Production of plant-derived punicic acid in engineered yeast platforms
Creators: Wang, Juli
Publisher: name: University of Alberta Library
Year: 2024

<p>Punicic acid (PuA) is a high-value edible conjugated fatty acid with strong bioactivities and has important potential applications in nutraceutical, pharmaceutical, and oleochemical industries. Since the production of PuA is severely limited by the fact that its primary natural source, pomegranate seed oil, is not readily available on a large scale, there is considerable interest in understanding the biosynthesis and accumulation of this plant-based unusual fatty acid in transgenic microorganisms to support the design of biotechnological approaches for PuA production via metabolic engineering and fermentation. <br>
In the first study, the effectiveness of genetic engineering and precursor supply in PuA production in the model yeast strain Saccharomyces cerevisiae was tested. The results revealed that the combination of precursor feeding and co-expression of selected genes in acyl channeling processes created a ‘Push-Pull’ approach to increase PuA content. Coupled with the deletion of a yeast lipid metabolism regulator, the feeding of 0.05% linoleic acid, and the introduction of PgFADX and other genes from pomegranate, PuA content was increased to 3.4% of total fatty acids.<br>
Due to the complexity of plant-derived unusual fatty acid biosynthetic pathways, individually testing each gene for pathway functionality and obtaining the best gene combination are time-consuming. A rapid workflow is necessary to facilitate the study of plant unusual fatty acid metabolism and the synthesis of plant-derived lipids in microorganisms. Therefore, in the second study, genes potentially contributing to PuA synthesis were directly shuffled within the yeast genome by targeting the yeast Ty retrotransposon, resulting in a recombinant yeast library with varying PuA content. The screening of 1752 strains led to the identification of a recombinant S. cerevisiae capable of accumulating 26.7% of total fatty acids as PuA without requiring linoleic acid precursor feeding. In shake flask cultivation, the PuA titer reached 424.6 mg/L. Subsequent analysis showed the combination of several upstream and downstream genes conducive to PuA accumulation. Moreover, PuA constituted over 22% of total fatty acids in the triacylglycerol (TAG) fraction of yeast single-cell oil, demonstrating a significant increase compared to PuA levels in the TAG fraction of transgenic plants. Following the increase of PuA production in yeast, substantial changes in the yeast lipidome, including TAG and major polar lipid species, were observed.<br>
Many non-conventional oleaginous yeasts have emerged as prominent candidates in biotechnological studies for their ability to produce single-cell oil and utilize cost-effective, renewable feedstocks. In the third study, the capability of oleaginous yeast Rhodosporidium toruloides to produce PuA was investigated. The initial expression of pomegranate PgFADX allowed R. toruloides to accumulate 3.7% of its total fatty acids as PuA. Subsequent genomic integration of genes encoding codon-optimized delta-12 acyl lipid desaturase (PgFAD2) or diacylglycerol acyltransferase 2 (PgDGAT2) significantly increased PuA levels. The engineered R. toruloides strain with PgFADX and PgFAD2 coexpression accumulated 12% of its lipids as PuA from glucose, which translated into a PuA titer of 451.6 mg/L in shake flask cultivation. The content of PuA achieved 6.4% when wood hydrolysate was used as the substrate, showcasing R. toruloides’ potential in the bioconversion of lignocellulosic feedstock into high-value PuA.<br>
In summary, the work included in this PhD thesis has led to two PuA-producing microbial platforms, including an engineered model yeast S. cerevisiae strain with a high PuA content, and a non-conventional oleaginous yeast R. toruloides strain that is capable of converting renewable agricultural and forestry waste substrate into high-value PuA. A novel Ty retrotransposon-targeted random gene shuffling workflow for efficiently engineering baker’s yeast for producing PuA was also developed. The findings of this thesis provide knowledge and valuable insights into the enrichment mechanism of PuA in yeast and will benefit the development of innovative microbial platforms for producing other plant-derived high-value fatty acids.</p>