초록
<P><B>Abstract</B></P> <P> <I>Yarrowia lipolytica</I>, 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 <I>via</I> the classic restriction digestion and ligation method. In contrast, the one-step <I>in vivo</I> pathway assembly method has been only rarely applied to <I>Y. lipolytica</I>. Here, with arachidonic acid biosynthesis as a case study, a one-step <I>in vivo</I> pathway assembly and integration method was used for engineering <I>Y. lipolytica</I>. 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 1kb, the method was able to rapidly assemble the arachidonic acid biosynthesis pathway (nearly 10kb) into the chromosome with high efficiency (nearly 23%). Meanwhile, the pathway assembled in <I>Y. lipolytica</I> demonstrated long-term genetic stability and the engineered strain exhibited robust growth. Furthermore, this study demonstrated that the codon-optimized genes from <I>Mortierella alpina</I> can function efficiently in <I>Y. lipolytica</I>: 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 <I>Y. lipolytica</I> for functional polyunsaturated fatty acids production. This method represents a powerful tool with potential for facilitating engineering applications in non-conventional yeasts.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A <I>in vivo</I> one-step pathway assembly was used for ARA biosynthesis in <I>Y. lipolytica</I>. </LI> <LI> The assembly efficiency was affected by the length of overlapping region. </LI> <LI> The assembled pathway performed long-term genetic stability in <I>Y. lipolytica</I>. </LI> <LI> The codon-optimized genes from <I>M. alpina</I> could function in <I>Y. lipolytica</I>. </LI> </UL> </P>