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.
Highlights
Cis, cis-Muconic acid, a C6 unsaturated dicarboxylic acid, is an industrially important precursor molecule employed for the synthesis of a broad range of economically valuable compounds of polymeric in nature (Salvachúa et al, 2018)
The C12O gene consisted of an open reading frame (ORF) spanning about 939 bp (Supplementary Figure S1A) that encodes a protein of 312 amino acid residues with a theoretical molecular mass of 34.1 kDa and pI of 4.84
Microbial synthesis of ccMA by the Isopropyl ß-D-1-thiogalactopyranoside (IPTG) induced recombinant E. coli expressed with C12O was optimized by growing them in M9 medium (100 ml) containing glycerol and different concentrations of catechol ranging from 10 to 50 mM to determine the efficiency of whole-cells catalyzed conversion of catechol to ccMA
Summary
Cis-Muconic acid (ccMA), a C6 unsaturated dicarboxylic acid, is an industrially important precursor molecule employed for the synthesis of a broad range of economically valuable compounds of polymeric in nature (Salvachúa et al, 2018). The production of an economically important chemical constituent by non-renewable sources appears miserable and such chemical methods require high energy inputs to produce ccMA, and liberate harmful greenhouse gases into the environment (Weber et al, 2012). Several attempts were made to develop microbial cell factories through genetic modifications, metabolic engineering by constructing artificial biosynthetic pathways, and process optimizations of metabolic pathways, which has resulted promising increase in the yield of ccMA (Xie et al, 2014; Wang et al, 2018; Choi et al, 2020) Such bioconversions require multiple enzyme-catalyzed reactions, the recovered yields were inadequate and their purification fold appeared to be exiguous due to other metabolic intermediates (Vardon et al, 2016). The recombinant E. coli was employed for the fed-batch fermentation with the continuous supply of catechol as a feedstock under optimistic conditions resulted in prodigious titre of ccMA, which was successfully recovered with maximum purity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
