Abstract
Regulating and ameliorating enzyme expression and activity greatly affects the performance of a given synthetic pathway. In this study, a new synthetic pathway for cis, cis-muconic acid (ccMA) production was reconstructed without exogenous induction by regulating the constitutive expression of the important enzyme catechol 1,2-dioxygenase (CatA). Next, new CatAs with significantly improved activities were developed to enhance ccMA production using structure-assisted protein design. Nine mutations were designed, simulated and constructed based on the analysis of the CatA crystal structure. These results showed that mutations at Gly72, Leu73 and/or Pro76 in CatA could improve enzyme activity, and the activity of the most effective mutant was 10-fold greater than that of the wild-type CatA from Acinetobacter sp. ADP1. The most productive synthetic pathway with a mutated CatA increased the titer of ccMA by more than 25%. Molecular dynamic simulation results showed that enlarging the entrance of the substrate-binding pocket in the mutants contributed to their increased enzyme activities and thus improved the performance of the synthetic pathway.
Highlights
The microbial production of platform chemicals, which can be either directly used or further processed for the production of industrial-scale and high value-added products in the chemical industry, has recently gained tremendous interest and attention, especially with recent rapid advances in synthetic biology and metabolic engineering[1,2,3]
The constitutive promoter-catA expression cassettes were constructed by either fusion polymerase chain reaction (PCR) or chemical synthesis and used to replace the inducible-catA expression cassette in pKD8.2925 to obtain four recombinant plasmids, pKD8.292K, pKD8.292C, pKD8.292T and pKD8.292PL25, with constitutive promoters Pkan, Pcm, Ptc, and PL25, respectively (Fig. S1B)
PKD8.292K, pKD8.292C, pKD8.292T and pKD8.292PL25 were each co-transformed with the plasmid pKD8.2435 containing 3-dehydroshikimate dehydratase and protocatechuate decarboxylase into E. coli AB2834 to obtain new engineered strains (E. coli WZK, WZC, WZT, and WZPL25, respectively) with ccMA heterologous synthetic pathways
Summary
The microbial production of platform chemicals, which can be either directly used or further processed for the production of industrial-scale and high value-added products in the chemical industry, has recently gained tremendous interest and attention, especially with recent rapid advances in synthetic biology and metabolic engineering[1,2,3]. Global microbial genome and environmental metagenome sequencing efforts are contributing ever-increasing genetic information on catA These studies facilitate screening efforts to find CatAs with higher activities, which are needed for the efficient production of ccMA15,17. ADP1, in which Ile[105], Pro[108], Phe[253], and Arg[221] are conserved in the catechol 1,2-dioxygenase family[18,19,20,21,22] This structural information lays a solid foundation for the rational redesign of CatA to improve its catalytic activities and other related functions. An increasing number of proteins have been engineered for altered substrate specificity/selectivity by ration design, the engineering of highly efficient enzymatic pathways for industrial-scale fuel and chemical production by increasing the catalytic activity of the key enzymes remains an overwhelming challenge and requires expanded efforts in metabolic engineering and synthetic biology[23]. Our results show that rational redesign can enable the improved performance of key enzymes and synthetic pathways and can be applied to many other metabolic engineering and synthetic biology studies
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