Engineering Yarrowia lipolytica for arachidonic acid production through rapid assembly of metabolic pathway

Abstract

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.

An Overview of Potential Oleaginous Microorganisms and Their Role in Biodiesel and Omega-3 Fatty Acid-Based Industries

“…Placing emphasis on differences among the microorganisms and aiming for the most suitable metabolic spot leads to improvements in lipid yields and modification of lipid profiles. The wild-type strains can be modified to improve the lipid accumulation by using the recent metabolic engineering tools [77,161,[228][229][230][231][232][233][234][235][236][237]. The methods of metabolic engineering technology interfere at parts or whole of the proteome, lipidome, transcriptome, genome, and metabolome of microorganisms.…”

Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals

“…As a proof of concept, an entire β-carotene biosynthesis pathway with multiple fragments (four genes with a total size of ∼11 kb) were assembled via in vivo homologous recombination (HR) into rDNA locus, which is a tandem repeat identified in Y. lipolytica ( Gao et al, 2014 ). Similarly, using rDNA as integrative sites, the biosynthetic pathway of arachidonic acid (ARA) was assembled and integrated to Y. lipolytica in one-step ( Liu et al, 2017 ). The resulting pathway showed long-term genetic stability and enabled the strain to produce ARA at 0.4% of total lipid.…”

“…As a first example of successful commercialization, Y. lipolytica has been used industrially to produce ω-3 eicosapentaenoic acid (EPA), making a breakthrough to replace an animal-derived product ( Xie et al, 2015 ). Recently, ARA, a typical omega-6 PUFA, was synthesized via the aerobic Δ-6 desaturation and elongation pathway in Y. lipolytica ( Liu et al, 2017 ). In the engineered strain, a high level of ARA production (0.4% of total FAs) was achieved.…”

“…Among oleaginous yeasts, Yarrowia lipolytica is the most-well studied one. Thanks to the availability of genetic tools ( Madzak, 2015 ; Schwartz et al, 2015 ), Y. lipolytica has been used for a variety of biotechnological applications, including the production of PUFAs ( Xue et al, 2013 ; Liu et al, 2017 ; Sun et al, 2017 ), citric acid ( Förster et al, 2007 ; Moeller et al, 2013 ; Tan et al, 2016 ), and alkanes ( Xu et al, 2016 ). Moreover, Y. lipolytica has also been shown to be rather robust and able to grow on a variety of substrates ( Papanikolaou et al, 2002 ; Ledesma-Amaro and Nicaud, 2016b ; Mirończuk et al, 2016 ).…”

Advancing metabolic engineering of Yarrowia lipolytica using the CRISPR/Cas system

“…Yarrowia lipolytica is a well-known non-conventional yeast, which is generally recognized as safe (Liu et al 2015 ). Due to its strong lipogenesis capability and high protein expression levels, Y. lipolytica is widely researched for the production of both bulk and fine chemicals, including organic acids, fatty acid-derived biofuels and chemicals, polyunsaturated fatty acids, single-cell proteins, terpenoids, and other valuable products (Rymowicz et al 2010 ; Cui et al 2011 ; Yin et al 2012 ; Xue et al 2013 ; Kamzolova et al 2014 ; Blazeck et al 2015 ; Sun et al 2016 ; Liu et al 2017a , b ; Gao et al 2017 ). Meanwhile, a large range of substrates can be effectively utilized by Y. lipolytica , including not only glucose and glycerol but also xylose, cellobiose, and other industrial wastes, which has made it into a hot topic of recent biorefinery research (Ledesma-Amaro and Nicaud 2016 ; Zeng et al 2018 ).…”