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Background <p>The oleaginous yeast <i>Yarrowia lipolytica</i> is an attractive chassis for sustainable production of long‑chain ω‑3 polyunsaturated fatty acids (PUFAs). Polyketide synthase (PKS)-like PUFA synthases bypass the canonical oxygen‑dependent desaturase/elongase route, yet the influence of precursor availability on PKS product selectivity in <i>Y. lipolytica</i> remains unclear.</p> Results <p>Here, we explored a panel of <i>Y. lipolytica</i> strains comprising single‑origin (<i>Aetherobacter fasciculatus</i>,<i> Minicystis rosea</i>) and hybrid PKS clusters. A domain‑shuffled producer, Hyb6, broadened the product spectrum to penta‑unsaturated ω‑3 species, yielding EPA (18.3 mg L<sup>-1</sup>), DPA (38.8 mg L<sup>-1</sup>) and trace DHA (1.5 mg L<sup>-1</sup>) in shake flasks. Time-resolved metabolomics revealed that ω-3 accumulation began in the stationary phase, when acetyl-CoA and malonyl-CoA pools were strongly reduced. <span>l</span>-lysine supplementation upon glycerol depletion was associated with elevated malonyl-CoA levels, accelerated EPA formation (4.6-fold vs. control), and maintenance of an EPA/DPA ratio &gt; 1.9. In contrast, a ketogenic amino-acid mix increased native lipids but reduced EPA selectivity. Transcriptomics revealed <span>l</span>-lysine‑dependent upregulation of acetyl‑CoA supply nodes (<i>ACL1</i>/<i>ACL2</i>, <i>ACS</i>, <i>ACC1</i>) and <span>l</span>-lysine catabolism (<i>KAT1</i>, <i>GCDH</i>, <i>UGA2</i>), together with induction of amino‑acid transporters and protein‑folding machinery. In fed‑batch processes, pulsed <span>l</span>-lysine selectively increased EPA to 405.5 mg L<sup>-1</sup> (11.8% selectivity), with DPA at 321.5 mg L<sup>-1</sup> and DHA at 14.0 mg L<sup>-1</sup>.</p> Conclusions <p>Changes in acetyl-CoA and malonyl-CoA availability are strongly associated with EPA selectivity. Coupling modular PKS design with targeted precursor remodeling provides a versatile strategy to fine-tune product spectra in <i>Y. lipolytica</i> and related microbial PUFA cell factories.</p>

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Acetyl- and malonyl-CoA availability drive EPA selectivity in polyketide synthase-engineered Yarrowia lipolytica

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月4日

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  • Hang Qi,
  • Fabian Ries,
  • Sofija Jovanovic Gasovic,
  • Demian Dietrich,
  • Katja Gemperlein,
  • Rolf Müller,
  • Michael Kohlstedt,
  • Christoph Wittmann

摘要

Background

The oleaginous yeast Yarrowia lipolytica is an attractive chassis for sustainable production of long‑chain ω‑3 polyunsaturated fatty acids (PUFAs). Polyketide synthase (PKS)-like PUFA synthases bypass the canonical oxygen‑dependent desaturase/elongase route, yet the influence of precursor availability on PKS product selectivity in Y. lipolytica remains unclear.

Results

Here, we explored a panel of Y. lipolytica strains comprising single‑origin (Aetherobacter fasciculatus, Minicystis rosea) and hybrid PKS clusters. A domain‑shuffled producer, Hyb6, broadened the product spectrum to penta‑unsaturated ω‑3 species, yielding EPA (18.3 mg L-1), DPA (38.8 mg L-1) and trace DHA (1.5 mg L-1) in shake flasks. Time-resolved metabolomics revealed that ω-3 accumulation began in the stationary phase, when acetyl-CoA and malonyl-CoA pools were strongly reduced. l-lysine supplementation upon glycerol depletion was associated with elevated malonyl-CoA levels, accelerated EPA formation (4.6-fold vs. control), and maintenance of an EPA/DPA ratio > 1.9. In contrast, a ketogenic amino-acid mix increased native lipids but reduced EPA selectivity. Transcriptomics revealed l-lysine‑dependent upregulation of acetyl‑CoA supply nodes (ACL1/ACL2, ACS, ACC1) and l-lysine catabolism (KAT1, GCDH, UGA2), together with induction of amino‑acid transporters and protein‑folding machinery. In fed‑batch processes, pulsed l-lysine selectively increased EPA to 405.5 mg L-1 (11.8% selectivity), with DPA at 321.5 mg L-1 and DHA at 14.0 mg L-1.

Conclusions

Changes in acetyl-CoA and malonyl-CoA availability are strongly associated with EPA selectivity. Coupling modular PKS design with targeted precursor remodeling provides a versatile strategy to fine-tune product spectra in Y. lipolytica and related microbial PUFA cell factories.