One-Pot Multienzymatic Transformation of NH3, CO2, and Ornithine into the Organic Nitrogen Plant Fertilizer
Citrulline Using a Single Recombinant Lysate of E.
coli
posted on 2019-04-19, 00:00authored byApostolos Alissandratos, Carol J. Hartley, Nigel G. French, Hye-Kyung Kim, Susan Allen, Gonzalo M. Estavillo, Colin Scott, Christopher J. Easton
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.