Neglected diseases have their importance in public health system, causing millions of deaths every year (MOREL, 2006). In Brazil dengue stands out; a disease which afflicted more than 1,350,406 people in 2015, according to data from the Brazilian Health Ministry epidemiological report. The microorganisms responsible for these diseases are usually transported by insects, thus, repellents represent a good strategy to fight these microorganisms (STEFANI et al., 2009; SORGE et al., 2007; FRADIN & DAY, 2002).

Besides the problem previously reported, it is interesting to highlight the toxicity associated to high concentrations of N,N-diethyl-m-toluamide (DEET), which is the molecule currently used on market. (CHEN-HUSSEY et al., 2014; ROBBINS et al., 1986; OSIMITZ et al., 2010; MCGREADY et al., 2001).

Repellents extracted from natural oils are an alternative to toxicity, but they have a shorter lifespan compared to DEET, necessitating more topical applications; between natural molecules with proved efficiency we highlight the D-limonene, that besides being safe to human skin it is also a GRAS (Generally recognized as safe) product (SUN, 2007; KARLBERG et al., 1991).

With the objective to prolong the duration of the natural repellent based on D-limonene, the use of synthetic biology to promote its continuous production was proposed in this project. However, previous efforts to produce D-limonene in bacterial chassi were not efficient due the insolubility of limonene synthase, enzyme responsible to convert geranyl pyrophosphate to limonene.

To make possible the production of D-limonene, the construction of a genetic circuit with three modules was proposed. The first module has a promoter responsible to different stresses (UspA), which induces the production of limonene synthase. The stresses create conditions that are likely to produce molecular chaperones, improving the efficiency in the process of protein folding (PURVIS et al., 2005; SHIMIZU et al., 2013). Combining this condition to the problem of storage of our product we chose to use osmotic stress, created by an ethylene glycol polymer (PEG). It showed to be efficient due to its flexible and hydrophilic features, which creates high osmotic pressures, and its chemical arrangement makes it unlikely to interact with other biological molecules present in the genetically modified microorganism (MONEY et al., 1989).

The second module is composed by three proteic chaperones, natural from Escherichia coli, overexpressed to obtain a better protein folding. The last module consists in a killswitch. It is a biosafety mechanism responsible for programmed cell death of the bacteria used in this project, after the required time to produce limonene. The killswitch’s function is associated to the action of znuABC operon, and to protein Zur. With this it is possible to control bacterial lifespan, depending on the concentration of zinc present at the final product.

References

MOREL, C. M. Inovação em saúde e doenças negligenciadas. Cad. Saúde Pública, Rio de Janeiro. 2006.

STEFANI G. P., PASTORINO A. C., CASTRO A. P. B. M., FOMIN A. B. F., JACOB C. M. A. Insect repellents: recommendations for use in children. Rev Paul Pediatr 2009

SORGE F., IMBERT P., LAURENT C., MINODIER P., BANERJEE A., KHELFAOUI F. Children arthropod bites protective measures: insecticides and repellents. Arch Pediatr 2007

FRADIN M. S., DAY J. F. Comparative efficacy of insect repellents against mosquito bites. N Engl J Med 2002

CHEN-HUSSEY V., BEHRENS R., LOGAN J. G. Assessment of methods used to determine the safety of the topical insect repellent N,N-diethyl-m-toluamide (DEET). Parasit Vectors. 2014

ROBBINS P. J., CHERNIAK M. G. Review of the biodistribution and toxicity of the insect repellent N, N-diethyl-m-toluamide (DEET). J Toxicol Environ Health. 1986

OSIMITZ T. G., MURPHY J. V., FELL L. A., PAGE B. Adverse events associated with the use of insect repellents containing N, N-diethyl-m-toluamide (DEET). Regul Toxicol Pharmacol. 2010

MCGREADY R., HAMILTON K. A., SIMPSON J. A., CHO T., LUXEMBURGER C., EDWARDS R., LOOAREESUWAN S., WHITE N. J., NOSTEN F., LINDSAY S. W. Safety of the insect repellent N, N-diethyl-m-toluamide (DEET) in pregnancy. Am J Trop Med Hyg. 2001

SUN J. D-Limonene: safety and clinical applications. Altern Med Rev. sep 2007

KARLBERG T., BOMAN A., MELINJ B. animal experiments on the allergenicity of d-limonene—the citrus solvent. AM. omip Hrg.. Vol 35. No. 4. pp. 419-426. 1991

PURVIS J. E., YOMANO L. P., INGRAM L. O. Enhanced Treahalose Production Improves Growth of Escherichia coli under Osmotic Stress. Appl. Environ. Microbiol. Jul 2005

SHIMIZU K. Regulation Systems of Bacteria such as Escherichia coli in Response to Nutrient Limitation and Environmental Stresses. Metabolites. Mar 2014

MONEY P. N. Osmotic Pressure of Aqueous Polyethylene Glycols, Relationship between Molecular Weight and Vapor Pressure Deficit. Plant Physiol. 91, 766-769, 1989.