Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content. Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials.

Microbial lipid production using renewable feedstock shows great promise for the biodiesel industry. In this study, the ability of a lipid-engineered Yarrowia lipolytica strain JMY4086 to produce lipids using molasses and crude glycerol under different oxygenation conditions and at different inoculum densities was evaluated in fed-batch cultures. The greatest lipid content, 31% of CDW, was obtained using a low-density inoculum, a constant agitation rate of 800 rpm, and an oxygenation rate of 1.5 L/min. When the strain was cultured for 450 h in a chemostat containing a nitrogen-limited medium (dilution rate of 0.01 h−1; 250 g/L crude glycerol), volumetric lipid productivity was 0.43 g/L/h and biomass yield was 60 g CDW/L. The coefficient of lipid yield to glycerol consumption (Y L/gly) and the coefficient of lipid yield to biomass yield (Y L/X ) were equal to 0.1 and 0.4, respectively. These results indicate that lipids may be produced using renewable feedstock, thus providing a means of decreasing the cost of biodiesel production. Furthermore, using molasses for biomass production and recycling glycerol from the biodiesel industry should allow biolipids to be sustainably produced.

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.

It was found that in the cells of Y. lipolytica the enzymes of pentose-phosphate pathways - transketolase and erythrose reductase were involved in the process o

Increasing interest of non-conventional yeasts has been observed for many years due to their biochemical characteristics and potential applications. Well-studied, oleaginous yeast Y. lipolytica is an attractive host for converting a low-cost glycerol, into value-added products such as erythritol (sweetener) or citric acid. Glycerol is an important renewable feedstock and is the main co-product of biodiesel production, which is nowadays applied on a large commercial scale. To this end, we engineered the yeast Y. lipolytica to increase the productivity of this strain. In this light, we enhanced glycerol assimilation by over-expression of the YALI0F00484g gene encoding glycerol kinase (GK) and gene YALI0B02948g encoding glycerol-3-P dehydrogenase (GDH). The modified strains have been tested for glycerol consumption rate and erythritol and citric acid synthesis under various conditions. Here, we show that the overexpression of GK and GDH, increased glycerol consumption resulting in rapid erythritol and citric acid synthesis. Next, we combined the two genes in the tandem gene construct for the simultaneous co-expression of GK and GDH, which further increased the desired product synthesis. The glycerol consumption was explored in a 5-L bioreactor and the engineered strains were able to utilize 150 g/L glycerol within 44–48 hours. The erythritol productivity for GK overexpression and co-expression of GK and DGH was 24 and 35 %, respectively, over the control strain. Moreover, we established conditions for the production of citric acid at pH 3.0, the engineered strains increased citric acid production 14-fold over the control. This work demonstrates the excellent capacity of the engineered strains as a starting platform for further modification for broad-range value-added product biosynthesis from glycerol. This study presents the highest reported titer citric acid at low pH to date. The process parameters such as productivity and yield of erythritol and citric acid were significantly elevated, what is valuable for industrial applications.