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

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. 2,3-Dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao) were expressed in recombinant E. coli and showed highest activity. The yield of 2-HTA was 108.97 ± 2.21 μg/L/mg protein in the whole-cell catalysis. 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 in the whole-cell catalysis by 24.7-fold, reaching 2.69 ± 0.029 mg/L/mg protein. 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 45.40 ± 0.28 μg/L 2-HTA at 36 h in the CO₂ environment.

Conclusions: De novo synthesis of 2-HTA has been achieved, using glucose as a raw material to generate shikimic acid, chorismic acid, and 3-HBA, and finally 2-HTA. We demonstrate the strong potential of hydroxybenzoate (de)carboxylase to produce terephthalic acid and its derivatives with CO₂ fixation.

Keywords: (De)carboxylase; 2-Hydroxyterephthalic acid; CO2 fixation; De novo biosynthetic pathway; Enzymatic Kolbe–Schmitt reaction; PBO fiber.

Fig. 1

HPLC–MS/MS analysis of 2-HTA and whole-cell reaction mixture of 2,3-DHBD_Ao. a Extracted ion chromatography of 2-HTA standard. b Secondary mass spectrum of 2-HTA standard. c Extracted ion chromatography of whole-cell reaction mixture of 2,3-DHBD_Ao. d Secondary mass spectrum of whole-cell reaction mixture of 2,3-DHBD_Ao

Fig. 2

HPLC–MS/MS analysis: a whole-cell reaction mixture of 2,3-DHBD_Ao, b 2-HTA standard, c 3-hydroxyphthalic acid standard, d 4-hydroxyphthalic acid standard

Fig. 3

2-HTA production by whole-cell reaction of E. coli BL21(DE3) harboring different 2,3-DHBD. a Reaction conditions: 30 mg/mL lyophilized whole cells, 10 mM substrate, 3 M KHCO₃, 30 ℃, 180 rpm. b Enzyme: 2,3-DHBD_Ao_opt: 2,3-DHBD_Ao with codon-optimized; 2,3-DHBD_Fo_opt: 2,3-DHBD_Fo with codon-optimized; sequences of native and codon-optimized genes were shown in Additional file 1: Table S3. c One-way analysis of variance (one-way ANOVA) was used to analyze the data

Fig. 4

Comparison of distances between residues 62/27 and 2-HTA. a Wild-type 2,3-DHBD_Ao (PDB ID:7WKM) with Thr62 (green stick); b 2,3-DHBD_Ao mutant with Ala62(yellow stick) ^a; c wild-type 2,3-DHBD_Ao with Phe27 (green stick); d the 2,3-DHBD_Ao mutant with Gly27 (blue stick) ^a. ^a Mutants were generated by Discovery Studio™ based on 7WKM

Fig. 5

2-HTA production by 2,3-DHBD_Ao mutants with single and double amino acid substitutions. ^aReaction conditions: 30 mg/mL lyophilized whole cells, 10 mM substrate, 3 M KHCO₃, 30 ℃, 180 rpm

Fig. 6

OD₆₀₀ of S. cerevisiae BY4741 in 1 M and 0.5 M KCHO₃ medium

Fig. 7

Effect of KHCO₃ concentration on the production of 2-HTA by S. cerevisiae BY-A. ^a Reaction conditions: 10 mM 3-HBA, 30 ℃, 180 rpm, 72 h

Fig. 8

Biosynthesis pathway of 2-HTA. PEP, phosphoenolpyruvate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-D-arabinoheptulosonate 7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SHK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvyl-shikimate 3-phosphate; CHA, chorismate; PPA, prephenate; L-Phe, L-phenylalanine; L-Trp, L-tryptophan; L-Tyr, L-tyrosine; Aro1, pentafunctional aromatic protein; Aro4, DAHP synthase; Hyg5, 3-hydroxybenzoate synthase; 2,3-DHBD, 2,3-dihydroxybenzoate decarboxylase; Aro7, chorismate mutase; Trp3, indole-3-glycerol-phosphate synthase

Fig. 9

Fermentation comparison of S. cerevisiae CEN.PK113-5D under CO₂ and O₂ environment. a Strain growth curve. b Accumulation of acetic acid, c accumulation of ethanol, d accumulation of glycerol

Fig. 10

Fermentation of S. cerevisiae CEN.PK113-5D in SC medium under CO₂ environment. a Concentration of 3-HBA. b Concentration of 4-HBA. c Concentration of 2-HTA

Fig. 11

Production of S. cerevisiae S288C in SC medium under CO₂ environment. a Growth curve. b Concentration of 3-HBA. c Concentration of 2-HTA