Biotransformation of ammonia and carbon dioxide to carbamoyl aspartate using in vitro multienzyme cascade
Creators: name: Fazelinejad, Somayeh
Publisher: name: The Australian National University
Year: 2023

description: The removal of ammonia from wastewater is a major worldwide concern. Ammonia is toxic for organisms living in water and results in eutrophication in aqueous environments. Many novel technologies have been applied for nitrogen removal from wastewater, and biological treatment is a potential effective treatment, yet it could not meet the effluent discharge standards. We have recently shown hyperthermophilic carbamate kinase (CKase) mitigates environmental pollutants ammonia and carbon dioxide into value-added products in a green process. CKase transforms ammonia and carbon dioxide into carbamoyl phosphate (CP) via carbamate in an adenosine triphosphate (ATP)-dependent manner. CP is the key metabolic intermediate in the urea cycle, pyrimidine and arginine pathway; yet it decomposes easily in aqueous solution and, therefore, is not feasible for industrial applications. Hence, using an alternative enzyme to utilise CP is beneficial. Aspartate transcarbamoylase (ATCase) is an alternative enzyme to utilise CP by carbamoylation of aspartate to produce carbamoyl aspartate, a very important precursor for pyrimidine pathway and production of nucleotides. We have developed a one-pot multienzymatic pathway to convert ammonia from wastewater and carbon dioxide from air to carbamoyl aspartate (CA) using one recombination lysate of Esherichia coli. We also established the immobilisation of CKase and ATCase on a solid carrier for easy usage and recovery in wastewater treatment. The following steps have been conducted in this project: First, the catalytic subunit of ATCase from three different hyperthermophilic microorganisms (the same as CKase) Thermococcus sibiricus, Thermococcus barophilus and Pyrococcus furiosus and one mesophilic microorganism, Enterococcus faecalis, have been cloned and expressed in E. coli Bl21 (DE3). The enzyme kinetics properties were studied to confirm if they have the same stability (pH and temperature) as CKase from hyperthermophile. Second, by confirming ATCase stability and activity at different pH and temperatures similar to hyperthermophilic CKase, ATCase and CKase pure enzyme were coupled to produce CA. Following that, the best combination of CKase and ATCase with respect to CA production yield was chosen for further study. In the next step, CKase crude lysate and pure ATCase were successfully coupled without adding any external ATP. CKase crude lysate contains sufficient endogenous acetate kinase that recycles cofactor ATP from adenosine monophosphate (AMP) (by engaging endogenous adenylate kinase) or adenosine diphosphate (ADP). Last, co-expressed crude lysate of recombinant CKase and ATCase from a single bacterial culture was employed to convert ammonia, carbon dioxide and aspartate to CA without using external ATP (there is enough in the lysate to initiate acetate kinase activity). The usage of lysate substantially decreases limitations related to protein production and purification. Third, we developed aspartate production from fumarate and ammonia (to remove more ammonia from wastewater) by using E. coli lysate as a source of aspartase. We demonstrated that co-expressed crude lysate of recombinant CKase and ATCase contains all required enzymes, including acetate kinase and aspartase, to convert ammonia and carbon dioxide into CA. Last, we immobilised CKase and ATCase on the Eupergit carrier to recover the enzymes and applied crude lysate of recombinant CKase and ATCase on wastewater to convert ammonia to CA.