De novo biosynthesis of 2-hydroxyterephthalic acid, the monomer for high-performance hydroxyl modified PBO fiber, by enzymatic Kolbe- Schmitt reaction with CO2 fixation

Abstract

Abstract Background High-performance poly(p-phenylenebenzobisoxazole) (PBO) fiber, with excellent mechanical properties (stiffness, strength, and toughness), high thermal stability combined and light weight, are widely employed in automotive and aerospace composites, body armor and sports goods. Hydroxyl modified PBO (HPBO) fiber shows better photostability and interfacial shear strength. 2-Hydroxyterephthalic acid (2-HTA), the monomer for the HPBO fiber, is usually synthesized by chemical method, which has poor space selectivity and high energy consumption. The enzymatic Kolbe-Schmitt reaction, which carboxylates phenolic substrates to generate hydroxybenzoic acids with bicarbonate/CO2, was applied in de novo biosynthesis of 2-HTA with CO2 fixation. Results The biosynthesis of 2-HTA was achieved by the innovative application of hydroxybenzoic acid (de)carboxylases to carboxylation of 3-hydroxybenzoic acid (3-HBA) at the para-position of the benzene carboxyl group, known as enzymatic Kolbe-Schmitt reaction. Three hydroxybenzoic acid (de)carboxylases from diverse microorganisms were expressed in recombinant E. coli. 2,3-Dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao) showed highest activity. In addition, two amino acid substitutions, F27G and T62A, proved to be of great help in improving 2,3-DHBD activity. The double site mutation F27G/T62A increased the production of 2-HTA by 28-fold up to 5.87 ± 0.06 mg/L in vitro. Moreover, de novo biosynthetic pathway of 2-HTA was constructed by co-expression of 2,3-DHBD_Ao and 3-hydroxybenzoate synthase Hyg5 in S. cerevisiae S288C with Ura3, Aro7 and Trp3 knockout. The engineered strain synthesized 45.4 ± 0.2 µg/L 2-HTA at 36 h in the CO2 environment. Conclusion De novo synthesis of 2-HTA has been achieved, which uses glucose as a raw material to generate shikimic acid, chorismic acid, and 3-HBA, and finally obtains 2-HTA. We demonstrate the strong potential of hydroxybenzoate (de)carboxylase to produce terephthalic acid and its derivatives with CO2 fixation.