Engineering Rhodosporidium toruloides with a membrane transporter facilitates production and separation of carotenoids and lipids in a bi-phasic culture

Abstract

The oleaginous yeast Rhodosporidium toruloides has great biotechnological potential. It accumulates a high amount of lipids which can be used for biofuels and also produces carotenoids which are valuable in the food and pharmaceutical industry. However, the location of these two hydrophobic products in the cell membrane prohibits its efficient harvesting and separation. Here, the transporter Pdr10 was engineered into R. toruloides and cultured in two-phase media containing oil. This enabled the production and in situ export of carotenoids into the oil and concurrent separation from intracellular lipids in the cells. When Pdr10 strain was cultured in the two-phase media, carotenoids and fatty acids yield increased from 1.9 to 2.9 μg/mg and 0.07 to 0.09 mg/mg, respectively. A total of 1.8 μg/mg carotenoids was exported by Pdr10 strain, as compared to 0.3 μg/mg in the wild type. In the Pdr10 strain, the composition of carotenoids and fatty acid it produced also changed. Torulene became the major carotene produced instead of torularhodin. Also, the unsaturated fatty acid C18:2 became the dominant fatty acid produced instead of the saturated C16:0, which was similar to the grape seed oil used in the two-phase media. This indicated that oil was being consumed by the cells, which was supported by the increased intracellular glycerol levels detected by gas chromatography-mass spectrometry (GC-MS). Our approach represents an easy and greener extraction method which could serve to increase the yield and facilitate separation of carotenoids and fatty acids.

Expression of phosphotransacetylase in Rhodosporidium toruloides leading to improved cell growth and lipid production

“…3 There are several strategies to improve the economic feasibility of MLs: rst of all, exploration of low cost substrates such as lignocellulosic biomass and various organic wastes; 4,5 second, developing more efficient culture strategies and product separation methods to reduce process costs; 6 in another scenario, manufacturing valuable (co-)products such as unsaturated fatty acids and carotenoids through metabolic engineering of oleaginous species. 7,8 However, the theoretical yield of per mole of hexose is limited to two moles acetyl-CoA, since the glycolysis loses a carbon equivalent for each C3 unit upon pyruvate decarboxylation. 9 Ideally, cell growth and lipid production may be further improved by devising more efficient acetyl-CoA generating pathways, but such efforts remain limited in oleaginous yeast.…”

Comparative metabolomics profiling of engineered Saccharomyces cerevisiae lead to a strategy that improving β-carotene production by acetate supplementation

“…cerevisiae strain (CEN.PK113-7D) can induce multidrug-resistant transporter synthesis, encoded by pleiotropic drug-resistance genes (Pdr10), and concluded that this can facilitate the secretion of carotenoids to the environment to decrease the toxicity within the cells. Based on these results, Lee et al [ 17 ] transformed Pdr10 from S . cerevisiae into Rhodosporidium toruloides , and found that Pdr10 strain cultivated in the two-phase media could obtain higher production of carotenoids due to continuous export of carotenoids in situ.…”

Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals

“…It is widely known that the oleaginous yeast R. toruloides can naturally accumulate high levels of carotenoids. Recently, a membrane transporter Pdr10 was introduced into R. toruloides to facilitate production and separation of carotenoids ( Lee et al, 2016 ). In the resulting strain, a total of 2.9 μg/mg carotenoids was produced, while a total of 1.8 μg/mg carotenoids was exported.…”