Establishing an Artificial Pathway for Efficient Biosynthesis of Hydroxytyrosol

Abstract

Hydroxytyrosol (HT) is a valuable natural phenolic compound with strong antioxidant activity and various physiological and pharmaceutical functions. In this study, we established an artificial pathway for HT biosynthesis. First, efficient enzymes were selected to construct a tyrosol biosynthetic pathway. Aro10 from Saccharomyces cerevisiae was shown to be a better ketoacid decarboxylase than Kivd from Lactococcus lactis for tyrosol production. While knockout of feaB significantly decreased accumulation of the byproduct 4-hydroxyphenylacetic acid, overexpression of alcohol dehydrogenase ADH6 further improved tyrosol production. The titers of tyrosol reached 1469 ± 56 mg/L from tyrosine and 620 ± 23 mg/L from simple carbon sources, respectively. The pathway was further extended for HT production by overexpressing Escherichia coli native hydroxylase HpaBC. To enhance transamination of tyrosine to 4-hydroxyphenylpyruvate, NHCl was removed from the culture media. To decrease oxidation of HT, ascorbic acid was added to the cell culture. To reduce the toxicity of HT, 1-dodecanol was selected as the extractant for in situ removal of HT. These efforts led to an additive increase in HT titer to 1243 ± 165 mg/L in the feeding experiment. Assembly of the full pathway resulted in 647 ± 35 mg/L of HT from simple carbon sources. This work provides a promising alternative for sustainable production of HT, which shows scale-up potential.

Dual pathway for metabolic engineering of E. coli metabolism to produce the highly valuable hydroxytyrosol

“…Moreover, when ALR-K was functioning, the levels of DOPA and tyrosol remained minimal showing that the biosynthetic flow from tyrosine to HT was running properly. The negative effect of feaB to remove intermediate metabolites from the introduced pathway to E. coli through AADC and TYR has been shown before [27,28,57], during previous attempts to heterologously produce HT from E. coli, which explains our status of data. Suggested deletion of feaB gene previously resulted in further increase of HT production.…”

“…The final titer of this metabolic construction was about 22 times higher than that of [27] and 1.3 times higher than that of Chung et al [28] (Table 3), while it was 12 times less than the precursor tyrosine reported to be produced by the tyrosine overproducer strain [29], a fact that dictates space for further optimization. At the time of submission, two new articles drawn our attention for the biosynthesis of hydroxytyrosol in E. coli, authored by Li et al [57] and Choo et al [63]. The performance of their system is of interest due to the efficacy they achieved, especially for the case of Li et al [57] who achieved a titer of 647 mg/L (Table 3).…”

“…At the time of submission, two new articles drawn our attention for the biosynthesis of hydroxytyrosol in E. coli, authored by Li et al [57] and Choo et al [63]. The performance of their system is of interest due to the efficacy they achieved, especially for the case of Li et al [57] who achieved a titer of 647 mg/L (Table 3). They ended up in such a high concentration following a different approach, directing the metabolic flow from 4-hydroxyphenylpyruvate to 4-hydroxyphenylacetaldehyde, tyrosol and finally to HT through the action of a ketoacid decarboxylase, an alcohol dehydrogenase and a 4hydroxyphenylacetic acid 3-hydroxylase, respectively.…”

Synergistic Neuroprotective Effect of Endogenously-Produced Hydroxytyrosol and Synaptic Vesicle Proteins on Pheochromocytoma Cell Line against Salsolinol

“…Keeping in mind the strongest neuroprotective effect of hydroxyltyrosol, ADH6 gene from S. cerevisiae was overexpressed in the PC12 and PC12+syn + +sep − cell lines to endogenously overproduce hydroxytyrosol from DOPAL and to strengthen the EADS of these cell lines. ADH6 has been previously reported for its efficient catalysis of 3,4-dihydroxyphenylacetaldehyde (DOPAL) into hydroxytyrosol [63,64]. Other studies have also reported the heterologous production of hydroxytyrosol from tyrosol in different bacterial species, such as Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, and Micrococcus luteus [83].…”

“…Through a literature survey, we discovered that alcohol dehydrogenase-6 (ADH6) from Saccharomyces cerevisiae catalyzes the conversion of 3,4-dihydroxyphenylacetaldehyde (DOPAL) into hydroxytyrosol with high efficiency [63,64]. DOPAL, an endogenous neurotoxin, is produced from the oxidative deamination of dopamine catalyzed by monoamine oxidases [65].…”