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

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.

Porting the synthetic D-glucaric acid pathway from Escherichia coli to Saccharomyces cerevisiae.

D-Glucaric acid can be produced as a value-added chemical from biomass through a de novo pathway in Escherichia coli. However, previous studies have identified pH-mediated toxicity at product concentrations of 5 g/L and have also found the eukaryotic myo-inositol oxygenase (MIOX) enzyme to be rate-limiting. We ported this pathway to Saccaromyces cerevisiae, which is naturally acid-tolerant and evaluate a codon-optimized MIOX homologue. We constructed two engineered yeast strains that were distinguished solely by their MIOX gene - either the previous version from Mus musculus or a homologue from Arabidopsis thaliana codon-optimized for expression in S. cerevisiae - in order to identify the rate-limiting steps for D-glucaric acid production both from a fermentative and non-fermentative carbon source. myo-Inositol availability was found to be rate-limiting from glucose in both strains and demonstrated to be dependent on growth rate, whereas the previously used M. musculus MIOX activity was found to be rate-limiting from glycerol. Maximum titers were 0.56 g/L from glucose in batch mode, 0.98 g/L from glucose in fed-batch mode, and 1.6 g/L from glucose supplemented with myo-inositol. Future work focusing on the MIOX enzyme, the interplay between growth and production modes, and promoting aerobic respiration should further improve this pathway.

Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway.

BACKGROUND: Vanillin is an industrially valuable molecule that can be produced from simple carbon sources in engineered microorganisms such as Saccharomyces cerevisiae and Escherichia coli. In E. coli, de novo production of vanillin was demonstrated previously as a proof of concept. In this study, a series of data-driven experiments were performed in order to better understand limitations associated with biosynthesis of vanillate, which is the immediate precursor to vanillin. RESULTS: Time-course experiments monitoring production of heterologous metabolites in the E. coli de novo vanillin pathway revealed a bottleneck in conversion of protocatechuate to vanillate. Perturbations in central metabolism intended to increase flux into the heterologous pathway increased average vanillate titers from 132 to 205 mg/L, but protocatechuate remained the dominant heterologous product on a molar basis. SDS-PAGE, in vitro activity measurements, and L-methionine supplementation experiments suggested that the decline in conversion rate was influenced more by limited availability of the co-substrate S-adenosyl-L-methionine (AdoMet or SAM) than by loss of activity of the heterologous O-methyltransferase. The combination of metJ deletion and overexpression of feedback-resistant variants of metA and cysE, which encode enzymes involved in SAM biosynthesis, increased average de novo vanillate titers by an additional 33% (from 205 to 272 mg/L). An orthogonal strategy intended to improve SAM regeneration through overexpression of native mtn and luxS genes resulted in a 25% increase in average de novo vanillate titers (from 205 to 256 mg/L). Vanillate production improved further upon supplementation with methionine (as high as 419 ± 58 mg/L), suggesting potential for additional enhancement by increasing SAM availability. CONCLUSIONS: Results from this study demonstrate context dependency of engineered pathways and highlight the limited methylation capacity of E. coli. Unlike in previous efforts to improve SAM or methionine biosynthesis, we pursued two orthogonal strategies that are each aimed at deregulating multiple reactions. Our results increase the working knowledge of SAM biosynthesis engineering and provide a framework for improving titers of metabolic products dependent upon methylation reactions.

Improvement of glucaric acid production in E. coli via dynamic control of metabolic fluxes.

D-glucaric acid can be used as a building block for biopolymers as well as in the formulation of detergents and corrosion inhibitors. A biosynthetic route for production in E. coli has been developed (Moon et al., 2009), but previous work with the glucaric acid pathway has indicated that competition with endogenous metabolism may limit carbon flux into the pathway. Our group has recently developed an E. coli strain where phosphofructokinase (Pfk) activity can be dynamically controlled and demonstrated its use for improving yields and titers of the glucaric acid precursor myo-inositol on glucose minimal medium. In this work, we have explored the further applicability of this strain for glucaric acid production in a supplemented medium more relevant for scale-up studies, both under batch conditions and with glucose feeding via in situ enzymatic starch hydrolysis. It was found that glucaric acid titers could be improved by up to 42% with appropriately timed knockdown of Pfk activity during glucose feeding. The glucose feeding protocol could also be used for reduction of acetate production in the wild type and modified E. coli strains.

Improving product yields on D-glucose in Escherichia coli via knockout of pgi and zwf and feeding of supplemental carbon sources.

The use of lignocellulosic biomass as a feedstock for microbial fermentation processes presents an opportunity for increasing the yield of bioproducts derived directly from glucose. Lignocellulosic biomass consists of several fermentable sugars, including glucose, xylose, and arabinose. In this study, we investigate the ability of an E. coli Δpgi Δzwf mutant to consume alternative carbon sources (xylose, arabinose, and glycerol) for growth while reserving glucose for product formation. Deletion of pgi and zwf was found to eliminate catabolite repression as well as the ability of E. coli to consume glucose for biomass formation. In addition, the yield from glucose of the bioproduct D-glucaric acid was significantly increased in a Δpgi Δzwf strain.