Carboxylic acid reductase-dependent biosynthesis of eugenol and related allylphenols

Abstract

Background: (Hydroxy)cinnamyl alcohols and allylphenols, including coniferyl alcohol and eugenol, are naturally occurring aromatic compounds widely utilised in pharmaceuticals, flavours, and fragrances. Traditionally, the heterologous biosynthesis of (hydroxy)cinnamyl alcohols from (hydroxy)cinnamic acids involved CoA-dependent activation of the substrate. However, a recently explored alternative pathway involving carboxylic acid reductase (CAR) has proven efficient in generating the (hydroxy)cinnamyl aldehyde intermediate without the need for CoA activation. In this study, we investigated the application of the CAR pathway for whole-cell bioconversion of a range of (hydroxy)cinnamic acids into their corresponding (hydroxy)cinnamyl alcohols. Furthermore, we sought to extend the pathway to enable the production of a variety of allylphenols and allylbenzene.

Results: By screening the activity of several heterologously expressed enzymes in crude cell lysates, we identified the combination of Segniliparus rugosus CAR (SrCAR) and Medicago sativa cinnamyl alcohol dehydrogenase (MsCAD2) as the most efficient enzymatic cascade for the two-step reduction of ferulic acid to coniferyl alcohol. To optimise the whole-cell bioconversion in Escherichia coli, we implemented a combinatorial approach to balance the gene expression levels of SrCAR and MsCAD2. This optimisation resulted in a coniferyl alcohol yield of almost 100%. Furthermore, we extended the pathway by incorporating coniferyl alcohol acyltransferase and eugenol synthase, which allowed for the production of eugenol with a titre of up to 1.61 mM (264 mg/L) from 3 mM ferulic acid. This improvement in titre surpasses previous achievements in the field employing a CoA-dependent coniferyl alcohol biosynthesis pathway. Our study not only demonstrated the successful utilisation of the CAR pathway for the biosynthesis of diverse (hydroxy)cinnamyl alcohols, such as p-coumaryl alcohol, caffeyl alcohol, cinnamyl alcohol, and sinapyl alcohol, from their corresponding (hydroxy)cinnamic acid precursors but also extended the pathway to produce allylphenols, including chavicol, hydroxychavicol, and methoxyeugenol. Notably, the microbial production of methoxyeugenol from sinapic acid represents a novel achievement.

Conclusion: The combination of SrCAR and MsCAD2 enzymes offers an efficient enzymatic cascade for the production of a wide array of (hydroxy)cinnamyl alcohols and, ultimately, allylphenols from their respective (hydroxy)cinnamic acids. This expands the range of value-added molecules that can be generated using microbial cell factories and creates new possibilities for applications in industries such as pharmaceuticals, flavours, and fragrances. These findings underscore the versatility of the CAR pathway, emphasising its potential in various biotechnological applications.

Keywords: Allylphenol; Bioconversion; Carboxylic acid reductase; Escherichia coli; Eugenol; Monolignol; Phenylpropanoid.

Fig. 1

Pathway for the biosynthesis of allylphenols and allylbenzene involving CoA-dependent activation (orange) or carboxylic acid reductase-mediated reduction (purple) of the (hydroxy)cinnamic acid substrate. Enzyme abbreviations: 4CL, 4-coumarate-CoA ligase; CCR, cinnamyl CoA reductase; CAR, carboxylic acid reductase; CAD, cinnamyl alcohol dehydrogenase; CFAT, coniferyl alcohol acyltransferase; EGS, eugenol synthase

Fig. 2

Combinatorial optimisation of the coniferyl alcohol biosynthesis pathway. A The pathway converting ferulic acid into coniferyl alcohol involves three genes – SrCAR, Sfp, and MsCAD2. A combinatorial library of plasmids was designed to encode this pathway, with variations in the origin of replication, order of pathway genes, and promoter parts. B Pathway variants that were tested for the in vivo production of coniferyl alcohol. E. coli NEB5α carrying the individual plasmids were grown at 30℃ for 20 h in TBP medium supplemented with 0.4% glycerol and 3 mM ferulic acid. Error bars represent standard deviations of three biological replicates. Cells were grown in the absence (−) and presence (+) of IPTG

Fig. 3

Bioconversion of ferulic acid into eugenol using the CAR-dependent pathway. A The eugenol biosynthesis pathway is split into two modules. The first module consists of SrCAR and MsCAD2 enzymes, catalysing the two-step reduction of ferulic acid, resulting in the formation of coniferyl alcohol. The second module comprises PhCFAT and ObEGS enzymes, catalysing the two-step conversion of coniferyl alcohol into eugenol. B Production of eugenol in E. coli NEB5α carrying SBC009876 in combination with one of the three indicated plasmids. At time point zero, cells were supplemented with 3 mM ferulic acid, and expression of pathway enzymes was induced by addition of IPTG. Error bars represent standard deviations of biological triplicates