Siyan Qiu, Jingru Li, Pengtian Xie, Chun Wei, Jie Sun
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Key strategies involved: (1) Knockout of <i>LCB4</i> (sphingoid long-chain base kinase), <i>SHM2</i> (serine hydroxymethyltransferase), and <i>CHA1</i> (l-serine deaminase) to block competitive pathways; (2) Overexpression of <i>TSC10</i> (3-ketosphingosine reductase), <i>SYR2</i> (sphingosine hydroxylase), and <i>LCB1/LCB2</i> (serine palmitoyltransferase) to amplify the PHS synthesis flux. Initial shake flask fermentation (96 h) yielded 15.31 mg/g DCW of PHS, with <i>ORM2</i> knockout providing a 73.6% productivity increase (26.54 mg/g DCW) despite inducing growth defects from sphingosine accumulation. We hypothesized that disrupted ORM2-mediated control of serine palmitoyltransferase activity might compromise ER homeostasis through sphingolipid imbalance, which was alleviated through <i>HAC1</i> overexpression to enhance unfolded protein response (UPR) capacity. Fed-batch fermentation under optimized conditions (40 mM serine, 0.5 mM palmitic acid, pH 5) demonstrated scalable production, delivering a 5.4-fold improvement over baseline. This work establishes UPR engineering as a critical strategy for resolving lipid toxicity constraints in yeast sphingolipid biosynthesis, while highlighting <i>S. cerevisiae</i>'s potential as an industrial PHS production platform through coordinated pathway and stress response manipulation.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1007/s13205-025-04417-z.</p>","PeriodicalId":7067,"journal":{"name":"3 Biotech","volume":"15 8","pages":"247"},"PeriodicalIF":2.6000,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12238450/pdf/","citationCount":"0","resultStr":"{\"title\":\"Construction of engineered <i>Saccharomyces cerevisiae</i> for producing phytosphingosine.\",\"authors\":\"Siyan Qiu, Jingru Li, Pengtian Xie, Chun Wei, Jie Sun\",\"doi\":\"10.1007/s13205-025-04417-z\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Phytosphingosine (PHS), a sphingolipid-derived bioactive compound, exhibits multifunctional properties including antimicrobial activity, skin moisturization, and hydration, rendering it highly valuable for cosmetic and pharmaceutical applications. Through systematic metabolic engineering of <i>Saccharomyces cerevisiae</i>, we achieved 82.62 mg/g DCW phytosphingosine (PHS) production via integrated pathway optimization and stress mitigation. Key strategies involved: (1) Knockout of <i>LCB4</i> (sphingoid long-chain base kinase), <i>SHM2</i> (serine hydroxymethyltransferase), and <i>CHA1</i> (l-serine deaminase) to block competitive pathways; (2) Overexpression of <i>TSC10</i> (3-ketosphingosine reductase), <i>SYR2</i> (sphingosine hydroxylase), and <i>LCB1/LCB2</i> (serine palmitoyltransferase) to amplify the PHS synthesis flux. Initial shake flask fermentation (96 h) yielded 15.31 mg/g DCW of PHS, with <i>ORM2</i> knockout providing a 73.6% productivity increase (26.54 mg/g DCW) despite inducing growth defects from sphingosine accumulation. We hypothesized that disrupted ORM2-mediated control of serine palmitoyltransferase activity might compromise ER homeostasis through sphingolipid imbalance, which was alleviated through <i>HAC1</i> overexpression to enhance unfolded protein response (UPR) capacity. Fed-batch fermentation under optimized conditions (40 mM serine, 0.5 mM palmitic acid, pH 5) demonstrated scalable production, delivering a 5.4-fold improvement over baseline. 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Construction of engineered Saccharomyces cerevisiae for producing phytosphingosine.
Phytosphingosine (PHS), a sphingolipid-derived bioactive compound, exhibits multifunctional properties including antimicrobial activity, skin moisturization, and hydration, rendering it highly valuable for cosmetic and pharmaceutical applications. Through systematic metabolic engineering of Saccharomyces cerevisiae, we achieved 82.62 mg/g DCW phytosphingosine (PHS) production via integrated pathway optimization and stress mitigation. Key strategies involved: (1) Knockout of LCB4 (sphingoid long-chain base kinase), SHM2 (serine hydroxymethyltransferase), and CHA1 (l-serine deaminase) to block competitive pathways; (2) Overexpression of TSC10 (3-ketosphingosine reductase), SYR2 (sphingosine hydroxylase), and LCB1/LCB2 (serine palmitoyltransferase) to amplify the PHS synthesis flux. Initial shake flask fermentation (96 h) yielded 15.31 mg/g DCW of PHS, with ORM2 knockout providing a 73.6% productivity increase (26.54 mg/g DCW) despite inducing growth defects from sphingosine accumulation. We hypothesized that disrupted ORM2-mediated control of serine palmitoyltransferase activity might compromise ER homeostasis through sphingolipid imbalance, which was alleviated through HAC1 overexpression to enhance unfolded protein response (UPR) capacity. Fed-batch fermentation under optimized conditions (40 mM serine, 0.5 mM palmitic acid, pH 5) demonstrated scalable production, delivering a 5.4-fold improvement over baseline. This work establishes UPR engineering as a critical strategy for resolving lipid toxicity constraints in yeast sphingolipid biosynthesis, while highlighting S. cerevisiae's potential as an industrial PHS production platform through coordinated pathway and stress response manipulation.
Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04417-z.
3 BiotechAgricultural and Biological Sciences-Agricultural and Biological Sciences (miscellaneous)
CiteScore
6.00
自引率
0.00%
发文量
314
期刊介绍:
3 Biotech publishes the results of the latest research related to the study and application of biotechnology to:
- Medicine and Biomedical Sciences
- Agriculture
- The Environment
The focus on these three technology sectors recognizes that complete Biotechnology applications often require a combination of techniques. 3 Biotech not only presents the latest developments in biotechnology but also addresses the problems and benefits of integrating a variety of techniques for a particular application. 3 Biotech will appeal to scientists and engineers in both academia and industry focused on the safe and efficient application of Biotechnology to Medicine, Agriculture and the Environment.