Frontiers in Bioengineering and Biotechnology · 2021 · 21 citations · 44 references
Cis, <i>cis</i>-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 <i>C12O</i> gene from a metabolically versatile <i>Paracoccus</i> sp. MKU1, which was cloned and expressed in <i>E. coli</i> to produce ccMA from catechol. From the <i>E. coli</i> transformant, recombinant C12O enzyme was purified and found to be a homotrimer with a subunit size of 38.6 kDa. The apparent <i>K</i> <sub>m</sub> and <i>V</i> <sub>max</sub> for C12O was 12.89 µM and 310.1 U.mg<sup>-1</sup>, 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 <i>E. coli</i> expressing <i>C12O</i> gene from <i>Paracoccus</i> 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 <i>E. coli</i> expressing high activity C12O from <i>Paracoccus</i> sp. MKU1 holds promise as a potential candidate for yielding high concentrations of ccMA at faster rates in low cost settings.
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