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Abstract
BackgroundCaffeic 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.ResultsThe 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.ConclusionsThe 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.
Highlights
Caffeic acid is industrially recognized for its antioxidant activity and its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent
In an earlier study Rodrigues et al demonstrated the two-step conversion of 3 mM l-tyrosine to caffeic acid in E. coli MG1655(DE3) expressing the enzymes R. glutinis tyrosine ammonia lyase (RgTAL) and CYP199A2 F185L N∆7 with redox partners, without reporting de novo production of caffeic acid from glucose (Fig. 1) [18]
The hydroxylation of p-coumaric acid to caffeic acid catalyzed by CYP199A2 F185L N∆7 appears to be a bottleneck in the pathway, since p-coumaric acid accumulates in the fermentation [18]
Summary
Caffeic acid is industrially recognized for its antioxidant activity and its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. For the biosynthesis of p-coumaric acid in E. coli, it was found that using l-tyrosine as a pathway precursor was superior over phenylalanine [25], since activity of the plant Cytochrome P450 enzyme C4H could not be reconstituted as of recently [26] Based on this finding, two major strategies have been devised to produce caffeic acid that employ microbial tyrosine ammonia lyases (TAL) to generate p-coumaric acid followed by either (1) a flavin-dependent HpaBC-type oxidoreductase complex (4-hydroxyphenylacetate 3-hydroxylase, PFAM PF03241) from Saccharothrix espanaensis [14,15,16,17,18], E. coli [19,20,21], Thermus thermophilus HB8 [20] or Pseudomonas aeruginosa [22, 23], or (2) a bacterial cytochrome P450 enzyme CYP199A2 F185L from Rhodopseudomonas palustris [14, 18, 24]. To our knowledge no de novo synthesis has been reported for pathways utilizing CYP199A2 F185L
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