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

Rhodotorula toruloides is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in R. toruloides. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes forR. toruloides. The results demonstrated that R. toruloides could be explored for efficient production of terpenoids.

[Advances in gene editing and natural product synthesis of Rhodotorula toruloides].

Rhodotorula toruloides is a non-conventional red yeast that can synthesize various carotenoids and lipids. It can utilize a variety of cost-effective raw materials, tolerate and assimilate toxic inhibitors in lignocellulosic hydrolysate. At present, it is widely investigated for the production of microbial lipids, terpenes, high-value enzymes, sugar alcohols and polyketides. Given its broad industrial application prospects, researchers have carried out multi-dimensional theoretical and technological exploration, including research on genomics, transcriptomics, proteomics and genetic operation platform. Here we review the recent progress in metabolic engineering and natural product synthesis of R. toruloides, and prospect the challenges and possible solutions in the construction of R. toruloides cell factory.

Metabolic engineering of Rhodotorula toruloides for resveratrol production.

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.