Microbial production of O-methylated flavanones from methylated phenylpropanoic acids in engineered Escherichia coli

Abstract

Methylated flavonoids possess improved bioactivities compared to their unmethylated counterparts. In this study, for the efficient production of O-methylated flavonoids from simple methylated phenylpropanoic acids, a recombinant Escherichia coli strain expressing 4-coumarate:coenzyme A ligase (4CL) from Oryza sativa and chalcone synthase (CHS) from Hordeum vulgare was constructed; this strain produced significant amount of homoeriodictyol (~ 52 mg/L) as well as a few amount of hesperetin (0.4 mg/L), respectively, from ferulic acid and 4-methylcaffeic acid. This demonstrates, for the first time, that the scarce but valuable methylated flavanones can be successfully produced from methylated phenylpropanoic acids in a microbial host via an artificial biosynthetic pathway consisting of 4CL and CHS that can accept O-methylated precursors.

Engineering Escherichia coli towards de novo production of gatekeeper (2S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol

“…Although we only produced 0.5 mg/l homoeriodictyol from our ΔPAL pathways, Cui et al ( 18 ) recently reported the production of 52 mg/l homoeriodictyol by feeding ferulic acid at lower concentrations. Therefore, we investigated the construction of pathways to allow the complete biosynthesis of homoeriodictyol from tyrosine, with the rationale that ferulic acid produced in situ would be quickly converted on to homoeriodictyol, preventing build-up that may inhibit pathway function.…”

“…Automated, worklist-driven liquid handling was implemented for bridging oligo pooling and ligase cycling reaction setup. Completed reactions were transformed into high-efficiency NEB 5-alpha cells, and correct plasmid assemblies identified by high-throughput, next-generation sequencing using Nanopore technology ( 18 ). Some assemblies ( Supplementary Table S3 ) were also verified using Sanger sequencing (GATC Biotech).…”

“…Feeding cinnamic acid has resulted in pinocembrin titers of 429 mg/l ( 16 ). Homoeriodictyol, methylated at the 3′ position, has been produced from eriodictyol by bioconversion using 3′-OMT from rice or tomatoes ( 17 ), and more recently from ferulic acid producing 52 mg/l homoeriodictyol ( 18 ). Bioproduction of eriodyctiol from caffeic acid has been previously reported (11 mg/l), and through extensive manipulation of malonyl-CoA biosynthesis combined with fine-tuning of E. coli metabolic pathways this has been improved to 50 mg/l ( 19 ) and 107 mg/l ( 20 ).…”

Engineering a plant polyketide synthase for the biosynthesis of methylated flavonoids

“…Recently, Cui et al . obtained hesperetin and homoeriodictyol at a low titer in engineered E. coli from fed isoferulic acid and ferulic acid 22 . The enzymes used in that study had previously been shown to have high substrate promiscuity in vitro 43,44 and were then used in vivo for the first time 22 .…”

“…The next day, 1mL of the starter culture was inoculated into 50 mL of fresh LB medium. IPTG (final concentration 1mM) was added to the culture broths when the optical density reached 0.4-0.6 for protein expression and the cultures were successively incubated at 30 °C for an additional 3 h. The cells were collected by centrifugation for 30 min at 3,700 rpm (Eppendorf 5920R, Germany) and 4 °C and then resuspended in 25 mL of fresh modified MOPS medium which included 1mM substrate, 1mM IPTG, and 20μg/mL cerulenin for further 32h fermentation 22 .…”