Bioproduction of methylated phenylpropenes and isoeugenol in Escherichia coli

Abstract

Phenylpropenes are a class of natural products that are synthesised by a vast range of plant species and hold considerable promise in the flavour and fragrance industries. Many in vitro studies have been carried out to elucidate and characterise the enzymes responsible for the production of these volatile compounds. However, there is a scarcity of studies demonstrating the in vivo production of phenylpropenes in microbial cell factories. In this study, we engineered Escherichia coli to produce methylchavicol, methyleugenol and isoeugenol from their respective phenylacrylic acid precursors. We achieved this by extending and modifying a previously optimised heterologous pathway for the biosynthesis of chavicol and eugenol. We explored the potential of six S-adenosyl l-methionine (SAM)-dependent O-methyltransferases to produce methylchavicol and methyleugenol from chavicol and eugenol, respectively. Additionally, we examined two isoeugenol synthases for the production of isoeugenol from coniferyl acetate. The best-performing strains in this study were able to achieve titres of 13 mg L^-1 methylchavicol, 59 mg L^-1 methyleugenol and 361 mg L^-1 isoeugenol after feeding with their appropriate phenylacrylic acid substrates. We were able to further increase the methyleugenol titre to 117 mg L^-1 by supplementation with methionine to facilitate SAM recycling. Moreover, we report the biosynthesis of methylchavicol and methyleugenol from l-tyrosine through pathways involving six and eight enzymatic steps, respectively.

Keywords: Biosynthesis; Escherichia coli; Methyleugenol; Phenylpropene; SAM-Dependent O-Methyltransferase.

Fig. 1

Phenylpropene biosynthesis pathway via CoA-dependent activation of the phenylacrylic acid substrate. Enzyme abbreviations and EC numbers: TAL, tyrosine ammonia-lyase (EC: 4.3.1.25); C3H, coumarate 3-hydroxylase (EC: 1.14.13.-); COMT, caffeic acid 3-O-methyltransferase (EC: 2.1.1.68); 4CL, 4-coumarate-CoA ligase (EC: 6.2.1.12); CCR, cinnamoyl-CoA reductase (EC: 1.2.1.44); CAD, cinnamyl-alcohol dehydrogenase (EC: 1.1.1.195); CFAT, coniferyl alcohol acyltransferase (EC: 2.3.1.84); EGS, eugenol synthase (EC: 1.1.1.318); IGS, isoeugenol synthase (EC: 1.1.1.319); OMT, (iso)eugenol O-methyltransferase (EC: 2.1.1.146).

Fig. 2

Microbial biosynthesis of phenylpropenes by whole-cell bioconversion of phenylacrylic acids. A Monolignol (green) and phenylpropene (yellow) biosynthesis pathways (constructs not drawn to scale). Enzyme abbreviations: SrCAR (Segniliparus rugosus carboxylic acid reductase); MsCAD (Medicago sativa cinnamyl alcohol dehydrogenase); PhCFAT (Petunia hybrida coniferyl alcohol acyltransferase); ObEGS (Ocimum basilicum eugenol synthase); OMT (O-methyltransferase). Plasmid SBC015869 (Hanko et al., 2023) was used for the biosynthesis of monolignols from phenylacrylic acids. The combinatorial plasmid library for phenylpropene production was constructed by inserting OMT gene candidates, either with or without a separate promoter, into plasmid SBC009876 (Robinson et al., 2020). B Library of strains tested for methylchavicol (left bar chart) and chavicol (right bar chart) production. C Library of strains tested for methyleugenol (left bar chart) and eugenol (right bar chart) production. Different OMT gene candidates used in each strain are represented by individual colours and the presence of the trc promoter is denoted by the arrow. Strain SBC009876 does not possess any OMT gene and was used as a control strain. All measurements were taken 24 h after induction and addition of the appropriate substrates. All experiments were performed using biological triplicates and error bars are representative of the standard deviations of these triplicates. Strains were grown in the absence (U) and presence (I) of IPTG.

Fig. 3

Methyleugenol and eugenol titres reported in the best-producing methyleugenol strain, SKF100_001080, in conditions of increasing trans-ferulic acid concentrations with and without additional 10 mM l-methionine supplementation. Methyleugenol, eugenol and unconverted trans-ferulic acid concentrations measured after 24 h after addition of the appropriate substrates. All strains were grown in the presence of IPTG, and measurements were taken 24 h after induction and addition of the appropriate substrates. All experiments were performed using biological triplicates and error bars are representative of the standard deviations of these triplicates.

Fig. 4

Extending microbial biosynthesis of methylated phenylpropenes from l-tyrosine. A The p-coumaric acid biosynthesis pathway (SBC007589 (Robinson et al., 2020)) or the trans-ferulic acid biosynthesis pathway (SBC010695 (Dunstan et al., 2020)) was introduced into the best methylchavicol producer strain, SKF100_001079, or the best methyleugenol producer strain, SKF100_001080, to create SKF100_001119 and SKF100_001120, respectively. Enzyme abbreviations: FjTAL (Flavobacterium johnsoniae tyrosine ammonia lyase); SeC3H (Saccharothrix espanensis coumarate 3-hydroxylase); PkCOMT (Populus kitakamiensis caffeate 3-O-methyltransferase). B Methylchavicol (orange) and chavicol (red) titres produced by SKF100_001119 with and without supplementation of 3 mM l-tyrosine. C Methyleugenol (yellow), eugenol (blue), methylchavicol (orange) and chavicol (red) titres produced by SKF100_001120 with and without the supplementation of 3 mM l-tyrosine. All measurements were taken 24 h after induction and addition of the appropriate substrates. All experiments were performed using biological triplicates and error bars are representative of the standard deviations of these triplicates. Strains were grown in the absence (U) and presence (I) of IPTG.

Fig. 5

Microbial biosynthesis of isoeugenol by whole-cell bioconversion of trans-ferulic acid. A Monolignol (green) and isoeugenol (yellow) biosynthesis pathways. Enzyme abbreviations: SrCAR (Segniliparus rugosus carboxylic acid reductase); MsCAD (Medicago sativa cinnamyl alcohol dehydrogenase); PhCFAT (Petunia hybrida coniferyl alcohol acyltransferase); IGS (isoeugenol synthase). The monolignol biosynthesis plasmids (SBC015863, SBC015866 and SBC015869 (Hanko et al., 2023)) were used in this study. B Combination of plasmids co-transformed to generate the isoeugenol production strains used in this study (constructs not drawn to scale). C Library of strains producing isoeugenol (left bar chart) and concentration of unconverted trans-ferulic acid (right bar chart). Measurements taken from strains expressing PhIGS and CbIGS are shown in pink and yellow, respectively. All experiments were performed using biological triplicates and error bars are representative of the standard deviations of these triplicates. Error bars are representative of standard deviations of biological triplicates. Strains were grown in the absence (U) and presence (I) of IPTG.