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Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast.

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Metabolically engineered yeast efficiently produces chiral amines via whole-cell bioconversion. Modifying the alanine-pyruvate node and replacing alanine aminotransferase (ALT1) with omega transaminase (cv-ATA) significantly enhanced production.

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Area of Science:

  • Biotechnology
  • Metabolic Engineering
  • Enzyme Catalysis

Background:

  • Baker's yeast is a versatile host for producing chiral amines through whole-cell bioconversion of prochiral ketones.
  • The alanine-pyruvate metabolic node plays a crucial role in regulating metabolic flux and can be modulated to enhance bioconversion efficiency.

Purpose of the Study:

  • To engineer baker's yeast for enhanced production of chiral amines by modulating the alanine-pyruvate metabolic node.
  • To investigate the impact of replacing endogenous alanine aminotransferase (ALT1) with a promiscuous omega transaminase (cv-ATA) on reductive amination.
  • To develop a CRISPR/cas9 method for rapid gene replacement in yeast for broader applicability.

Main Methods:

  • Metabolic engineering of baker's yeast by chromosomal integration of multiple copies of cv-ATA.
  • Gene knockout and replacement of ALT1 with cv-ATA using CRISPR/cas9 technology.
  • Physiological characterization in bioreactors under aerobic batch cultivation and NMR analysis of metabolic intermediates.

Main Results:

  • Engineered yeast expressing cv-ATA demonstrated active chiral amine production, specifically (S)-1-methyl-3-phenylpropylamine (MPPA), during post-diauxic growth on ethanol.
  • Replacing ALT1 with cv-ATA resulted in a 2.6-fold improvement in the reaction yield compared to the control strain.
  • Pyruvate formation during glucose metabolism was identified as an inhibitor of amine production, as indicated by NMR studies.

Conclusions:

  • Modulating the alanine-pyruvate node by replacing ALT1 with cv-ATA is an effective strategy for enhancing chiral amine production in metabolically engineered yeast.
  • The developed CRISPR/cas9 system facilitates rapid gene replacement, enabling efficient strain engineering for biocatalysis.
  • Optimal bioconversion conditions yielded 58% MPPA from benzylacetone, highlighting the potential of this engineered yeast system.