Two routes for tyrosol production by metabolic engineering of Corynebacterium glutamicum

Abstract

Background

The phenolic compound tyrosol is widely used in the pharmaceutical industry, owing to its beneficial effects on human health and its use as a precursor for key pharmaceuticals, including β₁-receptor blockers. Tyrosol can be found in olive oil, but despite its natural biosynthesis in plants, low extraction efficiencies render microbial production a more viable alternative.

Results

Here, we engineered the l-tyrosine overproducing Corynebacterium glutamicum strain AROM3 for the de novo production of tyrosol. Two routes were established and compared: one via 4-OH-phenylpyruvate as intermediate and the other via tyramine. We initially expected the first route to require heterologous expression of a prephenate dehydrogenase gene, given that C. glutamicum lacks this enzymatic function. However, heterologous expression of ARO10 from Saccharomyces cerevisiae (ARO10_Sc), which encodes a phenylpyruvate decarboxylase, was sufficient to establish tyrosol production in strain AROM3. We identified that 4-OH-phenylpyruvate is synthesized from l-tyrosine by native aminotransferases, which is subsequently decarboxylated by Aro10_Sc, and reduced to tyrosol by native alcohol dehydrogenases, leading to a titer of 9.4 ± 1.1 mM (1.30 ± 0.15 g/L). We identified the furfural dehydrogenase FudC as major enzyme involved in this pathway, as its gene deletion reduced tyrosol production by 75%. Given the instability of 4-OH-phenylpyruvate, the synthesis of tyrosol via the stable intermediate tyramine was pursued via the second route. Decarboxylation of l-tyrosine followed by oxidative deamination was accomplished by overexpression of the l-tyrosine decarboxylase gene tdc from Levilactobacillus brevis (tdc_Lb) and the tyramine oxidase gene tyo from Kocuria rhizophila (tyo_Kr). Using this route, tyrosol production was increased by 44% compared to the route via 4-OH-phenylpyruvate. With a division of labor approach by co-cultivating l-tyrosine producing strains that either express tdc_Lb or tyo_Kr, the highest titer of 14.1 ± 0.3 mM (1.95 ± 0.04 g/L) was achieved.

Conclusions

This study demonstrates the potential of endotoxin-free C. glutamicum as production host for the l-tyrosine-derived product tyrosol. Due to its l-arogenate pathway for l-tyrosine synthesis, the unstable 4-OH-phenylpyruvate could be excluded as intermediate in the Tdc–Tyo pathway, outcompeting the most often utilized production route via phenylpyruvate decarboxylases.