Biology subjects

Groves, M. R.

Publications and source records attributed to Groves, M. R..

4 recordsLinked to original sources

bioRxiv2025

De novo biosynthesis of alpinetin enhanced by directed evolution of 5-O-methyltransferase

Alpinetin ((2S)-7-Hydroxy-5-methoxyflavan-4-one) is a natural flavonoid found in various medicinal herbs and is frequently used in Chinese patent medicines. It exhibits a wide range of bioactivities, including anti-inflammatory, cardiovascular protective, lung protective, antiviral, hepatoprotective, and antitumor effects. Alpinetin features a 5-methyl group on the A ring, a rare characteristic among methylated flavonoids. The limited abundance of Alpinetin in plant biomass, its laborious extraction and purification from this biomass, and the toxicity and lack of regio- and chemoselectivity in organic synthesis render its biosynthesis in engineered microbes an attractive alternative. In this study, we aimed to achieve the de novo biosynthesis of alpinetin in Escherichia coli. In a series of optimization steps, we varied the selection of pathway enzymes, plasmid configurations, medium composition, and fermentation strategies. Lastly, we applied a directed evolution campaign to the 5-O-methyltransferase to successfully enable the de novo biosynthesis of alpinetin in E. coli for the first time. Moreover, we demonstrated the ability of O-methyltransferase to methylate a broad range of flavonoid substrates, leading to the production of valuable O-methylated flavonoids. Our study represents the first example of alpinetin biosynthesis in a heterologous host and paves the way to produce other valuable O-methylated flavonoids enzymatically.

biochemistry↗

bioRxiv2024

A biocatalytic platform for the production of substituted 2-quinolones and (thio)coumarins

2-quinolones are privileged scaffolds for drug discovery that are relatively rare in nature. Here, we characterise two promiscuous fungal polyketide synthases AthePKS and FerePKS, which we had previously found to produce quinolones in vitro. We challenged the enzymes with several substituted anthranilic acid derivatives, revealing their ability to produce precursors of pharmaceutically relevant quinolones. We also discovered that AthePKS and FerePKS accept other 2-substituted benzoic acids, leading to the formation of coumarin and thiocoumarin scaffolds. We applied AthePKS in an artificial enzymatic cascade towards an antimicrobial 4-methoxy-1-methyl-2-quinolone and demonstrated its in vivo feasibility by successfully expressing the pathway in Escherichia coli. Lastly, we determined the crystal structure of AthePKS, suggesting hotspots for enhancing its catalytic efficiency by enzyme engineering. Our results provide a framework for further engineering of enzymatic routes towards privileged heteroaromatic scaffolds and derivatives thereof.

biochemistry↗

bioRxiv2023

Structural characterization and extended substrate scope analysis of two Mg2+-dependent O-methyltransferases from bacteria

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis in terms of their enzymatic properties and substrate scope and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering. Table of contentsTwo promiscuous O-methyltransferases from bacteria were found to methylate a panel of catechol substrates towards high-value medicinal compounds. Surprisingly, the non-catechol substrates 5-hydroxyflavonoids and o-hydroxybenzoic acids/aldehydes were also methylated at low conversion rates. The crystal structures reveal potential target sites for enzyme engineering for biocatalytic applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/526015v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@4d9028org.highwire.dtl.DTLVardef@334ceborg.highwire.dtl.DTLVardef@1ff016forg.highwire.dtl.DTLVardef@b74b8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Engineering a plant polyketide synthase for the biosynthesis of methylated flavonoids

Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as complex mixtures of similar compounds that are difficult to separate. Synthetic biology offers the opportunity to produce various flavonoids in a targeted, bottom-up approach in engineered microbes with high product titers. However, the production of O-methylated flavonoids is currently still highly inefficient. In this study, we investigated and engineered a combination of enzymes that had previously been shown to support homoeriodictyol and hesperetin production in Escherichia coli from fed O-methylated cinnamic acids. We determined the crystal structures of the enzyme catalyzing the first committed step of the pathway, chalcone synthase from Hordeum vulgare, in three ligand-bound states. Based on these structures and a multiple sequence alignment with other chalcone synthases, we constructed mutant variants and assessed their performance in E. coli towards producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than previously reported. Our findings will facilitate the further engineering of this enzyme towards higher production of methylated flavonoids.

biochemistry↗