Multi‐modular engineering of Saccharomyces cerevisiae for high‐titre production of tyrosol and salidroside

Abstract

Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high-level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose-5phosphate ketol-isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 AE 0.41 mg l À1 in shake flasks, which were approximately 26-fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 AE 19.35 mg l À1 were accomplished in shake flasks. Furthermore, titres of 9.90 AE 0.06 g l À1 of tyrosol and 26.55 AE 0.43 g l À1 of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high valueadded aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae.

Systematic optimization of the yeast cell factory for sustainable and high efficiency production of bioactive ginsenoside compound K

“…High cell density fermentation in bioreactors could significantly boost the production of the target natural products, as proven for artemisinic acid [ 18 ], salidroside [ 25 ], and resveratrol [ 32 ] biosynthesis in engineered yeast. To further promote CK production and to compare the performance of engineered strains with or without UDP-glucose supply optimization in a scale-up condition, we performed fed-batch fermentation using the two CK-producing strains, WPK3 and WPK12, in a parallel bioreactor system.…”

“…The problems associated with the low glycosylation efficiency of PPD into CK may involve the activity and expression level of the key biopart (the glycosyltransferase UGTPg1) and the UDP-glucose supply in the yeast cell factory. Previous efforts to increase yeast UDP-glucose supply have been restricted to overexpressing the key genes involved in UDP-glucose biosynthesis, such as PGM2 and UGP1 [ 19 , 25 ]. Although the production of glycosylated products could be increased, UDP-glucose supply may be improved using other mechanisms besides overexpression.…”

Harnessing the Endogenous 2μ Plasmid of Saccharomyces cerevisiae for Pathway Construction

“…Taking into account the impact of the size of inserted fragment on the p2µM plasmid, we constructed three modules of different sizes using genes of the tyrosol biosynthetic pathway (Supplementary Figure 1D). The small module (mutation module, 3.8 kb) of ARO4 K229L and ARO7 G141S could efficiently relieve feedback inhibition and increase the production of tyrosol in S. cerevisiae (Liu H. et al, 2020), which was introduced to generate plasmid p2µM-ARO4 K229L -ARO7 G141S (p2µM-small-module). The rewiring module containing pentose phosphate pathway genes TKL1 and RKI1 could tune the flux of the precursor pathway (Walfridsson et al, 1996;Kondo et al, 2004;Bera et al, 2011).…”

“…The rewiring module containing pentose phosphate pathway genes TKL1 and RKI1 could tune the flux of the precursor pathway (Walfridsson et al, 1996;Kondo et al, 2004;Bera et al, 2011). The adjustment module that contains ARO2 and ARO10 could adjust the shikimate pathway and L-tyrosine branch by catalyzing the conversion of chorismate from EPSP and the decarboxylation of 4-HPP to 4-HPPA (Liu H. et al, 2020), respectively. The medium module (9.8 kb) composed of the rewiring module and the adjustment module was overexpressed by p2µM plasmid, resulting in plasmid p2µM-TKL1-RKI1-ARO10-ARO2 (p2µM-medium-module).…”

“…Yeast, especially Saccharomyces cerevisiae (S. cerevisiae), has been developed as a host organism for the heterologous production of high-value compounds (Luo et al, 2015;Suastegui and Shao, 2016;Gao et al, 2017;Cao M. et al, 2020;Cao X. et al, 2020;Liu H. et al, 2020;Liu Q. et al, 2020;Ren et al, 2020), free fatty acid (Zhang et al, 2019), soluble cytosolic proteins (Boulet et al, 2017;González et al, 2018;Huang et al, 2018;Zhang et al, 2019), and biofuels (Zhang et al, 2021). Many genetic manipulations of S. cerevisiae rely on the utilization of plasmids (Romanos et al, 1992).…”