Production of vanillate from lignin derivatives using genetically modifiedescherichia coli

Abstract

Vanillic acid is an important compound used in food, beverage, fragrance, and pharmaceutical industries. Vanillic acid can be produced by using either natural plant, native, and engineered microorganisms. However, the industrial scale production of vanillic acid compound still difficult due to low yielding production which can be improved by metabolic engineering of bacterial cells. Hence, the efficient biocatalyst, engineering, and biotechnology process can be a promising strategy to enhance vanillic acid production from lignin derivatives and strengthen the large-scale production for industrial approach. Among several lignin-derived compounds, ferulic acid is a widely used substrate for vanillic acid production. Recently, the Coenzyme A-independent production of vanillin from ferulic acid has been reported. To further develop this pathway for vanillic acid production, herein, the synthetic pathway consisting of four genes which are comt, padC, vdh, and ado was constructed and expressed in the recombinant E. coli strain BL21(DE3). Such strain showed an effective vanillic acid production with 96 ± 2 % vanillic acid yield when the growth-independent conversion was conducted with an initial OD600 of 40 in 100 mM Tris-HCl buffer pH 8, and ferulic acid 0.194 g/L (1 mM) as the substrate. The maximum production of vanillic acid was 0.95 ± 0.002 g/L (4.92 ± 0.01 mM, 58 ± 1 % molar yield) from 1.94 g/L (10 mM) of ferulic acid within 48 hours in shake flask scale. A two-step bioconversion process was established to produce vanillic acid from caffeic acid. The production of vanillic acid reached 0.168 ± 0.003 g/L (1.00 ± 0.02 mM, 100 ±2 % molar yield) from 0.180 g/L of caffeic acid (1 mM) within 48 hours. For the production of vanillic acid in 5-L fermenter, the maximum vanillic acid of 0.121 ± 0.006 g/L (0.72 ± 0.04 mM, 74 ± 4 % molar yield) was achieved from 0.194 g/L of ferulic acid within 67 hours. This study suggests this recombinant E. coli might be a practical strain for scaling up vanillic acid production and further development for industrial application.