Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450

Abstract

Background: Caffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented l-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far. Results: The herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 µM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even a virtually unproductive Cytochrome P450/redox system combination productive. Conclusions: The caffeic acid titer achieved in this study is about 10% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without l-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.

Attenuation of Anxiety-Like Behavior by Helichrysum stoechas (L.) Moench Methanolic Extract through Up-Regulation of ERK Signaling Pathways in Noradrenergic Neurons

“…The analysis of the methanolic extract led to the isolation of several components, in particular arzanol, a characteristic and novel pholoroglucinol α -pyrone isolated from Helichrysum spp., which possesses a variety of pharmacological activities including acting as an eicosanoid inhibitor on PGE 2 synthesis and the NF-κB pathway. Moreover, it has other important constituents including caffeic and quercetin derivates, which possess strong antioxidant and anti-inflammatory activities and they have been reported to have beneficial effects on mood disorders [ 14 , 15 , 16 ].…”

Synthetic Scaffold Systems for Increasing the Efficiency of Metabolic Pathways in Microorganisms

“…The co-localization of β-carotene ketolase and β-carotene hydroxylase on the membrane, via their fusion by the glycerol uptake facilitator protein, resulted in a 2.2-fold improvement in the astaxanthin yield [ 42 ]. The protein scaffold strategy using heterotrimeric DNA sliding clamp PCNA (Proliferating Cell Nuclear Antigen) of Sulfolobus solfataricus P2 was also utilized to improve the activity of cytochrome P450 enzyme on the production of caffeic acid [ 43 ].…”

Flavobacterium johnsoniae Tyrosine Ammonia Lyase (FjTAL) in-silico Structure Prediction and Molecular Docking to L-Tyrosine, p-Coumaric Acid (pHCA) and Caffeic Acid

“…In the recent methods that have been developed for the sustainable production of phenylpropanoids in recombinant E. coli , FjTAL has been proposed as one of the potential enzymes to convert L-tyrosine to p-coumaric acid 1 2 (Fig.1). Although it has been demonstrated that Flavobacterium johnsoniae Tyrosine Ammonia Lyase (FjTAL), has satisfactory affinity for L-tyrosine (K m ∼ 6.7 μM), little has been reported on the binding affinity of other structurally-related metabolites to FjTAL 3 4 .…”

“…Although it has been demonstrated that Flavobacterium johnsoniae Tyrosine Ammonia Lyase (FjTAL), has satisfactory affinity for L-tyrosine (Km ~ 6.7 μM), little has been reported on the binding affinity of other structurally-related metabolites to FjTAL 3 4 . It has been hypothesized by Sariaslani 5 (2007) and Haslinger and Prather (2020) that p-coumaric acid, the product of elimination reaction catalyzed by FjTAL, might inhibit the activity of the enzyme, exerting a kind of negative feedback response. Our goal in this study, was to computationally predict the 3D structure of FjTAL, paving the way to help other researchers to reasonably engineer the protein for the future studies.…”