Presentation Information
[P01-057]Harnessing Acetyl-CoA Metabolism in Lipomyces starkeyi for Enhanced Vitamin A Production
○Akari Kinoshita1, Rikako Sato2, Hibiki Higuchi1, Shunichi Kobayashi1, Kento Koketsu1, Taro Watanabe1, Hiroaki Takaku2 (1. Kirin Holdings Company, Limited (Japan), 2. Niigata University of Pharmacy and Medical and Life Sciences (Japan))
Keywords:
Acetyl-CoA,Vitamin A,Oleaginous yeasts
Vitamin A is an essential micronutrient required for vision, immunity, and skin function. Global demand for vitamin A continues to expand, particularly in animal nutrition, cosmetics, and pharmaceutical applications. However, current industrial vitamin A production relies primarily on petrochemical-based chemical synthesis, raising concerns about environmental sustainability. Microbial fermentation offers a promising alternative production strategy. Oleaginous yeasts possess a large cytosolic acetyl-CoA pool, providing a metabolic advantage for the biosynthesis of lipophilic compounds such as terpenoids. Lipomyces starkeyi is capable of accumulating lipids (mainly triacylglycerols, TAGs) to over 85% of its dry cell weight and assimilating a wide variety of carbon sources, indicating that L. starkeyi is a promising host for vitamin A production (Juanssilfero et al., 2018). In this study, we demonstrated, for the first time, vitamin A biosynthesis in L. starkeyi and discussed its potential as an industrial host.
Vitamin A is biosynthesized from β-carotene through oxidative cleavage reactions. β-Carotene is produced from acetyl-CoA via the mevalonate pathway. β-Carotene-producing strains were constructed by introducing McCarRP (lycopene cyclase/phytoene synthase) and McCarB (phytoene desaturase) from Mucor circinelloides, with additional expression of XdCrtE (geranylgeranyl diphosphate synthase) from Xanthophyllomyces dendrorhous. Vitamin A production was achieved by introducing MbBlh (β-carotene dioxygenase) from the uncultured marine bacterium 66A03. Furthermore, overexpression of the mevalonate pathway genes, ERG10 (acetyl-CoA acetyltransferase), ERG13 (HMG-CoA synthase), and tHMG1 (truncated HMG-CoA reductase) in L. starkeyi increased vitamin A production, suggesting that the activation of the mevalonate pathway enhances vitamin A productivity. In fed-batch fermentation, the engineered strain yielded 694 mg/L vitamin A. Notably, vitamin A production paralleled TAG accumulation in L. starkeyi, suggesting coordinated activation of lipid and terpenoid biosynthesis through shared acetyl-CoA utilization.
To further redirect acetyl-CoA flux toward vitamin A synthesis, fatty acid synthesis was attenuated via downregulation of FAS1 (fatty acid synthase), resulting in increased vitamin A yield. Moreover, cultivation under nitrogen-limited conditions, which promotes acetyl-CoA accumulation and TAG synthesis, enabled vitamin A production exceeding 1 g/L. These results demonstrate that both redistribution of acetyl-CoA flux and expansion of intracellular acetyl-CoA supply are effective strategies for enhancing vitamin A production. Overall, L. starkeyi is a promising platform for terpenoid production, including vitamin A, with potential for further metabolic engineering.
Vitamin A is biosynthesized from β-carotene through oxidative cleavage reactions. β-Carotene is produced from acetyl-CoA via the mevalonate pathway. β-Carotene-producing strains were constructed by introducing McCarRP (lycopene cyclase/phytoene synthase) and McCarB (phytoene desaturase) from Mucor circinelloides, with additional expression of XdCrtE (geranylgeranyl diphosphate synthase) from Xanthophyllomyces dendrorhous. Vitamin A production was achieved by introducing MbBlh (β-carotene dioxygenase) from the uncultured marine bacterium 66A03. Furthermore, overexpression of the mevalonate pathway genes, ERG10 (acetyl-CoA acetyltransferase), ERG13 (HMG-CoA synthase), and tHMG1 (truncated HMG-CoA reductase) in L. starkeyi increased vitamin A production, suggesting that the activation of the mevalonate pathway enhances vitamin A productivity. In fed-batch fermentation, the engineered strain yielded 694 mg/L vitamin A. Notably, vitamin A production paralleled TAG accumulation in L. starkeyi, suggesting coordinated activation of lipid and terpenoid biosynthesis through shared acetyl-CoA utilization.
To further redirect acetyl-CoA flux toward vitamin A synthesis, fatty acid synthesis was attenuated via downregulation of FAS1 (fatty acid synthase), resulting in increased vitamin A yield. Moreover, cultivation under nitrogen-limited conditions, which promotes acetyl-CoA accumulation and TAG synthesis, enabled vitamin A production exceeding 1 g/L. These results demonstrate that both redistribution of acetyl-CoA flux and expansion of intracellular acetyl-CoA supply are effective strategies for enhancing vitamin A production. Overall, L. starkeyi is a promising platform for terpenoid production, including vitamin A, with potential for further metabolic engineering.
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