Metabolic engineering of Rhodotorula toruloides for resveratrol production

Abstract

Background Resveratrol is a plant-derived phenylpropanoid with diverse biological activities and pharmacological applications. Plant-based extraction could not satisfy ever-increasing market demand, while chemical synthesis is impeded by the existence of toxic impurities. Microbial production of resveratrol offers a promising alternative to plant- and chemical-based processes. The non-conventional oleaginous yeast Rhodotorula toruloides is a potential workhorse for the production of resveratrol that endowed with an efficient and intrinsic bifunctional phenylalanine/tyrosine ammonia-lyase (RtPAL) and malonyl-CoA pool, which may facilitate the resveratrol synthesis when properly rewired. Results Resveratrol showed substantial stability and would not affect the R. toruloides growth during the yeast cultivation in flasks. The heterologus resveratrol biosynthesis pathway was established by introducing the 4-coumaroyl-CoA ligase (At4CL), and the stilbene synthase (VlSTS) from Arabidopsis thaliana and Vitis labrusca, respectively. Next, The resveratrol production was increased by 634% through employing the cinnamate-4-hydroxylase from A. thaliana (AtC4H), the fused protein At4CL::VlSTS, the cytochrome P450 reductase 2 from A. thaliana (AtATR2) and the endogenous cytochrome B5 of R. toruloides (RtCYB5). Then, the related endogenous pathways were optimized to affect a further 60% increase. Finally, the engineered strain produced a maximum titer of 125.2 mg/L resveratrol in YPD medium. Conclusion The non-conventional oleaginous yeast R. toruloides was engineered for the first time to produce resveratrol. Protein fusion, co-factor channeling, and ARO4 and ARO7 overexpression were efficient for improving resveratrol production. The results demonstrated the potential of R. toruloides for resveratrol and other phenylpropanoids production.

Enhanced microbial production of protocatechuate from engineered sorghum using an integrated feedstock-to-product conversion technology

“…1B) indicates it could be due to the activity of R. toruloides’ endogenous phenylalanine/tyrosine ammonia-lyase, which can catalyze the conversion of l -tyrosine to p -coumaric acid. 53,54 As mentioned before, further engineering of R. toruloides to increase carbon flow towards the aromatic biosynthetic pathway from central metabolism could be a promising strategy to increase PCA titers with the biomass conversion process developed in this study.…”

Dual-Regulation in Peroxisome and Cytoplasm toward Efficient Limonene Biosynthesis with Rhodotorula toruloides

“…Induction medium (IM) for ATMT was prepared as reported by Zhang et al that contains 200 μmmol/L acetosyringone in MM salt solution (10 mmol/L KH 2 PO 4 , 10 mmol/L K 2 HPO 4 , 2.5 mmol/L NaCl, 4 mmol/L (NH 4 ) 2 SO 4 , 0.7 mmol/L CaCl 2 , 2 mmol/L MgSO 4 , 9 μmol/L FeSO 4 , 10 mmol/L glucose, pH 7.0). 10 The ATMT induction was conducted at 25 °C for 36 h. The minimal medium (MM) contained 20 g/L glucose, 5 g/L (NH 4 ) 2 SO 4 , 3 g/L KH 2 PO 4 , 0.5 g/L MgSO 4 •7H 2 O, 1 mL/L vitamin solution, and 1 mL/L trace element solution. The vitamin and trace element solution were prepared by referring to Jensen et al's medium formulation.…”

“…toruloides has many attractive features conducive to microbial cell factory construction like fast growth, robustness against the toxic byproducts of biomass hydrolysates, and high-density fermentation capacity. , R. toruloides has been rewired for producing various value-added terpenoids, oleochemicals, and phenylpropanoids. , However, two main challenges are undermining the exploration efforts on R. toruloides toward high-level production of a specific product.…”

“…8,9 R. toruloides has been rewired for producing various value-added terpenoids, oleochemicals, and phenylpropanoids. 9,10 However, two main challenges are undermining the exploration efforts on R. toruloides toward high-level production of a specific product. One is the lack of free expression plasmids, which makes the construction of a desirable multiple metabolic route for products such as limonene highly dependent on the availability of selecting markers.…”

“…4,8 For example, when it was engineered toward resveratrol production, the critical intermediate product cinnamic acid was decomposed by unknown enzymes in R. toruloides. 10 Redirecting target metabolic pathways into organelles represents an attractive strategy for streamlining reaction cascades and averting cross-talk since the organelles can provide relatively higher local enzyme concentrations, isolated space, and specific microenvironments. 11−13 Moreover, organelle compartmentalization is also powerful in isolating toxic heterologous components away from the host cell cytosol, thereby relieving cytotoxicity, improving cell growth, and increasing product titer.…”