Engineering Escherichia coli for renewable benzyl alcohol production

Abstract

Benzyl alcohol is an aromatic hydrocarbon used as a solvent and an intermediate chemical in the pharmaceutical, cosmetics, and flavor/fragrance industries. The de novo biosynthesis of benzyl alcohol directly from renewable glucose was herein explored using a non-natural pathway engineered in Escherichia coli. Benzaldehyde was first produced from endogenous phenylpyruvate via three heterologous steps, including hydroxymandelate synthase (encoded by hmaS) from Amycolatopsis orientalis, followed by (S)-mandelate dehydrogenase (encoded by mdlB) and phenylglyoxylate decarboxylase (encoded by mdlC) from Pseudomonas putida ATCC 12633. The subsequent rapid and efficient reduction of benzaldehyde to benzyl alcohol occurred by the combined activity and native regulation of multiple endogenous alcohol dehydrogenases and/or aldo-keto reductases. Through systematic deletion of competing aromatic amino acid biosynthesis pathways to promote endogenous phenylpyruvate availability, final benzyl alcohol titers as high as 114±1 mg/L were realized, representing a yield of 7.6±0.1 mg/g on glucose and a ~5-fold improvement over initial strains.

Keywords: Aromatic chemicals; Benzaldehyde; Benzyl alcohol.

Fig. 1

Proposed pathway for benzaldehyde and benzyl alcohol biosynthesis from glucose by engineered E. coli. Dashed arrows indicate multiple steps. Black and gray arrows indicate native and heterologous pathway steps, respectively. Abbreviations: phosphoenolpyruvate (PEP), D-erythrose-4-phosphate (E4P), 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP).

Fig. 2

Demonstrating pathway function and monitoring metabolite flux via a whole resting cell biotransformation assay using exogenous phenylpyruvate and E. coli NST74 pHmaS-MdlC pMdlB. The conversion of 1 g/L phenylpyruvate (open diamonds) to phenylalanine (solid circles), (S)-mandelate (solid squares), phenylglyoxylate (open circles), benzaldehyde (open triangles), and benzyl alcohol (solid diamonds) was monitored over the course of 7 h. Error bars reported at one standard deviation from triplicate experiments.

Fig. 3

Growth response of E. coli NST74 to benzyl alcohol addition at concentrations of 0 g/L (solid squares), 0.25 g/L (open circles), 0.5 g/L (solid triangles), 0.75 g/L (open squares), 1 g/L (solid circles), and 2 g/L (open triangles). Error bars reported at one standard deviation from triplicate experiments.