(594c) Genomic Engineering of Escherichia coli for Improved Aromatic Aldehyde Stability

Aromatic aldehydes have notable applications as flavors, fragrances, reactive intermediates, and potential residues for click chemistry conjugations. Metabolic routes to aldehyde synthesis provide a green chemical alternative to petrochemical synthesis or costly native extraction methods, but microbial methods to aldehyde synthesis in E. coli have been limited by instability of aromatic aldehydes in a cellular context. In our prior work, we addressed the rapid reduction of most aromatic aldehydes in E. coli by creating the reduced aromatic aldehyde reduction (RARE) strain, which eliminated most of the reductive instability in aldehydes via genetic knockouts of aldo-keto reductases, alcohol dehydrogenases, and the regulator gene yqhC. However, for many aromatic aldehydes of commercial or synthetic interest, the RARE strain alone is insufficient for stability due to the oxidative activity of native aldehyde dehydrogenase enzymes. In this work, we have engineered an E. coli strain with knockouts of 6 key aldehyde dehydrogenase enzymes performed rapidly using multiplexed automated genome engineering (MAGE) resulting in further stability of a range of aromatic aldehydes over 24h in E. coli culture conditions. In addition, further genetic engineering using MAGE was utilized to expand aldehyde stabilization for specialized E. coli strains, namely the recoded C321.ΔA strain utilized for genetic code expansion. Here, we performed MAGE to create aldehyde stabilization knockouts, enabling the use of recoded C321.ΔA for use with normally unstable aldehydes. Using this strain, we incorporated an aldehyde-containing non-standard amino acid (nsAA) into proteins. Further use of this strain will enable incorporation of conjugatable, aldehyde-substituted nsAAs and stabilization of aldehydes for biosynthetic pathways relevant to genetic code expansion.