Current advances in alteration of fatty acid profile in Rhodotorula toruloides: a mini-review.

Microbial lipids are considered promising and environmentally friendly substitutes for fossil fuels and plant-derived oils. They alleviate the depletion of limited petroleum storage and the decrement of arable lands resulting from the greenhouse effect. Microbial lipids derived from oleaginous yeasts provide fatty acid profiles similar to plant-derived oils, which are considered as sustainable and alternative feedstocks for use in the biofuel, cosmetics, and food industries. Rhodotorula toruloides is an intriguing oleaginous yeast strain that can accumulate more than 70% of its dry biomass as lipid content. It can utilize a wide range of substrates, including low-cost sugars and industrial waste. It is also robust against various industrial inhibitors. However, precise control of the fatty acid profile of the lipids produced by R. toruloides is essential for broadening its biotechnological applications. This mini-review describes recent progress in identifying fatty synthesis pathways and consolidated strategies used for specific fatty acid-rich lipid production via metabolic engineering, strain domestication. In addition, this mini-review summarized the effects of culture conditions on fatty acid profiles in R. toruloides. The perspectives and constraints of harnessing R. toruloides for tailored lipid production are also discussed in this mini-review.

Functional characterization and overexpression of Δ12-desaturase in the oleaginous yeast Rhodotorula toruloides for production of linoleic acid-rich lipids.

Linoleic acid (LA) has garnered much attention due to its potential applications in the oleochemical and nutraceutical industries. The oleaginous yeast Rhodotorula toruloides has outstanding lipogenecity, and is considered a potential alternative to the current plant-based platforms for LA production. Δ12-fatty acid desaturases (Δ12-Fads) are involved in LA synthesis in various fungi and yeasts, but their functions in R. toruloides remain poorly understood. To achieve the production of LA-rich lipids in R. toruloides, we investigated the function of the native Δ12-FAD (RtFAD2). First, the overexpression of RtFAD2 and its co-overexpression with RtFAD1 (encoding R. toruloides Δ9-Fad) and their effects on LA production in R. toruloides were investigated. The function of RtFad2 was confirmed by heterologous expression in Saccharomyces cerevisiae. Overexpression of RtFAD2 significantly elevated the LA contents and titers in the wild-type strain R. toruloides DMKU3-TK16 (TK16) and in a thermotolerant derivative of TK16 (L1-1). Additionally, overexpression of RtFAD2 in R. toruloides strains also increased the lipid titer and content. Overexpression of RtFAD1 was down-regulated in the RtFAD1 and RtFAD2 co-overexpressing strains, suggesting that the elevated LA content may function as a key regulator of RtFAD1 expression to control C18 fatty-acid synthesis in R. toruloides. We characterized the function of RtFAD2 and showed that its overexpression in R. toruloides increased the lipid and LA production. These findings may assist in the rational design of metabolic engineering related to LA or polyunsaturated fatty acid production in R. toruloides.

Ethanol and H2O2 stresses enhance lipid production in an oleaginous Rhodotorula toruloides thermotolerant mutant L1-1.

Stress tolerance is a desired characteristic of yeast strains for industrial applications. Stress tolerance has been well described in Saccharomyces yeasts but has not yet been characterized in oleaginous Rhodotorula yeasts even though they are considered promising platforms for lipid production owing to their outstanding lipogenicity. In a previous study, the thermotolerant strain L1-1 was isolated from R. toruloides DMKU3-TK16 (formerly Rhodosporidium toruloides). In this study, we aimed to further examine the ability of this strain to tolerate other stresses and its lipid productivity under various stress conditions. We found that the L1-1 strain could tolerate not only thermal stress but also oxidative stress (ethanol and H2O2), osmotic stress (glucose) and a cell membrane disturbing reagent (DMSO). Our results also showed that the L1-1 strain exhibited enhanced ability to maintain ROS homeostasis, stronger cell wall strength and increased levels of unsaturated membrane lipids under various stresses. Moreover, we also demonstrated that ethanol-induced stress significantly increased the lipid productivity of the thermotolerant L1-1. The thermotolerant L1-1 was also found to produce a higher lipid titer under the dual ethanol-H2O2 stress than under non-stress conditions. This is the first report to indicate that ethanol stress can induce lipid production in an R. toruloides thermotolerant strain.

Delta-9 fatty acid desaturase overexpression enhanced lipid production and oleic acid content in Rhodosporidium toruloides for preferable yeast lipid production.

The oil plants provide a sufficient source of renewable lipid production for alternative fuel and chemical supplies as an alternative to the depleting fossil source, but the environmental effect from these plants' cropping is a concern. The high oleic acid (OA; C18:1) content in plant-derived products provide advantages of multiple uses with improved oxidative stability and a wide range of applicable temperature. Here we used a promising lipid producer, the oleaginous yeast Rhodosporidium toruloides, to attempt to obtain an OA-enriched lipid. Saccharomyces cerevisiae OLE1 (ScOLE1) gene encodes Δ9 fatty acid desaturase (Δ9FAD), which is generally known to synthesize palmitoleic acid (POA; C16:1) and OA, but the functions of putative R. toruloides Δ9FAD gene are not well understood. In a complementary test, the RtΔ9FAD gene rescued the survival of an OA-deficient Scole1Δ mutant, and we introduced the RtΔ9FAD gene into R. toruloides strains for the production of OA-enriched lipid. Increasing lipid production was observed in ScOLE1 and genomic RtΔ9FAD gene-overexpressing R. toruloides strains. The ScOLE1 transformant output fivefold more OA content in total amount, with >70% of total lipid. Different enhancing effects from the protein coding sequence and genomic sequence of RtΔ9FAD genes were also observed. Overall, this study resulted in ScOLE1 and RtΔ9FAD gene overexpression in R. toruloides to obtain OA-enriched lipid as a candidate source of designed biodiesel and lipid-related chemicals.

Isolation of a thermotolerant Rhodosporidium toruloides DMKU3-TK16 mutant and its fatty acid profile at high temperature.

Oleaginous yeast Rhodosporidium toruloides DMKU3-TK16 (TK16), which was isolated from Thailand, is considered a promising lipid producer for biodiesel production. For future industrial applications of this strain, thermotolerant traits are highly desired for their potential to reduce cooling costs in a commercial fermenter. Here, by using an adaptive breeding strategy, we isolated a thermotolerant R. toruloides mutant, L1-1. The isolated L1-1 strain exhibited better growth and higher lipid production at 37°C, and it was found to have significantly higher oleic acid (C18:1) content and yield compared with the wild-type TK16 when cultivated at 37°C. This is the first study to isolate a thermotolerant strain from the oleaginous yeast R. toruloides. The information gained herein will provide new clues for engineering lipid production and for studying the thermotolerance of R. toruloides.