Engineering of Hydroxytyrosol Production in Escherichia coli

Hydroxytyrosol (HTy) is one of the most powerful antioxidants with potential biological function as an anti-tumor, anti-atherogenic, anti-inflammatory and anti-platelet aggregation agent. It has a wide range of potential applications in industry, such as functional food, dietary supplement, cosmetics, and animal feed. Currently, HTy is produced from enriched olive extracts after chemical or enzymatic hydrolysis. There is an increasing demand for stable and sustainable production, and microbial fermentation can be a promising solution.

We have previously reported an engineered E. coli capable of producing HTy. Our synthetic HTy pathway consists of five heterologous genes for tyrosine hydroxylation and downstream conversion of L-DOPA to HTy. Although we have shown that this synthetic pathway is functional, the low titer from tyrosine or from glucose remained as an issue to be addressed. Here we report three metabolic engineering efforts to improve the efficiency of the synthetic HTy pathway. First, we have engineered the co-factor biosynthetic pathway for tyrosine hydroxylation. Next, we have identified inhibition of the tyrosine hydroxylation by downstream chemicals and applied co-culture strategy to overcome it. Lastly, the gene expression of pathway enzymes was optimized using metabolomics and proteomics data. With these engineering, we were able to improve the product yield from tyrosine about 3-fold. In addition, we have engineered the host strain to overproduce tyrosine. Using this engineered strain, we confirmed the production of L-DOPA from glucose without any external supplementation of tyrosine, and the resulting L-DOPA was further converted into HTy by co-culture strategy with an improved yield from the previous result.

This work demonstrates a potential industrial application of microbial HTy production and provides a good renewable microbial platform for the production of a wide range of chemicals that involve the hydroxylation of aromatic amino acid.

This work was supported by Ajinomoto (Study abroad program for research scientist) and the Joint BioEnergy Institute (http://www.jbei.org/), which is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.