Enhanced biosynthesis of poly(3‐hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl‐CoA availability
Abstract Malonyl‐coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value‐added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl‐CoA availability is limited by competit...
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| Format: | Article |
| Language: | English |
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Wiley
2024-11-01
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| Series: | Microbial Biotechnology |
| Online Access: | https://doi.org/10.1111/1751-7915.70044 |
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| author | Giusi Favoino Nicolas Krink Tobias Schwanemann Nick Wierckx Pablo I. Nikel |
| author_facet | Giusi Favoino Nicolas Krink Tobias Schwanemann Nick Wierckx Pablo I. Nikel |
| author_sort | Giusi Favoino |
| collection | DOAJ |
| description | Abstract Malonyl‐coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value‐added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl‐CoA availability is limited by competition in several essential metabolic pathways. To address this limitation, we modified a genome‐reduced Pseudomonas putida strain to increase acetyl‐CoA carboxylation while limiting malonyl‐CoA utilization. Genes involved in sugar catabolism and its regulation, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis were knocked‐out in specific combinations towards increasing the malonyl‐CoA pool. An enzyme‐coupled biosensor, based on the rppA gene, was employed to monitor malonyl‐CoA levels in vivo. RppA is a type III polyketide synthase that converts malonyl‐CoA into flaviolin, a red‐colored polyketide. We isolated strains displaying enhanced malonyl‐CoA availability via a colorimetric screening method based on the RppA‐dependent red pigmentation; direct flaviolin quantification identified four engineered strains had a significant increase in malonyl‐CoA levels. We further modified these strains by adding a non‐canonical pathway that uses malonyl‐CoA as precursor for poly(3‐hydroxybutyrate) biosynthesis. These manipulations led to increased polymer accumulation in the fully engineered strains, validating our general strategy to boost the output of malonyl‐CoA–dependent pathways in P. putida. |
| format | Article |
| id | doaj-art-099dbedf781e41d5a0161542a27cad6e |
| institution | Kabale University |
| issn | 1751-7915 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj-art-099dbedf781e41d5a0161542a27cad6e2024-11-27T11:56:36ZengWileyMicrobial Biotechnology1751-79152024-11-011711n/an/a10.1111/1751-7915.70044Enhanced biosynthesis of poly(3‐hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl‐CoA availabilityGiusi Favoino0Nicolas Krink1Tobias Schwanemann2Nick Wierckx3Pablo I. Nikel4The Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark Lyngby DenmarkThe Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark Lyngby DenmarkInstitute of Bio‐ and Geosciences IBG‐1: Biotechnology, Forschungszentrum Jülich GmbH Jülich GermanyInstitute of Bio‐ and Geosciences IBG‐1: Biotechnology, Forschungszentrum Jülich GmbH Jülich GermanyThe Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark Lyngby DenmarkAbstract Malonyl‐coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value‐added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl‐CoA availability is limited by competition in several essential metabolic pathways. To address this limitation, we modified a genome‐reduced Pseudomonas putida strain to increase acetyl‐CoA carboxylation while limiting malonyl‐CoA utilization. Genes involved in sugar catabolism and its regulation, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis were knocked‐out in specific combinations towards increasing the malonyl‐CoA pool. An enzyme‐coupled biosensor, based on the rppA gene, was employed to monitor malonyl‐CoA levels in vivo. RppA is a type III polyketide synthase that converts malonyl‐CoA into flaviolin, a red‐colored polyketide. We isolated strains displaying enhanced malonyl‐CoA availability via a colorimetric screening method based on the RppA‐dependent red pigmentation; direct flaviolin quantification identified four engineered strains had a significant increase in malonyl‐CoA levels. We further modified these strains by adding a non‐canonical pathway that uses malonyl‐CoA as precursor for poly(3‐hydroxybutyrate) biosynthesis. These manipulations led to increased polymer accumulation in the fully engineered strains, validating our general strategy to boost the output of malonyl‐CoA–dependent pathways in P. putida.https://doi.org/10.1111/1751-7915.70044 |
| spellingShingle | Giusi Favoino Nicolas Krink Tobias Schwanemann Nick Wierckx Pablo I. Nikel Enhanced biosynthesis of poly(3‐hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl‐CoA availability Microbial Biotechnology |
| title | Enhanced biosynthesis of poly(3‐hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl‐CoA availability |
| title_full | Enhanced biosynthesis of poly(3‐hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl‐CoA availability |
| title_fullStr | Enhanced biosynthesis of poly(3‐hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl‐CoA availability |
| title_full_unstemmed | Enhanced biosynthesis of poly(3‐hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl‐CoA availability |
| title_short | Enhanced biosynthesis of poly(3‐hydroxybutyrate) in engineered strains of Pseudomonas putida via increased malonyl‐CoA availability |
| title_sort | enhanced biosynthesis of poly 3 hydroxybutyrate in engineered strains of pseudomonas putida via increased malonyl coa availability |
| url | https://doi.org/10.1111/1751-7915.70044 |
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