Engineering the C3N Pathway as a Short Detour for <i>De Novo</i> NAD<sup>+</sup> Biosynthesis in <i>Saccharomyces cerevisiae</i>
As a life-essential coenzyme, nicotinamide adenine dinucleotide (NAD<sup>+</sup>) has been explored for more than a century. In <i>Saccharomyces</i>, the natural NAD<sup>+</sup><i>de novo</i> biosynthetic pathway initiating from tryptophan has been wel...
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MDPI AG
2023-09-01
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| Series: | Fermentation |
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| author | Xinli Li Yue Tang Yong Ding Pengwei Li Yihua Chen |
| author_facet | Xinli Li Yue Tang Yong Ding Pengwei Li Yihua Chen |
| author_sort | Xinli Li |
| collection | DOAJ |
| description | As a life-essential coenzyme, nicotinamide adenine dinucleotide (NAD<sup>+</sup>) has been explored for more than a century. In <i>Saccharomyces</i>, the natural NAD<sup>+</sup><i>de novo</i> biosynthetic pathway initiating from tryptophan has been well elucidated. To bypass this stringently controlled natural pathway in yeast, an economical C3N pathway that was developed in <i>Escherichia coli</i> previously was constructed in <i>Saccharomyces</i> as a short detour for <i>de novo</i> NAD<sup>+</sup> biosynthesis. After the functional expressions of the C3N genes were identified in <i>Saccharomyces cerevisiae</i> BY4741 by in vitro enzymatic assays, the C3N module was introduced into an NAD<sup>+</sup> auxotrophic <i>S. cerevisiae</i> strain BY01, in which the <i>BNA2</i> gene encoding tryptophan 2,3-dioxygenase was inactivated. The efficient NAD<sup>+</sup> synthesis via the C3N pathway was confirmed by both plate assays and fermentation analysis. The applicability of the C3N pathway in cofactor engineering was tested by introducing it into <i>S. cerevisiae</i> BY4741, which improved the cellular NAD(H) level considerably. Consequently, this study proved that the <i>de novo</i> NAD<sup>+</sup> biosynthetic pathway can be replaced by an artificial pathway in yeast, which paves a way to design more promising schemes in eukaryotes for rational manipulation of the cellular NAD(H) levels. |
| first_indexed | 2024-03-10T21:16:12Z |
| format | Article |
| id | doaj.art-f6fc1319ce4a47fc8bf9d35ce3b74441 |
| institution | Directory Open Access Journal |
| issn | 2311-5637 |
| language | English |
| last_indexed | 2024-03-10T21:16:12Z |
| publishDate | 2023-09-01 |
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| series | Fermentation |
| spelling | doaj.art-f6fc1319ce4a47fc8bf9d35ce3b744412023-11-19T16:26:10ZengMDPI AGFermentation2311-56372023-09-0191088610.3390/fermentation9100886Engineering the C3N Pathway as a Short Detour for <i>De Novo</i> NAD<sup>+</sup> Biosynthesis in <i>Saccharomyces cerevisiae</i>Xinli Li0Yue Tang1Yong Ding2Pengwei Li3Yihua Chen4State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaAs a life-essential coenzyme, nicotinamide adenine dinucleotide (NAD<sup>+</sup>) has been explored for more than a century. In <i>Saccharomyces</i>, the natural NAD<sup>+</sup><i>de novo</i> biosynthetic pathway initiating from tryptophan has been well elucidated. To bypass this stringently controlled natural pathway in yeast, an economical C3N pathway that was developed in <i>Escherichia coli</i> previously was constructed in <i>Saccharomyces</i> as a short detour for <i>de novo</i> NAD<sup>+</sup> biosynthesis. After the functional expressions of the C3N genes were identified in <i>Saccharomyces cerevisiae</i> BY4741 by in vitro enzymatic assays, the C3N module was introduced into an NAD<sup>+</sup> auxotrophic <i>S. cerevisiae</i> strain BY01, in which the <i>BNA2</i> gene encoding tryptophan 2,3-dioxygenase was inactivated. The efficient NAD<sup>+</sup> synthesis via the C3N pathway was confirmed by both plate assays and fermentation analysis. The applicability of the C3N pathway in cofactor engineering was tested by introducing it into <i>S. cerevisiae</i> BY4741, which improved the cellular NAD(H) level considerably. Consequently, this study proved that the <i>de novo</i> NAD<sup>+</sup> biosynthetic pathway can be replaced by an artificial pathway in yeast, which paves a way to design more promising schemes in eukaryotes for rational manipulation of the cellular NAD(H) levels.https://www.mdpi.com/2311-5637/9/10/886C3N pathwayNAD<sup>+</sup> <i>de novo</i> biosynthesis<i>Saccharomyces cerevisiae</i>eukaryotes |
| spellingShingle | Xinli Li Yue Tang Yong Ding Pengwei Li Yihua Chen Engineering the C3N Pathway as a Short Detour for <i>De Novo</i> NAD<sup>+</sup> Biosynthesis in <i>Saccharomyces cerevisiae</i> Fermentation C3N pathway NAD<sup>+</sup> <i>de novo</i> biosynthesis <i>Saccharomyces cerevisiae</i> eukaryotes |
| title | Engineering the C3N Pathway as a Short Detour for <i>De Novo</i> NAD<sup>+</sup> Biosynthesis in <i>Saccharomyces cerevisiae</i> |
| title_full | Engineering the C3N Pathway as a Short Detour for <i>De Novo</i> NAD<sup>+</sup> Biosynthesis in <i>Saccharomyces cerevisiae</i> |
| title_fullStr | Engineering the C3N Pathway as a Short Detour for <i>De Novo</i> NAD<sup>+</sup> Biosynthesis in <i>Saccharomyces cerevisiae</i> |
| title_full_unstemmed | Engineering the C3N Pathway as a Short Detour for <i>De Novo</i> NAD<sup>+</sup> Biosynthesis in <i>Saccharomyces cerevisiae</i> |
| title_short | Engineering the C3N Pathway as a Short Detour for <i>De Novo</i> NAD<sup>+</sup> Biosynthesis in <i>Saccharomyces cerevisiae</i> |
| title_sort | engineering the c3n pathway as a short detour for i de novo i nad sup sup biosynthesis in i saccharomyces cerevisiae i |
| topic | C3N pathway NAD<sup>+</sup> <i>de novo</i> biosynthesis <i>Saccharomyces cerevisiae</i> eukaryotes |
| url | https://www.mdpi.com/2311-5637/9/10/886 |
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