A Yarrowia lipolytica Strain Engineered for Pyomelanin Production

The yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine.

Valorization of Crude Glycerol, Residue Deriving from Biodiesel- Production Process, with the Use of Wild-type New Isolated Yarrowia lipolytica Strains: Production of Metabolites with Pharmaceutical and Biotechnological Interest

Background & Objective:Crude glycerol (Glol), used as substrate for screening eleven natural Yarrowia lipolytica strains in shake-flask experiments. Aim of this study was to assess the ability of the screened strains to produce biomass (dry cell weight; X), lipid (L), citric acid (Cit), mannitol (Man), arabitol (Ara) and erythritol (Ery), compounds presenting pharmaceutical and biotechnological interest, in glycerol-based nitrogen-limited media, in which initial glycerol concentration had been adjusted to 40 g/L.Methods:Citric acid may find use in biomedical engineering (i.e. drug delivery, tissue engineering, bioimaging, orthopedics, medical device coating, wound dressings). Polyols are considered as compounds with non-cariogenic and less calorigenic properties as also with low insulin-mediated response. Microbial lipids containing polyunsaturated fatty acids (PUFA) are medically and dietetically important (selective pharmaceutical and anticancer properties, aid fetal brain development, the sight function of the eye, hormonal balance and the cardio-vascular system, prevent reasons leading to type-2 diabetes, present healing and anti-inflammatory effects).Results:All strains presented satisfactory microbial growth (Xmax=5.34-6.26 g/L) and almost complete substrate uptake. The principal metabolic product was citric acid (Citmax=8.5-31.7 g/L). Production of cellular lipid reached the values of 0.33-0.84 g/L. Polyols were also synthesized as strain dependent compounds (Manmax=2.8-6.1 g/L, Aramax ~2.0 g/L, Erymax= 0.5-3.8 g/L). The selected Y. lipolytica strain ACA-DC 5029 presented satisfactory growth along with synthesis of citric acid and polyols, thus, was further grown on media presenting an increased concentration of Glol~75 g/L. Biomass, lipid and citric acid production presented significant enhancement (Xmax=11.80 g/L, Lmax=1.26 g/L, Citmax=30.8 g/L), but conversion yield of citric acid produced per glycerol consumed was decreased compared to screening trials. Erythritol secretion (Erymax=15.6 g/L) was highly favored, suggesting a shift of yeast metabolism from citric acid accumulation towards erythritol production. Maximum endopolysaccharides (IPS) concentration was 4.04 g/L with yield in dry weight 34.2 % w/w.Conclusion:Y. lipolytica strain ACA-YC 5029 can be considered as a satisfactory candidate grown in high concentrations of crude glycerol to produce added-value compounds that interest pharmaceutical and biotechnology industries.

Production of Added-Value Chemical Compounds through Bioconversions of Olive-Mill Wastewaters Blended with Crude Glycerol by a Yarrowia lipolytica Strain

Olive mill wastewaters (OMW) are the major effluent deriving from olive oil production and are considered as one of the most challenging agro-industrial wastes to treat. Crude glycerol is the main by-product of alcoholic beverage and oleochemical production activities including biodiesel production. The tremendous quantities of glycerol produced worldwide represent a serious environmental challenge. The aim of this study was to assess the ability of Yarrowia lipolytica strain ACA-DC 5029 to grow on nitrogen-limited submerged shake-flask cultures, in crude glycerol and OMW blends as well as in media with high initial glycerol concentration and produce biomass, cellular lipids, citric acid and polyols. The rationale of using such blends was the dilution of concentrated glycerol by OMW to (partially or fully) replace process tap water with a wastewater stream. The strain presented satisfactory growth in blends; citric acid production was not affected by OMW addition (Citmax~37.0 g/L, YCit/Glol~0.55 g/g) and microbial oil accumulation raised proportionally to OMW addition (Lmax~2.0 g/L, YL/X~20% w/w). Partial removal of color (~30%) and phenolic compounds (~10% w/w) of the blended media occurred. In media with high glycerol concentration, a shift towards erythritol production was noted (Erymax~66.0 g/L, YEry/Glol~0.39 g/g) simultaneously with high amounts of produced citric acid (Citmax~79.0 g/L, YCit/Glol~0.46 g/g). Fatty acid analysis of microbial lipids demonstrated that OMW addition in blended media and in excess carbon media with high glycerol concentration favored oleic acid production.