The problem: hypertension

High blood pressure (HBP) or hypertension constitutes a major cause of cardiovascular disease and mortality throughout the world, estimated to affect more than 65% of people over 65 years of age (1). Underlying HBP is the increase in peripheral resistance associated with the progressive decrease in the bioavailability of nitric oxide (NO) observed in aging (2). NO plays an important role in regulating blood pressure as it mediates vascular dilatation and therefore represents a therapeutic target.

Our approach: an L-arginine-overproducing device

Our team designed Biobricks which could be used to create an L-arginine overproducing strain which could potentially be applied as a probiotic counteracting hypertension.

L-Arginine (2-amino-5-guanidinovaleric acid) is a conditionally essential amino acid, particularly important in people with hypertension and type II diabetes as these conditions are accompanied by an increase in the activity of the enzyme that degrades L-arginine (known as arginase), resulting in L-arginine deficiency. This amino acid is very versatile because it acts as a precursor of numerous molecules including NO, urea, agmatine, glutamate, proline and creatine (3). One interesting property of L-arginine is its potential to increase protein solubility and suppress protein aggregation (due to the change in the surface tension of proteins caused by interaction with proteins or the water surrounding them) - it has been shown to destabilize and remove mature oral biofilms preventing plaque, elevate dental plaque pH preventing tooth demineralization as well as the growth of aciduric bacteria and diminish infectivity of envelope viruses, e.g. herpes simplex and influenza. It has been indicated that L-arginine intake in doses larger than standard dietary consumption can improve symptoms of cardiovascular disease (4) and insulin sensitivity (5), aid in the healing of wounds (6) and gastric ulcers (7) and relieve stress (8).

The amino acid is synthesized in the so-called linear pathway (Fig. 1) from L-glutamate. The initial enzyme in the pathway, N-acetylglutamate synthase (NAGS), is inhibited by L-arginine in a negative feedback loop (9). Another source of inhibition of the pathway is ArgR, the common repressor of L-arginine biosynthetic genes for which the amino acid acts as a co-repressor (10).

Our team designed Biobricks: BBa_K1954006, BBa_K1954008 and BBa_K1954009, to overcome the limitations of the linear pathway. The mutagenized version of NAGS is resistant to inhibition by L-arginine. The ARG box is a binding sequence for ArgR and could be introduced into a high-copy number plasmid to titrate the repressor away from the regulatory sequences of biosynthetic genes. In addition, our team made a Biobrick for the exporter of L-arginine under a constitutive promoter to maximize the transport of the amino acid out of the cell.

References

1. Lionakis N. Hypertension in the elderly. World J Cardiol. 2012;4(5):135.
2. Förstermann U, Münzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 2006 Apr 4;113(13):1708–14.
3. Gad MZ. Anti-aging effects of l-arginine. J Adv Res. 2010 Jul;1(3):169–77.
4. Böger RH, Bode-Böger SM. The clinical pharmacology of L-arginine. Annu Rev Pharmacol Toxicol. 2001;41:79–99.
5. Carvalho DS, Diniz MM, Haidar AA, de Fátima Cavanal M, da Silva Alves E, Carpinelli AR, et al. L-Arginine supplementation improves insulin sensitivity and beta cell function in the offspring of diabetic rats through AKT and PDX-1 activation. Eur J Pharmacol. 2016 Oct 4;
6. Sax HC. Arginine stimulates wound healing and immune function in elderly human beings. JPEN J Parenter Enteral Nutr. 1994 Dec;18(6):559–60.
7. Brzozowski T, Konturek SJ, Drozdowicz D, Dembiński A, Stachura J. Healing of chronic gastric ulcerations by L-arginine. Role of nitric oxide, prostaglandins, gastrin and polyamines. Digestion. 1995;56(6):463–71.
8. Gupta V, Gupta A, Saggu S, Divekar HM, Grover SK, Kumar R. Anti-stress and Adaptogenic Activity of l-Arginine Supplementation. Evid-Based Complement Altern Med ECAM. 2005 Mar;2(1):93–7.
9. Rajagopal BS, DePonte J, Tuchman M, Malamy MH. Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains. Appl Environ Microbiol. 1998 May;64(5):1805–11.
10. Tuchman M, Rajagopal BS, McCann MT, Malamy MH. Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains. Appl Environ Microbiol. 1997 Jan;63(1):33–8.