic and metabolic analyses, revealed that, following a duplication event, this BGC evolved two tissue-specifically expressed sub-clusters, containing eight pairs of paralogs that contribute to related but distinct biochemical processes; this sub- functionalisation is tightly associated with epigenetic features and the local chromatin environment. The two sub-clusters appear strictly isolated from each other at the chromatin structural level, each forming a highly self-interacting chromatin domain with tissue-dependent levels of condensation. This correlates with gene expression exclusively in either above- or below-ground tissue, thus spatially separating the production of different withanolide compounds. By comparative phylogenomic analysis, we show that the withanolide BGC most likely evolved before the diversification of the Solanaceae family and underwent lineage- specific diversifications and losses. The tissue-specific sub-functionalisation is common to species of the Physalideae tribe but distinct from other, independent duplication events outside of this clade. In sum, our study reports on the first instance of an epigenetically modulated sub-functionalisation within a BGC and sheds light on the biosynthesis of withanolides, a highly diverse group of steroidal triterpenoids important in plant defence and amenable to pharmaceutical applications due to their anti-inflammatory, antibiotic and anti- cancer properties.

plant biology↗

bioRxiv2023

The lactonase BxdA mediates metabolic adaptation of maize root bacteria to benzoxazinoids

Root exudates contain secondary metabolites that affect the plants root microbiome. How microbes cope with these bioactive compounds, and how this ability shapes root microbiomes remain largely unknown. We investigated how maize root bacteria metabolise benzoxazinoids, the main specialised metabolites of maize. Diverse and abundant bacteria metabolised the major compound (6-methoxy-benzoxazolin-2-one, MBOA) in the maize rhizosphere to 2-amino-7-methoxyphenoxazin-3-one (AMPO). By contrast, bacteria isolated from Arabidopsis, which does not produce benzoxazinoids, were unable to metabolise MBOA. Among Microbacteria strains, this differential metabolisation allowed to identify a conserved gene cluster containing the lactonase bxdA. BxdA converts MBOA to AMPO in vitro and we show that this capacity provided bacteria a growth benefit under carbon-limiting conditions. Together these results reveal that maize root bacteria - through BxdA - are metabolically adapted to the benzoxazinoids of their host. We propose that metabolic adaptation to plant-specialised compounds shapes root bacterial communities across the plant kingdom.

microbiology↗

bioRxiv2022

Transcriptional response of a target plant to benzoxazinoid and diterpene allelochemicals highlights commonalities in detoxification

Plants growing in proximity to other plants are exposed to a variety of metabolites that these neighbors release into the environment. Some species produce allelochemicals to inhibit growth of neighboring plants, which in turn have evolved ways to detoxify these compounds. In order to understand how the allelochemical-receiving target plants respond to chemically diverse compounds, we performed whole-genome transcriptome analysis of Arabidopsis thaliana exposed to either the benzoxazinoid derivative 2-amino-3H-phenoxazin-3-one (APO) or momilactone B. These two allelochemicals belong to two very different compound classes, benzoxazinoids and diterpenes, respectively, produced by different cereal crop species. Despite their distinct chemical nature, we observed similar molecular responses of A. thaliana to these allelochemicals. In particular, many of the same or closely related genes belonging to the three-phase detoxification pathway were upregulated in both treatments. Further, we observed an overlap between genes upregulated by allelochemicals and those involved in herbicide detoxification. Our findings highlight the overlap in the transcriptional response of a target plant to natural and synthetic phytotoxic compounds and illustrate how herbicide resistance could arise via pathways involved in plant-plant interaction.

plant biology↗