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.

Keywords: Caffeic acid; Cytochrome P450; PUPPET; Recombinant pathway; Tethering.

Fig. 1

Aromatic amino acid anabolism and recombinant caffeic acid pathway with l-tyrosine as a branchpoint, and TAL and CYP199A2 F185L N∆7 catalyzing the two pathway steps

Fig. 2

Titers of p-coumaric acid and caffeic acid produced from glucose without (a) and with (b) L-Tyr supplementation in selected strains (stacked histograms, error bars = standard deviation of biological replicates, n ≥ 3)

Fig. 3

The choice of redox partners and tethering strategies for redox partners leads to higher caffeic acid titers from p-coumaric acid (a–c) and from glucose (d). Pictograms of tether design I and tether design II (e). a–c caffeic acid titers from 3 mM p-coumaric acid 72 h p. i.: untethered/free redox partners (a), tether design I analogous to PUPPET [35] (b), tether design II (c). d Stacked histograms of p-coumaric and caffeic acid titers after 72 h of fermentation for select strains expressing the two-step pathway. (Error bars = standard deviation of biological replicates, n ≥ 3; Pictograms of tether designs: CYP cytochrome P450 enzyme (CYP199A2 F185L N∆7), Fdx ferredoxin (Pux or Pdx), FdR ferredoxin reductase (PuR or PdR))

Fig. 4

Duplication of the pux gene copy number further increases caffeic acid titers. Stacked histograms of p-coumaric and caffeic acid titers after 72 h of fermentation with glucose as the only carbon source for select strains expressing the two-step pathway (a). Titers plotted over time of a 96 h fermentation of s18 (b). (Error bars = standard deviation of biological replicates, n ≥ 3.)