Influence of Agitation and Aeration on Single Cell Oil Production by Rhodotorula glutinis from Glycerol

In the present study, the yeast Rhodotorula glutinis has been assessed with the aim of producing microbial lipids from glycerol under different aeration conditions. For such a purpose, experiments were carried out in shake flasks, under different conditions of agitation (150 to 250 rpm) and aeration (2.5 to 5.0 of flask volume-to-medium volume ratio). Furthermore, their influence on fermentative parameters (lipid and cell concentration, biomass yield;lipid yield;and lipid volumetric productivity) has been investigated using a 22 full factorial design. The statistical analysis has revealed a strong influence of both variables on substrate consumption, lipid accumulation, cell growth and lipid productivity. As a whole, results suggest that higher aeration levels provide greater cell and lipid concentrations, and lipid volumetric productivity. The best results (4.5 g/L of lipids and QP = 0.95 g/L⋅day) were achieved at the highest aeration (5.0 flask volume-to-medium volume ratio) and agitation (250 rpm) levels. Their fatty acid profile showed that oleic acid was produced in greater quantity (53.5%), followed by linoleic acid (18.7%), palmitic acid (6.8%) and stearic acid (9.9%). The microbial oil presented viscosity of 39.3 cP at 50°C and free fatty acid content of 1.93% ± 0.08%. These are significant results and contribute to establishing operational conditions that maximize single-cell oil production from glycerol by Rhodotorula glutinis, i.e. an alternative source as renewable raw material for lipid-based biorefineries.

Engineering of Yarrowia lipolytica for production of astaxanthin

Astaxanthin is a red-colored carotenoid,used as food and feed additive.Astaxanthin is mainly produced by chemical synthesis,however,the process is expensive and synthetic astaxanthin is not approved for human consumption.In this study,we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation.First,we screened 12 different Y.lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis:bi-functional phytoene synthase/lycopene cyclase(crtYB)and phytoene desaturase(crtI)from the red yeast Xanthophyllomyces dendrorhous.The best strain produced 31.1±0.5 mg/L β-carotene.Next,we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMG1)and geranylgeranyl diphosphate synthase(GGS1/crtE)in the best producing strain and obtained 453.9±20.2 mg/L β-carotene.Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1±57.2 mg/L.Then we introduced β-carotene ketolase(crtW)from Paracoccus sp.N81106 and hydroxylase(crtZ)from Pantoea ananatis to convert β-carotene into astaxanthin.The constructed strain accumulated 10.4±0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates,5.7±0.5 mg/L canthaxanthin,and 35.3±1.8 mg/L echinenone.Finally,we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW(54.6 mg/L)of astaxanthin in a microtiter plate cultivation.Our study for the first time reports engineering of Y.lipolytica for the production of astaxanthin.The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y.lipolytica-based fermentation process for astaxanthin production.