An Engineered Plant Metabolic Pathway Results in High Yields of Hydroxytyrosol Due to a Modified Whole-Cell Biocatalysis in Bioreactor

Abstract

Hydroxytyrosol (HT) is a phenolic substance primarily present in olive leaves and olive oil. Numerous studies have shown its advantages for human health, making HT a potentially active natural component with significant added value. Determining strategies for its low-cost manufacturing by metabolic engineering in microbial factories is hence still of interest. The objective of our study was to assess and improve HT production in a one-liter bioreactor utilizing genetically modified Escherichia coli strains that had previously undergone fed-batch testing. Firstly, we compared the induction temperatures in small-scale whole-cell biocatalysis studies and then examined the optimal temperature in a large volume bioreactor. By lowering the induction temperature, we were able to double the yield of HT produced thereby, reaching 82% when utilizing tyrosine or L-DOPA as substrates. Hence, without the need to further modify our original strains, we were able to increase the HT yield.

Keywords: HPLC-MS/MS; aldehyde reductase; aromatic acetaldehyde synthase; bioreactor; hydroxytyrosol; metabolic engineering; microbial biotechnology; synthetic biology; tyrosinase; whole cell biocatalysis.

Figure 1

Whole-cell biocatalysis experiments in a 1 L bioreactor. (a) Schematic representation of the experimental procedure used. Induction of heterologously expressed proteins in strain BL^DOPA→HT and biocatalysis were performed at 30 °C. (b) Diagram showing the bioconversion of DOPA into hydroxytyrosol (HT). An average HT yield of 45.5% ± 12 was reached as estimated by the initial DOPA concentration added. Three biological replicates were used for the experiments.

Figure 2

Whole-cell biocatalysis experiments for the production of hydroxytyrosol (HT) from various substrates. (a) Schematic presentation of the experimental procedure used. Two different temperatures, 18 °C and 30 °C, were used for the induction of the heterologously expressed protein in each strain. Two different strains of E. coli were used for the production of HT, one from tyrosine (strain BL^Tyr→HT) and one from DOPA (strain BL^DOPA→HT). (b) Diagram showing the average yield of HT production from various substrates at the two different temperatures used. In all cases, higher yields of HT were observed when induction was performed at 18 °C. Statistical analysis was performed with one-way ANOVA (* p < 0,05; 2–3 biological replicates were used for each experiment).

Figure 3

Whole-cell biocatalysis experiments in a 1 L bioreactor. (a) Schematic representation of the experimental procedure used. Induction of heterogously expressed proteins in each strain was performed at 18 °C while biocatalysis was performed at 30 °C. Two different strains of E. coli were used for the production of hydroxytyrosol (HT), one from tyrosine (strain B^Tyr→HT) and one from DOPA (strain B^DOPA→HT). (b) Diagram showing the production of HT vs. the substrate for strain BL^DOPA→HT and strain BL^Tyr→HT. (c) The average yield for strain BL^DOPA→HT (two independent experiments) was 82% ± 9 (two independent experiments), for strain BL^Tyr→HT, it was 81% ± 14 (two independent experiments) as estimated by the initial precursor concentration added.