Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast
Abstract Metabolically engineered baker’s yeast can be used to produce chiral amines through whole-cell bioconversion of prochiral ketones. This study investigates the modulation of the alanine-pyruvate metabolic node to enhance reductive amination, using the stereoselective conversion of benzylacet...
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Nature Portfolio
2025-05-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-025-01182-0 |
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| author | Arne Hagman Olof Stenström Göran Carlström Mikael Akke Carl Grey Magnus Carlquist |
| author_facet | Arne Hagman Olof Stenström Göran Carlström Mikael Akke Carl Grey Magnus Carlquist |
| author_sort | Arne Hagman |
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| description | Abstract Metabolically engineered baker’s yeast can be used to produce chiral amines through whole-cell bioconversion of prochiral ketones. This study investigates the modulation of the alanine-pyruvate metabolic node to enhance reductive amination, using the stereoselective conversion of benzylacetone to (S)-1-methyl-3-phenylpropylamine (MPPA) as a model reaction. Chromosomal integration of multiple copies of the promiscuous omega transaminase from Chromobacterium violaceum (cv-ATA) resulted in an active yeast catalyst. Physiological characterization in bioreactors under aerobic batch cultivation revealed that amine production occurred only under post-diauxic growth on ethanol. To reduce native alanine utilization, the endogenous alanine aminotransferase (ALT1) was knocked out and replaced with cv-ATA. To rapidly employ this strategy in other strains, a simple CRISPR/cas9 method for universal gene replacement was developed. The replacement of ALT1 with cv-ATA improved the reaction by 2.6-fold compared to the control strain with intact ALT1. NMR measurements of metabolites originating from 15N L-alanine and 13C glucose indicated that pyruvate formation during growth on glucose inhibited amine production. Under optimal conditions, the biocatalytic bioconversion of benzylacetone to MPPA reached a yield of 58%. |
| format | Article |
| id | doaj-art-2c4cddb4d8e54ca7a661bf317433f1dd |
| institution | OA Journals |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj-art-2c4cddb4d8e54ca7a661bf317433f1dd2025-08-20T01:51:31ZengNature PortfolioScientific Reports2045-23222025-05-0115111410.1038/s41598-025-01182-0Biocatalytic reductive amination with CRISPR-Cas9 engineered yeastArne Hagman0Olof Stenström1Göran Carlström2Mikael Akke3Carl Grey4Magnus Carlquist5Division of Biotechnology and Applied Microbiology, Lund UniversityDivision of Biophysical Chemistry, Center for Molecular Protein Science, Lund UniversityDivision of Biophysical Chemistry, Center for Molecular Protein Science, Lund UniversityDivision of Biophysical Chemistry, Center for Molecular Protein Science, Lund UniversityDivision of Biotechnology and Applied Microbiology, Lund UniversityDivision of Biotechnology and Applied Microbiology, Lund UniversityAbstract Metabolically engineered baker’s yeast can be used to produce chiral amines through whole-cell bioconversion of prochiral ketones. This study investigates the modulation of the alanine-pyruvate metabolic node to enhance reductive amination, using the stereoselective conversion of benzylacetone to (S)-1-methyl-3-phenylpropylamine (MPPA) as a model reaction. Chromosomal integration of multiple copies of the promiscuous omega transaminase from Chromobacterium violaceum (cv-ATA) resulted in an active yeast catalyst. Physiological characterization in bioreactors under aerobic batch cultivation revealed that amine production occurred only under post-diauxic growth on ethanol. To reduce native alanine utilization, the endogenous alanine aminotransferase (ALT1) was knocked out and replaced with cv-ATA. To rapidly employ this strategy in other strains, a simple CRISPR/cas9 method for universal gene replacement was developed. The replacement of ALT1 with cv-ATA improved the reaction by 2.6-fold compared to the control strain with intact ALT1. NMR measurements of metabolites originating from 15N L-alanine and 13C glucose indicated that pyruvate formation during growth on glucose inhibited amine production. Under optimal conditions, the biocatalytic bioconversion of benzylacetone to MPPA reached a yield of 58%.https://doi.org/10.1038/s41598-025-01182-0 |
| spellingShingle | Arne Hagman Olof Stenström Göran Carlström Mikael Akke Carl Grey Magnus Carlquist Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast Scientific Reports |
| title | Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast |
| title_full | Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast |
| title_fullStr | Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast |
| title_full_unstemmed | Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast |
| title_short | Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast |
| title_sort | biocatalytic reductive amination with crispr cas9 engineered yeast |
| url | https://doi.org/10.1038/s41598-025-01182-0 |
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