A Novel Microbial Consortia Catalysis Strategy for the Production of Hydroxytyrosol from Tyrosine

Abstract

Hydroxytyrosol, a valuable plant-derived phenolic compound, is increasingly produced from microbial fermentation. However, the promiscuity of the key enzyme HpaBC, the two-component flavin-dependent monooxygenase from Escherichia coli, often leads to low yields. To address this limitation, we developed a novel strategy utilizing microbial consortia catalysis for hydroxytyrosol production. We designed a biosynthetic pathway using tyrosine as the substrate and selected enzymes and overexpressing glutamate dehydrogenase GdhA to realize the cofactor cycling by coupling reactions catalyzed by the transaminase and the reductase. Additionally, the biosynthetic pathway was divided into two parts and performed by separate E. coli strains. Furthermore, we optimized the inoculation time, strain ratio, and pH to maximize the hydroxytyrosol yield. Glycerol and ascorbic acid were added to the co-culture, resulting in a 92% increase in hydroxytyrosol yield. Using this approach, the production of 9.2 mM hydroxytyrosol was achieved from 10 mM tyrosine. This study presents a practical approach for the microbial production of hydroxytyrosol that can be promoted to produce other value-added compounds.

Keywords: HpaBC; enzyme promiscuity; microbial consortia; synthetic biology.

Figure 1

Production of hydroxytyrosol from tyrosine by whole-cell bioconversion. (A) Biosynthesis of hydroxytyrosol from tyrosine via the designed pathway. (B) Production of hydroxytyrosol by strains with different aminotransferases. (C) Production of hydroxytyrosol by strains with different 4-hydroxyphenylpyruvate decarboxylases. (D) Production of hydroxytyrosol by strains with different reductases. 10 mM tyrosine was supplemented as substrate. Data are expressed as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001, compared to each control.

Figure 2

Optimization of cofactor involved in the production of hydroxytyrosol from tyrosine. (A) Scheme of hydroxytyrosol production by introducing GdhA and adding L-glutamate, nicotinic acid or nicotinamide to optimize cofactor supply. (B) Optimization of hydroxytyrosol production by introducing GdhA and addition of glutamate. (C) Optimization of hydroxytyrosol production by addition of nicotimic acid. (D) Optimization of hydroxytyrosol production by addition of nicotinamide. 10 mM tyrosine was supplemented as substrate. Data are expressed as the mean ± SD (n = 3). * p < 0.05, compared to the control.

Figure 3

The promiscuity of HpaBC and design of a microbial consortia catalysis strategy. (A) The color change of strain with pRSF-HT plasmid, CCHT-1, and CCHT-2 cultured with total 10 mM substrate (tyrosin and tyrosol). (B) Catalytic activities of HpaBC towards different substrates. (C) Design of a microbial consortia catalysis strategy by dividing the biosynthetic pathway into two individual strains. Data are expressed as the mean ± SD (n = 3). **** p < 0.0001, compared to the control.

Figure 4

Fermentation time course of CCHT-1 and CCHT-2. (A) Time course of tyrosine, 4-hydroxyphenylpyruvate, 4-hydroxyphenylacetaldehyde and tyrosol during CCHT-1 fermentation. The orange closed circle, blue closed square, magenta closed triangle, and green closed triangle represents tyrosine, 4-hydroxyphenylpyruvate, 4-hydroxyphenylacetaldehyde, and tyrosol, respectively. (B) Time course of tyrosol and hydroxytyrosol during CCHT-2 fermentation. The green closed circle and purple closed square represent tyrosol and hydroxytyrosol, respectively. Data are expressed as the mean ± SD (n = 3).

Figure 5

Optimization of the production of hydroxytyrosol from tyrosine. (A) Optimization of the ratio of two strains and the time of addition. (B) Optimization of buffer concentration and pH. (C) Optimization of the concentration of glycerol and ascorbic acid. 10 mM tyrosine was supplemented as substrate. Data are expressed as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, compared to each control.

Figure 6

Diagram of the two-stage microbial consortia catalysis strategy.