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Yarrowia lipolytica, a non-conventional oleaginous yeast with special traits, has attracted increasing interest for producing value-added products. Generally, the DNA fragments of these heterologous metabolic pathways are constructed via the classic restriction digestion and ligation method. In contrast, the one-step in vivo pathway assembly method has been only rarely applied to Y. lipolytica. Here, with arachidonic acid biosynthesis as a case study, a one-step in vivo pathway assembly and integration method was used for engineering Y. lipolytica. Using rDNA as integrative locus, this study showed that there was a relation between the assembly efficiency and the length of overlapping region. Especially, with an overlap up to 1 kb, the method was able to rapidly assemble the arachidonic acid biosynthesis pathway (nearly 10 kb) into the chromosome with high efficiency (nearly 23%). Meanwhile, the pathway assembled in Y. lipolytica demonstrated long-term genetic stability and the engineered strain exhibited robust growth. Furthermore, this study demonstrated that the codon-optimized genes from Mortierella alpina can function efficiently in Y. lipolytica: a high level arachidonic acid production (0.4% of total fatty acids) was produced in the engineered strain. To our knowledge, this is the first time that this method is applied to Y. lipolytica for functional polyunsaturated fatty acids production. This method represents a powerful tool with potential for facilitating engineering applications in non-conventional yeasts.

γ-linolenic acid (GLA) has various well-documented beneficial physiological effects and high biological significance. Because the natural supply of GLA is insufficient, microbial GLA production is a promising method for pharmaceutical and nutraceutical purposes. To establish and develop a biotechnological process for GLA production by Yarrowia lipolytica, the codon-optimized △6-desaturase from Mortierella alpina was introduced into this yeast under the control of the strong hp4d promoter. A recombinant Y. lipolytica strain was constructed, which produced 4.6% GLA in total fatty acids. By using a temperature-shift strategy of cultivation, consisting in preliminary growth at 28 °C followed by 6-day culture at 20 °C, optimal levels of dry cell weight (DCW), lipid content and GLA concentration were obtained from the recombinant strain: 18.55 g/L, 1.16 g/L and 71.6 mg/L, respectively. These DCW, lipid and GLA values were respectively 25.7%, 19.6% and 60.9% higher than those obtained in the control cultivation experiment at the standard constant temperature of 28 °C. This work demonstrates the excellent capacity of Y. lipolytica for GLA production, by combining metabolic engineering with a temperature-shift strategy.

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L −1 . To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/ L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.

The microbial fermentation process has been used as an alternative pathway to the production of value-added natural products. Of the microorganisms, Yarrowia lipolytica, as an oleaginous platform, is able to produce fatty acid-derived biofuels and biochemicals. Nowadays, there are growing progresses on the production of value-added fatty acid-based bioproducts in Y. lipolytica. However, there are fewer reviews performing the metabolic engineering strategies and summarizing the current production of fatty acid-based bioproducts in Y. lipolytica. To this end, we briefly provide the fatty acid metabolism, including fatty acid biosynthesis, transportation, and degradation. Then, we introduce the various metabolic engineering strategies for increasing bioproduct accumulation in Y. lipolytica. Further, the advanced progress in the production of fatty acid-based bioproducts by Y. lipolytica, including nutraceuticals, biofuels, and biochemicals, is summarized. This review will provide attractive thoughts for researchers working in the field of Y. lipolytica.

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As the first nucleoside antibiotic discovered in fungi, cordycepin, with its various biological activities, has wide applications. At present, cordycepin is mainly obtained from the natural fruiting bodies of Cordyceps militaris. However, due to long production periods, low yields, and low extraction efficiency, harvesting cordycepin from natural C. militaris is not ideal, making it difficult to meet market demands. In this study, an engineered Yarrowia lipolytica YlCor-18 strain, constructed by combining metabolic engineering strategies, achieved efficient de novo cordycepin production from glucose. First, the cordycepin biosynthetic pathway derived from C. militaris was introduced into Y. lipolytica. Furthermore, metabolic engineering strategies including promoter, protein, adenosine triphosphate, and precursor engineering were combined to enhance the synthetic ability of engineered strains of cordycepin. Fermentation conditions were also optimized, after which, the production titer and yields of cordycepin in the engineered strain YlCor-18 under fed-batch fermentation were improved to 4362.54 mg/L and 213.85 mg/g, respectively, after 168 h. This study demonstrates the potential of Y. lipolytica as a cell factory for cordycepin synthesis, which will serve as the model for the green biomanufacturing of other nucleoside antibiotics using artificial cell factories.