Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates

Abstract

Whole-cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco-efficient route to produce phenolic amines. Here, a novel cell growth-based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from Chromobacterium violaceum (Cv-ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp-SpuC-II and Cv-ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94- and 92-fold increased specific transaminase activity, respectively. Whole-cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp-SpuC-II and Cv-ATA, respectively. Still, amine production was limited by a substantial re-assimilation of the product and formation of the by-products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv-ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re-assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement.

Fig. 1

Biochemical route for conversion of ferulic acid to vanillylamine, and major by‐products vanillyl alcohol and vanillic acid. The first step is the synthesis of vanillin from ferulic acid by the two enzymes Feruloyl‐CoA‐synthetase and Enoyl‐CoA‐hydratase/aldolase encoded by the genes fcs and ech, respectively. The reversible transamination of vanillin is accomplished by an amine transaminase (ATA). The amino group is transferred from the amino donor (l‐alanine) to vanillin with the formation of the corresponding amine and release of a co‐product (pyruvate). Alanine dehydrogenase (AlaDH) recycles l‐alanine from pyruvate consuming NADH and NH₃. Conversion of vanillin to vanillyl alcohol, and to vanillic acid is prevented by the deletion of vanillin dehydrogenase (vdh), and the aldehyde dehydrogenases (PP_2680 and PP_0545) and bdh (PP_1948; Graf and Altenbuchner, 2014), as well as areA (calA) encoding a vanillin reductase (García‐Hidalgo et al., 2020). Black arrows: Not modified; Green arrows: overexpressed genes; Red arrows: deleted genes.

Fig. 2

Growth of P. putida KT2440 overexpressing (A) Pp‐SpuC‐II (TMB‐NM011) or (B) Cv‐ATA (TMB‐JH002) in M9 medium supplemented with different concentrations of vanillylamine (0–25 mM) as sole carbon source. Growth curves are representative figures from one of the two biological replicates. After fitting the data in (A–B) to a logistic growth curve (described in Experimental Procedures) the estimated Carrying capacity (K; Panel C) and the maximal growth rates (r; Panel D) for each concentration are plotted against vanillylamine concentration for both Pp‐SpuC‐II (red line) and Cv‐ATA (black line).

Fig. 3

M9 plates after 3 days incubated at 30 °C with 5 mM of vanillylamine as the sole carbon source, streptomycin (100 µg ml⁻¹) for plasmid selection and IPTG (1 mM) to induce gene expression. A: KT2440 cells with the empty pSEVA424 plasmid; B: TMB‐JH002 cells expressing Cv‐ATA gene.

Fig. 4

Effect of l‐alanine regeneration on whole‐cell bioconversion of vanillin to vanillylamine using metabolically engineered P.putida GN442ΔPP_2426 strains as biocatalysts. Bioconversions were performed with a high cell density (OD₆₂₀ = 10) for 24 h at 30 °C and 180 rpm in M9 medium supplemented with 5 mM vanillin, 10 g/L of glucose for NADH regeneration and 200 mM of NH₄Cl. l‐Alanine was omitted for A and B and 50 mM of l‐alanine was added to the medium for C and D. A and C: TMB‐JH006 (Cv‐ATA + AlaDH) and B and D: TMB‐JH004 (Cv‐ATA). Error bars indicate ± SD of two biological replicates.

Fig. 5

Whole‐cell bioconversion of ferulic acid to vanillylamine using metabolically engineered P.putida GN442ΔPP_2426 strains as biocatalysts. Bioconversions were performed with high cell density (OD₆₂₀ = 10) for 24 h at 30 °C and 180 rpm in M9 medium supplemented with 3.5 mM ferulic acid, 10 g l⁻¹ of glucose for NADH regeneration, 50 mM of l‐Alanine and 200 mM of NH₄Cl. A: TMB‐JH006 strain (Cv‐ATA + AlaDH) and B: TMB‐JH004 strain (Cv‐ATA). Error bars indicate ± SD of two biological replicates.