Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli

Abstract

Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. 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 expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, 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. 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. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

“…At the same time, the enzymatic background of bacterial cells rapidly reduces aldehydes ( b ) to the corresponding alcohols ( c ) which minimizes the oxidative and electrophilic stress responses induced by the reactive carbonyl group in aldehydes. [39,41] To circumvent b reduction, we opted for E. coli K‐12 MG1655 RARE42 – a knock‐out strain with r educed aromatic a ldehyde re duction – as a host for whole cell mediated aldehyde preparation. Despite this deliberate choice, initial experiments towards the reduction of the model substrate cinnamic acid 1a to 1b showed significant amounts of the undesired alcohol 1c .…”

Microbial Engineering for Aldehyde Synthesis

“…After deletion of six genes that encode enzymes with confirmed activity on benzaldehyde in vitro (dkgA, dkgB, yeaE, yahK, ahr, and yqhD), the engineered E. coli strain accumulated benzaldehyde and vanillin with minimal alcohol formation and was thus dubbed "RARE" for displaying reduced aromatic aldehyde reduction (Addgene catalog no. 61440) (43). Each targeted gene was capable of causing reduction of benzaldehyde and vanillin in vivo when individually overexpressed in the RARE background.…”

“…Each targeted gene was capable of causing reduction of benzaldehyde and vanillin in vivo when individually overexpressed in the RARE background. However, the use of deletion subset strains and quantitative reverse transcription-PCR (qRT-PCR) revealed that deletions of dkgB and yeaE did not contribute to aldehyde accumulation under the conditions tested due to low native expression of these genes (43).…”

“…Cultures of the RARE strain expressing a recombinant mutant PDC were able to produce L-PAC using exogenously supplied benzaldehyde and metabolized pyruvate with minimal benzyl alcohol formation. Under the conditions tested, the use of wild-type E. coli expressing the same PDC produced no detectable L-PAC (43). In addition to PDC, other enzymes capable of catalyzing chiral carboligations of aldehyde substrates have been discussed (57).…”

PRODUÇÃO DE ALDEÍDOS A PARTIR DO CULTIVO HETEROTRÓFICO DE Phormidium autumnale COM MONOSSACARÍDEOS COMO FONTE DE CARBONO EXÓGENA

“…A Figura 1 apresenta os cromatogramas obtidos por GC/MS do cultivo de Phormidium com monossacarídeos no tempo 96 horas com seus respectivos aldeídos em destaque. A diminuição da concentração se explica pela rápida conversão de aldeídos em álcoois por uma numerosa quantidade de enzimas endógenas presente em micro-organismos (Kunjapur et al, 2014), justificando a formação de álcoois significativamente alta no final de 144h. Dos componentes identificados em ambos os cultivos, 3-metilbutanal (2,2 µg/mg) destaca-se como majoritário no período de 144h, seguido pelo 3-octenal (2,1 µg/mg).…”