Yarrowia lipolytica as a Platform for Punicic Acid Production.

Punicic acid (PuA) is a polyunsaturated fatty acid with significant medical, biological, and nutraceutical properties. The primary source of punicic acid is the pomegranate seed oil obtained from fruits of trees that are mainly cultivated in subtropical and tropical climates. To establish sustainable production of PuA, various recombinant microorganisms and plants have been explored as platforms with limited efficiencies. In this study, the oleaginous yeast Yarrowia lipolytica was employed as a host for PuA production. First, growth and lipid accumulation of Y. lipolytica were evaluated in medium supplemented with pomegranate seed oil, resulting in the accumulation of lipids up to 31.2%, consisting of 22% PuA esterified in the fraction of glycerolipids. In addition, lipid-engineered Y. lipolytica strains, transformed with the bifunctional fatty acid conjugase/desaturase from Punica granatum (PgFADX), showed the ability to accumulate PuA de novo. PuA was detected in both polar and neutral lipid fractions, especially in phosphatidylcholine and triacylglycerols. Promoter optimization for PgFADX expression resulted in improved accumulation of PuA from 0.9 to 1.8 mg/g of dry cell weight. The best-producing strain expressing PgFADX under the control of a strong erythritol-inducible promoter produced 36.6 mg/L PuA. These results demonstrate that the yeast Y. lipolytica is a promising host for PuA production.

Insertional mutagenesis in Chlamydomonas reinhardtii: An effective strategy for the identification of new genes involved in the DNA damage response.

The formation of double-strand breaks in DNA represents a serious stress for all types of organisms and requires a precisely regulated and organized DNA damage response (DDR) to maintain genetic information and genome integrity. Chlamydomonas reinhardtii possesses the characteristics of both plants and animals and is therefore suitable for the identification of novel genes connected to a wide spectrum of metabolic pathways, including DDR. One very effective tool for the detection and subsequent characterization of new mutants in C. reinhardtii is insertional mutagenesis. We isolated several insertion mutants sensitive to DNA-damaging agents that had disrupted or completely deleted genes with putative functions in the DDR. In most of the analysed mutants, we identified various changes at both ends and even inside the inserted cassette. Using recent information from databases, we were also able to supplement the characteristics of the previously described mutant with a pleiotropic phenotype. In addition, we confirmed the effectiveness of hairpin-PCR as a strategy for the identification of insertion flanking sites and as a tool for the detection of changes at the site of insertion, thus enabling a better understanding of insertion events.