Computational design of carboxylase for the synthesis of 4-hydroxyisophthalic acid from p-hydroxybenzoic acid by fixing CO2.

Carbon dioxide (CO 2) emissions constitute the primary contribution to global climate change. Synthetic CO 2 fixation represents an exceptionally appealing and sustainable method for carbon neutralization. Unlike the limitations of chemical catalysis, biological CO 2 fixation displays high selectivity and the ability to operate under mild conditions. The superfamily of amidohydrolases has demonstrated the ability to synthesize a range of aromatic monocarboxylic acids. However, there is a scarcity of reported carboxylases capable of synthesizing aromatic dicarboxylic acids. Among these, 4-hydroxyisophthalic acid holds significant potential for applications across various fields, yet no enzyme has been reported for its synthesis. In this study, we developed for the first time that exhibits starting activity in fixing CO 2 to synthesize 4-hydroxyisophthalic acid. Furthermore, we have devised a computational strategy that effectively enhances the catalytic activity of this enzyme. A focused library comprising only 13 variants was generated. Experimental validation confirmed a threefold improvement in the carboxylation activity of the optimal variant (L47M). The computational enzyme design strategy proposed in this paper demonstrates broad applicability in developing carboxylases for synthesizing other aromatic dicarboxylic acids. This lays the groundwork for leveraging biocatalysis in industrial synthesis for CO 2 fixation. • Demonstration that carboxylase can synthesize aromatic dicarboxylic acid by fixing CO 2. • 5-Carboxyvanillate carboxylase was found to catalyze the synthesis of 4-HIPA. • Develop an enzyme design strategy combing energy optimization and MD simulation. • The designed variant exhibited a 3-fold increase in carboxylation activity. [ABSTRACT FROM AUTHOR]