Engineering a non-model yeast Rhodotorula mucilaginosa for terpenoids synthesis

Terpenoids have tremendous biological activities and are widely employed in food,healthcare and pharmaceutical industries.Using synthetic biology to product terpenoids from microbial cell factories presents a promising alternative route compared to conventional methods such as chemical synthesis or phytoextraction.The red yeast Rhodotorula mucilaginosa has been widely studied due to its natural production capacity of carotenoid and lipids,indicating a strong endogenous isoprene pathway with readily available metabolic intermediates.This study constructed several engineered strains of R.mucilaginosa with the aim of producing different terpenoids.Monoterpeneα-terpineol was produced by expressing theα-terpineol synthase from Vitis vinifera.The titer ofα-terpineol was further enhanced to 0.39 mg/L by overexpressing the endogenous rate-limiting gene of the MVA pathway.Overexpression ofα-farnesene synthase from Malus domestica,in combination with MVA pathway rate-limiting gene resulted in significant increase inα-farnesene production,reaching a titer of 822 mg/L.The carotenoid degradation productβ-ionone was produced at a titer of 0.87 mg/L by expressing theβ-ionone synthase from Petunia hybrida.This study demonstrates the potential of R.mucilaginosa as a platform host for the direct biosynthesis of various terpenoids and provides insights for further development of such platforms.

Product Variability of the ‘Cineole Cassette＇ Monoterpene Synthases of Related Nicotiana Species

Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the ＇cineole cassette＇ comprising 1,8-cineole, limonene, myrcene, α-pinene, β-pinene, sabinene, and α-terpineol. We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes, which produced the characteristic monoter- penes of this ＇cineole cassette＇ with α-terpineol being most abundant in the volatile spectra. The amino acid sequences of both terpineol synthases were 99% identical. The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum. The cyclization reactions （α-terpineol to 1,8-cineole） of the terpineol synthases of N. alata and N. langsdorfii were less efficient compared to the ＇cineole cassette＇ monoterpene synthases of Arabidopsis thaliana, N. suaveolens, Salvia fruticosa, Salvia officinalis, and Citrus unshiu. The terpineol synthases of IV. alata and N. langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals. The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N. alata was highest at the transition from day to night （diurnal rhythm）.