(576b) Synthesis and Accumulation of Aromatic and Aliphatic Aldehydes in an Engineered Strain of Escherichia coli

Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and precursors to pharmaceuticals. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expressing a recombinant carboxylic acid reductase in the RARE strain and supplying the strain with benzoate during growth, benzaldehyde remained in the culture after 24 hours, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol even after 48 hours. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Next, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose using growing E. coli cells that expressed a recombinant mutant pyruvate decarboxylase from Zymomonas mobilis. When the wild-type host strain was used, all supplied benzaldehyde was converted to benzyl alcohol within 2 hours and no production of L-PAC occurred. Finally, we show that the RARE strain also enables the accumulation of aliphatic aldehydes ranging from at least C4-C8. Beyond allowing accumulation of aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced via aldehyde intermediates using metabolic engineering.