Abstract
BackgroundLimonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc. The objective of this work was to engineer Rhodosporidium toruloides as a cell factory for the production of limonene.ResultsBy overexpressing the limonene synthase (LS), neryl pyrophosphate synthase (NPPS)/geranyl pyrophosphate synthase and the native hydroxy-methyl-glutaryl-CoA reductase (HMGR), we established a baseline for limonene production based on the mevalonate route in Rhodosporidium toruloides. To further enhance the limonene titer, the acetoacetyl-CoA thiolase/HMGR (EfMvaE) and mevalonate synthase (EfMvaS) from Enterococcus faecalis, the mevalonate kinase from Methanosarcina mazei (MmMK) and the chimeric enzyme NPPS-LS were introduced in the carotenogenesis-deficient strain. The resulting strains produced a maximum limonene titer of 393.5 mg/L.ConclusionIn this study, we successfully engineered the carotenogenesis yeast R. toruloides to produce limonene. This is the first report on engineering R. toruloides toward limonene production based on NPP and the fusion protein SltNPPS-CltLS. The results demonstrated that R. toruloides is viable for limonene production, which would provide insights into microbial production of valuable monoterpenes.
Highlights
Limonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc
Since it involves multiple enzymes for constructing an efficient limonene biosynthetic pathway based on nerol diphosphate (NPP) (Fig. 2A; Additional file 1: Fig S1), we employed the 2A peptidemediated multiple proteins production, which has been demonstrated successful in building a carotenoids biosynthesis route (Fig. 2B) [24]
geranyl diphosphate (GPP) and NPP are the direct precursors for monoterpenes, which are biosynthesized by condensing isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) [21]
Summary
Limonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc. Limonene is a widely used natural monoterpene in food, beverage, cosmetics, biomaterials, pharmaceuticals, and advanced biofuels [1,2,3]. It stands as a versatile platform chemical that can be decorated to form various value-added fine chemicals, including linalool, carveol, menthol, limonene-1,2-epoxide, α-terpineol, perillyl alcohol, perillic acid and limonene-1,2-diol, by various natural or engineered microorganisms, and chemical processes [4, 5]. Limonene produced through microbial technology can be regarded as a natural alternative to that extracted from plants.
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