Improving the Production of Punicic Acid in Baker’s Yeast by Engineering Genes in Acyl Channeling Processes and Adjusting Precursor Supply

Abstract

Punicic acid (PuA) is a high-value edible conjugated fatty acid with strong bioactivities and has important potential applications in nutraceutical, pharmaceutical, feeding, and oleochemical industries. Since the production of PuA is severely limited by the fact that its 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 rational design of biotechnological approaches for PuA production via fermentation. Here, we tested the effectiveness of genetic engineering and precursor supply in PuA production in the model yeast strain Saccharomyces cerevisiae. The results revealed that the combination of precursor feeding and co-expression of selected genes in acyl channeling processes created an effective "push−pull" approach to increase PuA content, which could prove valuable in future efforts to produce PuA in industrial yeast and other microorganisms via fermentation.

Engineering Yarrowia lipolytica for Sustainable Production of the Pomegranate Seed Oil-Derived Punicic Acid

“…13 Expressing PgFADX in S. cerevisiae with the addition of C18:2Δ 9,12 only resulted in trace amounts of punicic acid (<0.5%), possibly due to a low level of PgFADX expression. 24 It is presumed that this is because the concentration of punicic acid is too low to detect in the gas phase. Subsequently, a second copy of PgFADX was introduced into the IntF locus of the Po1f-Δku70-X strain to increase the PgFADX expression level.…”

“…S. cerevisiae cannot accumulate punicic acid because C18:2Δ 9,12 is a non-native fatty acid in S. cerevisiae. 24 Therefore, strengthening the supply of C18:2Δ 9,12 is beneficial to punicic acid synthesis. The higholeic oil Y. lipolytica strain 2Pg2E could accumulate 82% C18:1Δ 9,34 which can be converted by YlFad2 to produce C18:2Δ 9,12 as an important precursor for punicic acid.…”

“…When 0.05% exogenous linoleic acid was added, the engineered strain produced 7.2 mg/L of punicic acid, accounting for 3.4% of total fatty acids (TFAs). 24 PgFADX and PgFAD2 were also expressed in Schizosaccharomyces pombe, boosting the punicic acid content to 25.1% of TFAs, with a titer of 38.7 mg/L. 25 The production of punicic acid is restrained by the low oil production capacity and limited precursor supply in these microorganisms.…”

Engineering Yarrowia lipolytica to produce nutritional fatty acids: Current status and future perspectives

“…Nevertheless, some progress has been made by selected plant-derived acyl-editing enzymes to improve the accumulation of PUFAs in microorganisms. The expression of PgPDAT and PgLPCAT from Punica granatum in S. cerevisiae successfully created an acyl channeling process to produce punicic acid, an important conjugated linolenic acid-derived nutritive fatty acid [ 95 ].…”

Heterologous Production of Calendic Acid Naturally Found in Calendula officinalis by Recombinant Fission Yeast

“…In addition, we have previously shown that the fission yeast is more suitable for the accumulation of punicic acid (an isomer of CLNAs) compared to S. cerevisiae. , Indeed, CA shows potent anti-cancer properties. ,, The fission yeast S. pombe is often used to study the cell cycle as a unicellular eukaryotic model organism. , Thus, recombinant fission yeast cells producing CA can represent a valuable tool for understanding the bioactive properties of CA.…”