Carbon dioxide (CO2) emissions constitute the primary contribution to global climate change. Synthetic CO2 fixation represents an exceptionally appealing and sustainable method for carbon neutralization. Unlike the limitations of chemical catalysis, biological CO2 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 CO2 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 CO2 fixation.

The carbon isotopic composition (δ13C) of foliage is often used as proxy for plant performance. However, the effect of N O 3 - vs. N H 4 + supply on δ13C of leaf metabolites and respired CO2 is largely unknown. We supplied tobacco plants with a gradient of N O 3 - to N H 4 + concentration ratios and determined gas exchange variables, concentrations and δ13C of tricarboxylic acid (TCA) cycle intermediates, δ13C of dark-respired CO2, and activities of key enzymes nitrate reductase, malic enzyme and phosphoenolpyruvate carboxylase. Net assimilation rate, dry biomass and concentrations of organic acids and starch decreased along the gradient. In contrast, respiration rates, concentrations of intercellular CO2, soluble sugars and amino acids increased. As N O 3 - decreased, activities of all measured enzymes decreased. δ13C of CO2 and organic acids closely co-varied and were more positive under N O 3 - supply, suggesting organic acids as potential substrates for respiration. Together with estimates of intra-molecular 13C enrichment in malate, we conclude that a change in the anaplerotic reaction of the TCA cycle possibly contributes to 13C enrichment in organic acids and respired CO2 under N O 3 - supply. Thus, the effect of N O 3 - vs. N H 4 + on δ13C is highly relevant, particularly if δ13C of leaf metabolites or respiration is used as proxy for plant performance.