Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast.

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.