Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates.

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.

[Biosynthesis of isocitric acid by the yeast yarrowia lipolytica and its regulation].

We studied the biosynthesis of isocitric acid from rapeseed (canola) oil by the yeast Yarrowia lipolytica and its regulation. We determined a fundamental possibility for directed biosynthesis of isocitric acid by Y lipolytica yeast, with only minimal amounts of citric acid byproduct, when grown on a medium containing canola oil. Wild type strains of Y lipolytica were mutagenized by UV irradiation and treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NG). Subsequent selection on media with acetate and isocitrate resulted in isolation of a UV/NG Y lipolytica UV/NG mutant that synthesized isocitrate and citrate at a ratio of 2.7:1. In the parent strain, this ratio is 1:1. Inhibition of isocitrate lyase, a key enzyme in the metabolism of isocitric acid, by the addition of itaconic acid resulted in increased synthesis of isocitrate with a ratio of isocitrate to citrate reaching 6:1. Culturing of the Y lipolytica UV/NG mutant in a pilot industrial fermenter in the presence of itaconic acid resulted in the production of 88.7 g/L of isocitric acid with a yield of 90%.

A shift to 50°C provokes death in distinct ways for glucose- and oleate-grown cells of Yarrowia lipolytica.

Based on the observation that shocks provoked by heat or amphiphilic compounds present some similarities, this work aims at studying whether cells grown on oleate (amphiphilic pre-stress) acquire a tolerance to heat shock. In rich media, changing glucose for oleate significantly enhanced the cell resistance to the shock, however, cells grown on a minimal oleate medium lost their ability to grow on agar with the same kinetic than glucose-grown cells (more than 7-log decrease in 18 min compared with 3-log for oleate-grown cells). Despite this difference in kinetics, the sequence of events was similar for oleate-grown cells maintained at 50°C with a (1) loss of ability to form colonies at 27°C, (2) loss of membrane integrity and (3) lysis (observed only for some minimal-oleate-grown cells). Glucose-grown cells underwent different changes. Their membranes, which were less fluid, lost their integrity as well and cells were rapidly inactivated. But, surprisingly, their nuclear DNA was not stained by propidium iodide and other cationic fluorescent DNA-specific probes but became stainable by hydrophobic ones. Moreover, they underwent a dramatic increase in membrane viscosity. The evolution of lipid bodies during the heat shock depended also on the growth medium. In glucose-grown cells, they seemed to coalesce with the nuclear membrane whereas for oleate-grown cells, they coalesced together forming big droplets which could be released in the medium. In some rare cases of oleate-grown cells, lipid bodies were fragmented and occupied all the cell volume. These results show that heat triggers programmed cell death with uncommon hallmarks for glucose-grown cells and necrosis for methyl-oleate-grown cells.

[Obtaining of the mutant Yarrowia lipolytica strains producing citric acid from glucose].

The possibility of obtaining mutant yeasts Yarrowia lipolytica VKM Y-2373 with increased ability to synthesize citric acid from glucose by using UV irradiation and N-methyl-NT-nitro-N-nitrosoguanidine was studied. Of 1500 colonies of the Y. lipolytica treated with either UV or N-methyl-NT-nitro-N-nitrosoguanidine, three mutants were selected that displayed higher (by 23%) biosynthetic ability as compared with the initial strain. Additionally, three mutants were selected from 1000 colonies of the Y. lipolytica exposed to a combined action of UV and N-methyl-NT-nitro-N-nitrosoguanidine; their biosynthetic activity exceeded that of the initial strain by 43.9%. The selective media with citrate and acetate were developed for a rapid selection of mutants as well as the express methods for the detection of active citric acid producers on the solid media with chalk and bromocresol containing a limiting concentration of amine nitrogen and an excess of glucose.