Optimization of naringenin andp-coumaric acid hydroxylation using the nativeE. colihydroxylase complex, HpaBC

Abstract

Flavonoids are a growing class of bioactive natural products with distinct and interesting bioactivity both in vitro and in vivo. The extraction of flavonoids from plant sources is limited by their low natural abundance and commonly results in a mixture of products that are difficult to separate. However, due to recent advances, the microbial production of plant natural products has developed as a promising alternative for flavonoid production. Through optimization of media, induction temperature, induction point, and substrate delay time, we demonstrate the highest conversion of naringenin to eriodictyol (62.7 ± 2.7 mg/L) to date, using the native E. coli hydroxylase complex, HpaBC. We also show the first evidence of in vivo HpaBC activity towards the monohydroxylated flavan-3-ol afzelechin with catechin product titers of 34.7 ± 1.5 mg/L. This work confirms the wide applicability of HpaBC towards realizing efficient de novo production of various orthohydroxylated flavonoids and flavonoid derived products in E. coli.

Complete Biosynthesis of Anthocyanins Using E. coli Polycultures

“…Recent efforts from two labs have, however, enabled the near-gram-scale de novo production of both p -coumaric and caffeic acids (18, 19). The development of the tyrosine-overproducing E. coli strain rpoA14(DE3) represents a major milestone for the de novo production of phenylpropanoic acids (20, 21), while the discovery and optimization of the natively silenced E. coli non-P450 hydroxylase HpaBC enabled, for the first time, efficient production of caffeic acid through the ortho-hydroxylation of p -coumaric acid (18, 22–24). Building off of these efforts, we first set out to develop a phenylpropanoic acid production module that was compatible with our previously described “C5” and “p168” modules to enable the de novo production of flavan-3-ols in vivo (9).…”

“…The corresponding plasmid was sequence verified (Genewiz, Inc.) and used together with pETM6-HpaBC (no. 12 in Table S1) (22) to create pETM6- Rg TAL syn -HpaB-HpaC through standard ePathBrick cloning protocols (37). …”

“…S1 in the supplemental material. Product titers were determined using authentic standards, while (+)-afzelechin was quantified using the (+)-catechin standard curve in accordance with previous literature, because (+)-afzelechin is not commercially available (22, 40). All experiments were performed in at least biological duplicate, with key high-titer conditions reproduced in biological and experimental triplicate.…”

Production of three phenylethanoids, tyrosol, hydroxytyrosol, and salidroside, using plant genes expressing in Escherichia coli

“…We tested the HpaBC gene, encoding 4-hydroxyphenylacetate 3-hydroxylase, from E. coli 25 and Sam5 from Saccharothrix espanaensis 26 in order to convert tyrosol into hydroxytyrosol. HpaBC and Sam5 were used to modify other phenolic compounds such as p -coumaric acid, tyrosine, and flavonoids 27–29 . The HpaBC or Sam5 genes were coexpressed with PcAAS in E. coli , and each transformant was tested for the production of hydroxytyrosol.…”

Techniques for chromosomal integration and expression optimization in Escherichia coli

“…Additional strategies and methods, described in the following sections, are being explored to enhance the expression level. After optimization for chromosomal integration of target genes or metabolic pathways, at least for some proteins, the capability of protein expression from chromosomal integration surpasses high copy plasmid‐based expression systems (Englaender et al, ; Jones, Collins, Vernacchio, Lachance, & Koffas, ; Wang et al, ).…”