High-Level Production of Hydroxytyrosol in Engineered Saccharomyces cerevisiae

Abstract

Hydroxytyrosol (HT) is a valuable aromatic compound with numerous applications. Herein, we enabled the efficient and scalable de novo HT production in engineered Saccharomyces cerevisiae (S. cerevisiae) from glucose. Starting from a tyrosol-overproducing strain, six HpaB/HpaC combinations were investigated, and the best catalytic performance was acquired with HpaB from Pseudomonas aeruginosa (PaHpaB) and HpaC from Escherichia coli (EcHpaC), resulting in 425.7 mg/L HT in shake flasks. Next, weakening the tryptophan biosynthetic pathway through downregulating the expression of TRP2 (encoding anthranilate synthase) further improved the HT titer by 27.2% compared to the base strain. Moreover, the cytosolic NADH supply was improved through introducing the feedback-resistant mutant of the TyrA (the NAD + -dependent chorismate mutase/prephenate dehydrogenase, TyrA*) from E. coli, which further increased the HT titer by 36.9% compared to the base strain. The best performing strain was obtained by optimizing the biosynthesis of HT in S. cerevisiae through a screening for an effective HpaB/HpaC combination, biosynthetic flux rewiring, and cofactor engineering, which enabled the titer of HT reaching 1120.0 mg/L in the shake flask. Finally, the engineered strain produced 6.97 g/L of HT by fed-batch fermentation, which represents the highest titer for de novo HT biosynthesis in microorganisms reported to date.

Yeast: A platform for the production of L‐tyrosine derivatives

“…The best combination Pa HpaB‐ Ec HpaC results more than 2‐fold production of hydroxytyrosol than others (H. Liu, Wu, et al, 2022). On the other hand, enriching the tyrosol's supply represents an efficient method for producing hydroxytyrosol (Bisquert et al, 2022; Y. Liu, Liu, et al, 2022; H. Liu, Wu, et al, 2022). The Lyu group compared different sugars in the production of hydroxytyrosol, and the production of hydroxytyrosol was improved by three‐fold with the usage of sucrose and glycerol as carbon source than that of glucose (Y. Liu, Liu, et al, 2022).…”

“…However, the HpaBC‐based reaction has a low yield of hydroxytyrosol in yeast (Y. Liu, Song, et al, 2022). To improve the conversion rate, researchers tested the effects of different combination of HpaB and HpaC (Y. Liu, Liu, et al, 2022; H. Liu, Wu, et al, 2022). The best combination Pa HpaB‐ Ec HpaC results more than 2‐fold production of hydroxytyrosol than others (H. Liu, Wu, et al, 2022).…”

“…To improve the conversion rate, researchers tested the effects of different combination of HpaB and HpaC (Y. Liu, Liu, et al, 2022; H. Liu, Wu, et al, 2022). The best combination Pa HpaB‐ Ec HpaC results more than 2‐fold production of hydroxytyrosol than others (H. Liu, Wu, et al, 2022). On the other hand, enriching the tyrosol's supply represents an efficient method for producing hydroxytyrosol (Bisquert et al, 2022; Y. Liu, Liu, et al, 2022; H. Liu, Wu, et al, 2022).…”

“…The Lyu group compared different sugars in the production of hydroxytyrosol, and the production of hydroxytyrosol was improved by three‐fold with the usage of sucrose and glycerol as carbon source than that of glucose (Y. Liu, Liu, et al, 2022). Our group improved the cofactor supply by introducing the mutant of TyrA M53I A354V , and 6.97 g L −1 production of hydroxytyrosol in fed‐batch was achieved (H. Liu, Wu, et al, 2022). Taking that Escherichia coli may be a better platform to hydroxylate tyrosol, the Lyu group adopted a S. cerevisiae ‐ E. coli coculture strategy and placed the hydroxylation of tyrosol in E. coli with 435 mg L −1 hydroxytyrosol produced in fermentation (Y. Liu, Gue, et al, 2022).…”

Production of 3-Hydroxytyrosol from Glucose by Chromosomally Engineered Escherichia coli by Fed-Batch Cultivation in a Jar Fermenter

“…A recent study using chromosomally engineered S. cerevisiae and glucose produced HT with a titer of 6.97 g/L in fed-batch cultivation, which is extremely high . However, the net titer of HT from glucose was unclear because a small amount of Tyr, which is a source of HT via the Ehrlich pathway, was supplied to the culture with the peptone and yeast extract.…”