Abstract
Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics. Production of FA by eco‐friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p-coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as caffeic acid O-methytransferase. Fermentation result revealed JM109(DE3) as a more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 or p15A), and the combination p15a-T5 works best. To further improve FA production, E. coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also overexpressed. Based on the flask fermentation data which show that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E. coli K-12 strains reported to the best of our knowledge.
Highlights
p-Hydroxy-3-methoxycinnamic acid (FA) is a ubiquitous phenolic acid naturally presents in plant cell walls, mainly cross linked with polysaccharides and lignin (Harris and Trethewey 2009)
The construct was introduced to E. coli BL21(DE3) and JM109(DE3), and the recombinant E. coli strains were fermented in Terrific Broth (TB) medium with additional 1 g/L L-tyrosine and 2% (v/v) glycerol at 28 °C and 250 rpm for 3 days
The data showed that the strain based on the E. coli JM109(DE3) chassis produced significantly more FA than the strain based on BL21(DE3) (60 vs. 14 mg/L, Fig. 2B)
Summary
FA is a ubiquitous phenolic acid naturally presents in plant cell walls, mainly cross linked with polysaccharides and lignin (Harris and Trethewey 2009). It is the main effective component of Angelica sinensis, Ligusticum chuanxiong Hort., Ferula foetida and other traditional Chinese medicinal herbs (Liang et al 2018). FA is an important plant antioxidant that acts as a free radical scavenger. Chemical synthesis of FA from vanillin via the Wittig–Horner reaction or Knoevenagel reaction offer a short producing. (2021) 8:70 cycle, low cost, and large output, but these approaches produce a mixture of trans- and cis-ferulic acids, which are challenging to separate Bioprocess. (2021) 8:70 cycle, low cost, and large output, but these approaches produce a mixture of trans- and cis-ferulic acids, which are challenging to separate
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