Juli Wang | ALES Graduate Seminar

A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES PhD Final Exam Seminar by Juli Wang. This seminar is open to the general public to attend, either in person or online:

https://ualberta-ca.zoom.us/j/8411257438?pwd=eXNuVE9Mc1BjdlZWQWptbmRrNlZsdz09&omn=95331950126

Meeting ID: 841 125 7438 | Passcode: InnerPeace | Find your local number: https://ualberta-ca.zoom.us/u/acjBjB2ejc

Thesis Topic: Production of plant-derived punicic acid in engineered yeast platforms

PhD with Dr. Gavin Chen.

Seminar Abstract:

Punicic acid (PuA) is a high-value edible conjugated fatty acid with strong bioactivities. It has potential applications in the nutraceutical and oleochemical industries. Since the production of PuA is 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. In this study, the model yeast strain Saccharomyces cerevisiae was initially used to test the effectiveness of PuA production in microorganisms. 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 yeast lipid metabolism regulator, the feeding of 0.05% linoleic acid, and the introduction of bifunctional fatty acid desaturase and conjugase (PgFADX) and other enzymes from pomegranate, PuA content was increased to 3.4% of total fatty acids. Due to the complexity of plant-derived unusual fatty acid synthetic 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, genes potentially contributing to PuA synthesis were further shuffled within the yeast genome by targeting 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 425 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 fraction of yeast single-cell oil, demonstrating a significant increase compared to the levels obtained in transgenic plants. Following the increase of PuA production in yeast, substantial changes in the yeast lipidome, including triacylglycerol and major polar lipid species, were observed. Since many non-conventional oleaginous yeasts have emerged as prominent candidates in biotechnological studies for their ability to produce single-cell oil from low-cost feedstocks, the capability of the 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 or diacylglycerol acyltransferase 2 significantly increased PuA levels. The engineered R. toruloides strain accumulated up to 12% of its lipids as PuA from glucose, which translated into a PuA titer of 452 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. In summary, the work included in this study has led to two PuA-producing microbial platforms, including an engineered conventional 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.