MilliporeSigma
  • Home
  • Search Results
  • Synthesis and accumulation of aromatic aldehydes in an engineered strain of Escherichia coli.

Synthesis and accumulation of aromatic aldehydes in an engineered strain of Escherichia coli.

Journal of the American Chemical Society (2014-07-31)
Aditya M Kunjapur, Yekaterina Tarasova, Kristala L J Prather
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Vanillic acid, certified reference material, TraceCERT®
Benzaldehyde, European Pharmacopoeia (EP) Reference Standard
Supelco
Benzaldehyde, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Vanillin, Pharmaceutical Secondary Standard; Certified Reference Material
Vanillin, European Pharmacopoeia (EP) Reference Standard
Supelco
Vanillin, certified reference material, TraceCERT®
Sigma-Aldrich
Vanillin, ReagentPlus®, 99%
Sigma-Aldrich
Benzaldehyde, ≥98%, FG, FCC
Sigma-Aldrich
Benzaldehyde, natural, FCC, FG
Sigma-Aldrich
Benzaldehyde, purified by redistillation, ≥99.5%
Sigma-Aldrich
Vanillin, natural, ≥97%, FCC, FG
Sigma-Aldrich
Benzaldehyde, ReagentPlus®, ≥99%
Sigma-Aldrich
Vanillin, ≥97%, FCC, FG
Sigma-Aldrich
Benzaldehyde, puriss. p.a., ≥99.0% (GC)
Sigma-Aldrich
Vanillic acid, purum, ≥97.0% (HPLC)
Supelco
Benzaldehyde, analytical standard
Sigma-Aldrich
Vanillin, tested according to Ph. Eur.
Sigma-Aldrich
3-Hydroxy-4-methoxybenzaldehyde, 99%
Sigma-Aldrich
Vanillic acid, 97%
Sigma-Aldrich
Vanillyl alcohol, ≥98%, FG
Sigma-Aldrich
Vanillic acid, ≥97%, FG
Supelco
Vanillin melting point standard, Pharmaceutical Secondary Standard; Certified Reference Material
USP
Vanillin Melting Point Standard, United States Pharmacopeia (USP) Reference Standard
Supelco
Mettler-Toledo Calibration substance ME 51143093, Vanillin, traceable to primary standards (LGC)
Sigma-Aldrich
Dimethyl sulfoxide, sterile-filtered, BioPerformance Certified, meets EP, USP testing specifications, suitable for hybridoma
Sigma-Aldrich
Sodium benzoate, purum p.a., ≥99.0% (NT)
Sigma-Aldrich
Dimethyl sulfoxide, anhydrous, ≥99.9%
Sigma-Aldrich
Dimethyl sulfoxide, BioUltra, for molecular biology, ≥99.5% (GC)
Supelco
Benzyl alcohol, analytical standard
Sigma-Aldrich
Sodium benzoate, puriss., meets analytical specification of Ph. Eur., BP, FCC, E211, 99.0-100.5% (calc. to the dried substance), powder