One-Pot Multienzymatic Transformation of NH₃, CO₂, and Ornithine into the Organic Nitrogen Plant Fertilizer Citrulline Using a Single Recombinant Lysate of E. coli

Biocatalytic transformation of carbamate formed readily from CO₂ and NH₃ provides attractive green routes for mitigation of these important environmental pollutants. Accordingly, a coupled-enzyme system was developed for the one-pot production of citrulline through carbamoylation of ornithine in aqueous solutions of CO₂ and NH₃. Hyperthermophilic ornithine carbamoyltransferases are produced recombinantly in E. coli with carbamate kinases known to have a propensity for carbamoyl phosphate synthesis. Importantly, in vitro biocatalysis is carried out by E. coli cell lysate prepared through coexpression of the required recombinant enzymes in a single bacterial culture, greatly reducing limitations normally associated with protein production and purification. Acetate kinase that is endogenous in the lysate also recycles the required ATP cofactor, which would otherwise have been required in costly stoichiometric amounts. Recombinant lysates catalyze the production of carbamoyl phosphate with substoichiometric ATP (>300 turnovers) as well as its in situ reaction with ornithine to give citrulline in high yield (>95%) and g L^-1 h^-1 titers. The system is active over a wide range of NH₃ concentrations (2.5 mM - 2 M), and >90% conversions of NH₃ may be reached within 1.5 h. Aqueous NH₃ used to sequester CO₂ gas (10% v/v) may be directly used as the biocatalyst feedstock. In preliminary studies, citrulline is found to be an effective organic nitrogen fertilizer of the wheat grass Brachypodium distachyon. Therefore, lysates described here constitute a cost-effective biocatalytic platform for one-pot production of a promising organic nitrogen fertilizer, under mild reaction conditions, from environmental pollutants as feedstock.