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

Abstract

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.

Efficient One-Pot Synthesis of Uridine Diphosphate Galactose Employing a Trienzyme System

“…The dilution of the reaction solution caused by the liquid substrate addition resulted in a decrease in UDP-Gal yield during 3–6 h. The failure to feed repeatedly was due to the inactivation of Tth0595 under high ATP concentration and the decline of Tte0732′s activity triggered by excess accumulation of ADP. The regeneration of ATP is an effective means to relieve ADP and ATP inhibition by catalyzing the reproduction of ATP from the byproduct ADP via polyphosphate kinase, pyruvate kinase, , or acetate kinase. − Furthermore, ATP regeneration is a more promising approach to synthesizing UDP-Gal on an industrial scale with lower initial feeding of ATP and higher cost-effectiveness.…”

DNA amplification with in situ nucleoside to dNTP synthesis, using a single recombinant cell lysate of E. coli

“…The application of cell lysates for multi-enzymatic cascades has gained increasing popularity due to its potential for efficient and inexpensive production of useful bio-chemicals 22–27 . Previously, we have employed this approach to produce nucleotides from cheap starting materials in quantitative yields with endogenous cofactor recycling driving multiple ATP-dependent steps (>10 2 turnovers) 26,28,29 . Even though the only dNK known to be present in E. coli is thymidine kinase 30 , we have observed that kinase promiscuity in lysates may accommodate non-substrate nucleotide to catalyze the initial phosphorylations 14 .…”

“…Nonetheless, we investigated the possibility of replacing it altogether with ADP or AMP. We previously found both to be suitable replacements in lysate-catalyzed biosyntheses, as they are rapidly converted by endogenous kinases to ATP with acetyl phosphate 14,26,28,29 . Indeed, when either was applied to the lysate PCR in place of ATP, uninhibited DNA synthesis was observed (Supplementary Information, Fig.…”

In vitro multi-enzymatic cascades using recombinant lysates of E. coli: an emerging biocatalysis platform

“…The utility of this approach is not limited to the synthesis of nucleotides and nucleic acids. Recently, a cell-free extract was developed for catalysis of an in vitro synthetic cascade that transforms pervasive environmental pollutants (ammonia and carbon dioxide) into nitrogen-rich citrulline (Alissandratos et al 2019). The reported cascade (Fig.…”