Engineering a Plant Polyketide Synthase for the Biosynthesis of Methylated Flavonoids

Abstract

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 hydroxycinnamic 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 toward producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234 V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than reported previously. Our findings will facilitate further engineering of this enzyme toward higher production of methylated flavonoids.

Novel Catechol O-methyltransferases from Lentinula edodes Catalyze the Generation of Taste-Active Flavonoids

“…The majority of reported flavonoid-specific OMTs were described for plants, such as soybean (Glycine max), rice (Oryza sativa), or periwinkle (Catharanthus roseus), which showed biocatalytic activities for the modification of i.a. naringenin, quercetin, and eriodictyol. − Moreover, synthetic pathways have recently been developed by heterologous expression of plant genes in bacterial cell hosts to produce O -methylated flavonoids. , Flavonoid-specific OMTs outside the plant kingdom have been reported only to a lesser extent. − ,,, In particular, the presence of OMTs from higher fungi, such as Basidiomycota, has been scarcely described. However, results from genome sequencing of various white-rot fungi revealed the presence of genes putatively encoding OMTs, which may be involved in the O -methylation of monolignols to inhibit oxidative stress from their free hydroxy groups and facilitate peroxidase activity for lignin degradation .…”

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein

“…One of the greatest challenges in the enhanced production of flavonoids via enzymatic engineering is the lack of fundamental understanding of the structure and function of the participating enzymes in the target species [ 41 , 42 ]. This study comprehensively characterized CHS, CHIL, and CHI in the flavonoid pathway of two closely related monocots, sorghum and switchgrass, both of which are gaining prominence due to their resilience to weather extremes anticipated as a result of climate change.…”