Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random Mutagenesis
Squalene is an important polyunsaturated triterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Currently, the main method for squalene production is extraction from oil-producing plants, but the scale is limited. The microbial fermentation with Saccharomyces cerevi...
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Frontiers Media S.A.
2022-01-01
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| Series: | Frontiers in Chemical Engineering |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fceng.2021.790261/full |
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| author | Lu Xia Lu Xia Yunbin Lv Yunbin Lv Song Liu Song Liu Song Liu Shiqin Yu Weizhu Zeng Weizhu Zeng Jingwen Zhou Jingwen Zhou Jingwen Zhou Jingwen Zhou |
| author_facet | Lu Xia Lu Xia Yunbin Lv Yunbin Lv Song Liu Song Liu Song Liu Shiqin Yu Weizhu Zeng Weizhu Zeng Jingwen Zhou Jingwen Zhou Jingwen Zhou Jingwen Zhou |
| author_sort | Lu Xia |
| collection | DOAJ |
| description | Squalene is an important polyunsaturated triterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Currently, the main method for squalene production is extraction from oil-producing plants, but the scale is limited. The microbial fermentation with Saccharomyces cerevisiae still needs improvement to be economically viable. This study aimed to improve squalene production by metabolic engineering and random mutagenesis. First, the mevalonate (MVA) pathway was enhanced, by integrating tHMG1 and IDI1 into multi-copy site Ty2. Subsequently, the ACL gene from Yarrowia lipolytica, encoding citrate lyase was introduced and the β-oxidation pathway was enhanced with multiple copies of key genes. In addition, a high throughput screening strategy based on Nile red staining was established for high squalene-producer screening. After treatment with ARTP mutagenesis, a higher-producing mutant was obtained, with squalene production enhanced by 18.4%. A two-stage fermentation of this mutant in a 5 L bioreactor produced 8.2 g/L of squalene. These findings may facilitate the development of industrial squalene production by fermentation and potentially, other terpenoids. |
| first_indexed | 2024-04-11T17:17:28Z |
| format | Article |
| id | doaj.art-e45ad787352041699c7640b7d9fcbcf6 |
| institution | Directory Open Access Journal |
| issn | 2673-2718 |
| language | English |
| last_indexed | 2024-04-11T17:17:28Z |
| publishDate | 2022-01-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Chemical Engineering |
| spelling | doaj.art-e45ad787352041699c7640b7d9fcbcf62022-12-22T04:12:37ZengFrontiers Media S.A.Frontiers in Chemical Engineering2673-27182022-01-01310.3389/fceng.2021.790261790261Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random MutagenesisLu Xia0Lu Xia1Yunbin Lv2Yunbin Lv3Song Liu4Song Liu5Song Liu6Shiqin Yu7Weizhu Zeng8Weizhu Zeng9Jingwen Zhou10Jingwen Zhou11Jingwen Zhou12Jingwen Zhou13National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, ChinaSchool of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, ChinaNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, ChinaSchool of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, ChinaNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, ChinaScience Center for Future Foods, Jiangnan University, Wuxi, ChinaJiangsu Provisional Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi, ChinaNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, ChinaNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, ChinaSchool of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, ChinaNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, ChinaSchool of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, ChinaScience Center for Future Foods, Jiangnan University, Wuxi, ChinaJiangsu Provisional Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi, ChinaSqualene is an important polyunsaturated triterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Currently, the main method for squalene production is extraction from oil-producing plants, but the scale is limited. The microbial fermentation with Saccharomyces cerevisiae still needs improvement to be economically viable. This study aimed to improve squalene production by metabolic engineering and random mutagenesis. First, the mevalonate (MVA) pathway was enhanced, by integrating tHMG1 and IDI1 into multi-copy site Ty2. Subsequently, the ACL gene from Yarrowia lipolytica, encoding citrate lyase was introduced and the β-oxidation pathway was enhanced with multiple copies of key genes. In addition, a high throughput screening strategy based on Nile red staining was established for high squalene-producer screening. After treatment with ARTP mutagenesis, a higher-producing mutant was obtained, with squalene production enhanced by 18.4%. A two-stage fermentation of this mutant in a 5 L bioreactor produced 8.2 g/L of squalene. These findings may facilitate the development of industrial squalene production by fermentation and potentially, other terpenoids.https://www.frontiersin.org/articles/10.3389/fceng.2021.790261/fullSaccharomyces cerevisiaepromotor substitutionmulti-copy site integrationhigh-throughput screeningβ-oxidation |
| spellingShingle | Lu Xia Lu Xia Yunbin Lv Yunbin Lv Song Liu Song Liu Song Liu Shiqin Yu Weizhu Zeng Weizhu Zeng Jingwen Zhou Jingwen Zhou Jingwen Zhou Jingwen Zhou Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random Mutagenesis Frontiers in Chemical Engineering Saccharomyces cerevisiae promotor substitution multi-copy site integration high-throughput screening β-oxidation |
| title | Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random Mutagenesis |
| title_full | Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random Mutagenesis |
| title_fullStr | Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random Mutagenesis |
| title_full_unstemmed | Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random Mutagenesis |
| title_short | Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random Mutagenesis |
| title_sort | enhancing squalene production in saccharomyces cerevisiae by metabolic engineering and random mutagenesis |
| topic | Saccharomyces cerevisiae promotor substitution multi-copy site integration high-throughput screening β-oxidation |
| url | https://www.frontiersin.org/articles/10.3389/fceng.2021.790261/full |
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