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Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast

Hagman, Arne LU ; Stenström, Olof LU ; Carlström, Göran LU orcid ; Akke, Mikael LU orcid ; Grey, Carl LU orcid and Carlquist, Magnus LU (2025) In Scientific Reports 15.
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... (More)

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%.

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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Scientific Reports
volume
15
article number
16972
pages
14 pages
publisher
Nature Publishing Group
external identifiers
  • scopus:105005285058
  • pmid:40374732
ISSN
2045-2322
DOI
10.1038/s41598-025-01182-0
language
English
LU publication?
yes
additional info
Publisher Copyright: © The Author(s) 2025.
id
6c0d869b-71ef-44c2-b101-7ad44414374f
date added to LUP
2025-06-17 19:27:51
date last changed
2025-07-15 22:21:19
@article{6c0d869b-71ef-44c2-b101-7ad44414374f,
  abstract     = {{<p>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 <sup>15</sup>N L-alanine and <sup>13</sup>C 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%.</p>}},
  author       = {{Hagman, Arne and Stenström, Olof and Carlström, Göran and Akke, Mikael and Grey, Carl and Carlquist, Magnus}},
  issn         = {{2045-2322}},
  language     = {{eng}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Scientific Reports}},
  title        = {{Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast}},
  url          = {{http://dx.doi.org/10.1038/s41598-025-01182-0}},
  doi          = {{10.1038/s41598-025-01182-0}},
  volume       = {{15}},
  year         = {{2025}},
}