Catechol 1,2-Dioxygenase From Paracoccus sp. MKU1-A Greener and Cleaner Bio-Machinery for cis, cis-Muconic Acid Production by Recombinant E. coli

Abstract

Cis, cis-muconic acid (ccMA) is known for its industrial importance as a precursor for the synthesis of several biopolymers. Catechol 1,2-dioxygenase (C12O) is involved in aromatic compounds catabolism and ccMA synthesis in a greener and cleaner way. This is the first study on C12O gene from a metabolically versatile Paracoccus sp. MKU1, which was cloned and expressed in E. coli to produce ccMA from catechol. From the E. coli transformant, recombinant C12O enzyme was purified and found to be a homotrimer with a subunit size of 38.6 kDa. The apparent K _m and V _max for C12O was 12.89 µM and 310.1 U.mg^-1, respectively, evidencing high affinity to catechol than previously reported C12Os. The predicted 3D-structure of C12O from MKU1 consisted of five α-helices in N-terminus, one α-helix in C-terminus, and nine β-sheets in C-terminus. Moreover, a unique α-helix signature 'EESIHAN' was identified in C-terminus between 271 and 277 amino acids, however the molecular insight of conservative α-helix remains obscure. Further, fed-batch culture was employed using recombinant E. coli expressing C12O gene from Paracoccus sp. MKU1 to produce ccMA by whole-cells catalyzed bioconversion of catechol. With the successive supply of 120 mM catechol, the transformant produced 91.4 mM (12.99 g/L) of ccMA in 6 h with the purity of 95.7%. This single step conversion of catechol to ccMA using whole-cells reactions of recombinants did not generate any by-products in the reaction mixtures. Thus, the recombinant E. coli expressing high activity C12O from Paracoccus sp. MKU1 holds promise as a potential candidate for yielding high concentrations of ccMA at faster rates in low cost settings.

Keywords: Paracoccus sp.; bioplastics; catechol 1, 2-dioxygenase; cis, cis-muconic acid; fed-batch culture; recombinant E. coli.

FIGURE 1

Over-expression of C12O from Paracoccus sp. MKU1 in E. coli. (A) SDS-PAGE analysis of IPTG induced cell lysate. Lane M—Protein Ladder; Lane 1—Uninduced; Lane 2—0.2 mM IPTG; Lane 3—0.4 mM IPTG; Lane 4—0.6 mM IPTG; Lane 5—0.8 mM IPTG; Lane 6—1.0 mM IPTG. (B) SDS-PAGE analysis of Ni-NTA column purified recombinant C12O protein after 0.2 mM IPTG induction. M—Marker, Lane 1—Crude extract, Lane 2—Eluate 1, Lane 3—Eluate 2, Lane 4—Eluate 3. (C)—Size exclusion chromatography analysis of purified of C12O and reference proteins. (D) Calibration graph for C12O.

FIGURE 2

Effect of pH, temperature and metal ions on recombinant C12O activity. (A) pH optima, (B) pH stability, (C) Temperature optima, (D) Temperature stability, (E) Effect of metal ions, (F) Determination of enzyme kinetic constants. Inlet: Lineweaver-Burk plot for the determination of K _m and V _max ^. The residual relative activity of enzyme was determined by the standard assay and compared with the highest activity of the enzyme displayed at different conditions. All the assays were performed in triplicate and the values are presented as mean ± SEM.

FIGURE 3

Structural insights in to catechol binding sites in C12O enzyme from Paracoccus sp. MKU1. (A) 3D structure of C12O with catechol, (B) Enlarged image of molecular interaction of C12O with catechol, (C) 2D interaction of catechol with specific amino acids of C12O enzyme.

FIGURE 4

ccMA production by fed-batch fermentation. (A) Determination of substrate concentration for catalytic conversion by whole-cells catalyzed reactions at 1 h, (B) Bacterial growth profile and media pH during fed-batch fermentation, (C) ccMA production and catechol reduction followed by successive supply of 20 mM catechol at 1 h interval up to 6 h.