The global market for high-value fatty acids production, mainly omega-3/6, hydroxy fatty-acids, waxes and their derivatives, has seen strong development in the last decade. The reason for this growth was the increasing utilization of these lipids as significant ingredients for cosmetics, food and the oleochemical industries. The large demand for these compounds resulted in a greater scientific interest in research focused on alternative sources of oil production - among which microorganisms attracted the most attention. Microbial oil production offers the possibility to engineer the pathways and store lipids enriched with the desired fatty acids. Moreover, costly chemical steps are avoided and direct commercial use of these fatty acids is available. Among all microorganisms, the oleaginous yeasts have become the most promising hosts for lipid production - their efficient lipogenesis, ability to use various (often highly affordable) carbon sources, feasible large-scale cultivations and wide range of available genetic engineering tools turns them into powerful micro-factories. This review is an in-depth description of the recent developments in the engineering of the lipid biosynthetic pathway with oleaginous yeasts. The different classes of valuable lipid compounds with their derivatives are described and their importance for human health and industry is presented. The emphasis is also placed on the optimization of culture conditions in order to improve the yield and titer of these valuable compounds. Furthermore, the important economic aspects of the current microbial oil production are discussed.

Human milk fat has a distinctive stereoisomeric structure where palmitic acid is esterified to the middle (sn-2) position on the glycerol backbone of the triacylglycerol and unsaturated fatty acids to the outer (sn-1/3) positions. This configuration allows for more efficient nutrient absorption in the infant gut. However, the fat used in most infant formulas originates from plants, which exclude palmitic acid from the sn-2 position. Oleaginous yeasts provide an alternative source of lipids for human nutrition. However, these yeasts also exclude palmitic acid from the sn-2 position of their triacylglycerol. Here we show that Yarrowia lipolytica can be engineered to produce triacylglycerol with more than 60% of the palmitic acid in the sn-2 position, by expression of lysophosphatidic acid acyltransferases with palmitoyl-Coenzyme A specificity. The engineered Y. lipolytica strains can be cultured on glycerol, glucose, palm oil or a mixture of substrates, under nitrogen limited condition, to produce triacylglycerol with a fatty acid composition that resembles human milk fat, in terms of the major molecular species (palmitic, oleic and linoleic acids). Culture on palm oil or a mixture of glucose and palm oil produced the highest lipid titre and a triacylglycerol composition that is most similar with human milk fat. Our data show that an oleaginous yeast can be engineered to produce a human milk fat substitute (β-palmitate), that could be used as an ingredient in infant formulas.

The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We investigated the role of hexose transporters and hexokinase (native and fructophilic) in this preference. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Interestingly, combined overexpression of either hexokinase with hexose transporters significantly accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L).

Yarrowia lipolytica is a non-conventional yeast producing valuable compounds, such as citric acids, from renewable raw materials. This study investigated the impact of citrate synthase overexpression on the biosynthesis of citric and isocitric acid in Y. lipolytica. Two transformants of Y. lipolytica A101.1.31 strain (efficient citric acid producer), overexpressing CIT1 or CIT2 gene (encoding proteins with citrate synthase activity), were constructed. The results revealed that overexpression of either of these genes enhances citrate synthase activity. Additionally, the cit1 knockout strain was unable to use propionate as the sole carbon source, which proves that CIT1 gene encodes a dual activity protein–citrate and 2-methylcitrate synthase. In the overexpressing mutants, a significant increase in isocitric acid biosynthesis was observed. Both CIT1 and CIT2 overexpressing strains produced citric and isocitric acid from vegetable oil in a ratio close to 1 (CA/ICA ratio for wild-type strain was 4.12).