Process and applications

The proposed process for the plasmolysis induction by polyethylene glycol is promising in many aspects; among them, the conservation of bacteria at ambient temperature, and also to aid in the process of solubilizing heterologous proteins. It happens because in a osmotic stress situation the bacteria, during the process of cytoplasmic reduction, produce diverse compounds capable to protect its proteins, from chaperones to foldases (SUSIN et al., 2006; MAGER et al., 2000; OGANESYAN et al., 2007; GUNASEKERA et al., 2008). They can aid in proper folding of hard expression proteins (PURVIS et al., 2005; SHIMIZU et al., 2013); conditions already demonstrated in our results, by the conciliation between our promoter responsible to stresses and proteins of hard expression, like limonene synthase.

Figure 1: Project module I comprising the partial circuit to limonene synthase expression as conceived.

Limonene

The use of terpenes in insect control emerged from the necessity of repellents less harmful to the environment and health when compared to synthetic compounds (IBRAHIM et al., 2001). Monoterpenes represent one of the largest and more diverse families of natural compounds, and occur in many forms: acyclic, monocyclics, bicyclics, tricyclics and derivatives (CROTEAU, 1987). They can still exist at hydrocarbon forms or oxygenated radicals, such as: aldehydes, alcohols, ketones, esters, ethers and other functional groups (WEIDENHAMER et al., 1993).

The monoterpenes more broadly distributed in the vegetable kingdom are the main component of volatile oils, responsible for the characteristic smell of many plants, like limonene in citric fruits (SCHUTTE, 1984).

Limonene, a hydrocarbon labeled as monocyclic terpene, is a chiral molecule, present at the environment in both enantiomeric forms D(+)–Limonene and L(-)–Limonene (FERRARINI et al., 2008). L-Limonene is found mainly in a variety of trees and herbs such as Mentha spp.; while D-Limonene is the main component of peel oil from Citrus species (DUETZ et al., 2003).

Figure 2: Limonene molecule.

Monoterpenes can be toxic to insects by its penetration through the cuticle; respiratory and digestive system (PRATES et al., 1998). There are indications that they act affecting octopamine receptors (KOSTYUKOVSKY et al., 2002). The insecticide effect of limonene was observed with success for insect control in domestic animals (TONELLI, 1987), as well in haematophagous insects, showing a DL50, on mosquitoes’ larvae, of 53.80 ppm after 24h and 32.52 ppm after 48 hours (KASSIR et al., 1989).

D-Limonene is considered a GRAS (Generally recognized as safe) compound, present at fruit juices, ice creams and sodas, as flavoring agent. It has a low toxicity in humans, even after repetitive exposures in a year. It is clinically used to dissolve gallstones containing cholesterol. Due to its neutralizing effect of gastric acid and assistance in normal peristalsis, it has also been used to relieve heartburn and acid reflux. Even after being experimentally proved that D-Limonene on its natural state does not exhibit significant reactions on human skin, the products of its oxidation does (SUN, 2007). Thinking of this, our objective is to produce limonene at the moment of its use, to not present any risks to health.

The limonene features anti-carcinogenic activity, by inhibiting isoprenylation. It acts by inhibiting the interaction of hydrophobic molecules with G protein, responsible for controlling cellular growth, such as p21ras (CROWELL & GOULD, 1994). With proteic isoprenylation the protein docking to the cellular membrane is facilitated, disabling it in a manner that the control of cellular growth is affected. The limonene also acts in others anti-carcinogenic pathways, like in apoptosis induction (TSUDA et al., 2004) and tumor cell differentiation (GOULD, 1997).

Other important feature is that limonene does not have any relations with sunburn, the damage caused by citric oils to the stratum corneum, by exposition to sun, is caused by the presence of furanocoumarins (JUNIOR, 2009).

Limonene Synthase

Limonene synthase is an enzyme of lyases family responsible to catalyze the reaction that produces limonene from geranyl pyrophosphate. The enzyme shows an optimum pH for its activity between 6-7, and its isoelectric point is at pH of 4,35. Its action mechanism requires \(Mn^{2+}\), but can be replaced by \(Mg^{2+}\). When there is one of these ions and geranyl pyrophosphate the enzyme is capable to catalyze the synthesis of D-limonene. Apparently the activity of this enzyme is inhibited by sodium phosphate or sodium pyrophosphate; also by the application of compounds against histidine, cysteine and methionine, suggesting that at least one of these residues are in the catalytic site (RAJAONARIVONY et al., 1992).

This enzyme was produced in Escherichia coli by Colby et al. (1993). However, the production of limonene was detected only with the use of the recombinant enzyme in vitro, not in vivo. The team of Wisconsin University exhibited, on iGEM 2012, a project to produce limonene using the limonene synthase with the amino acid sequence featured by Colby et al (1993). They produced limonene synthase, but not limonene. It is possible that the constitutive expression of the gene promoted interchain interactions, resulting in insoluble proteic clumps. Thus, possibly, a lower transcription rate would allow the process of folding to happen slowly, and this could provide the correct conformation for the protein.

Figure 3: Limonene synthase structure. For complete study acess the link.

This project aimed the elaboration of a genetic circuit seeking to overcome the problems related to the recombinant expression of limonene synthase and, for last, produce limonene.

Osmotic stresses represent a simple manner to induce the production of chaperones and foldases, which allows a better protein folding. The polyethylene glycol (PEG) represents an important molecule used to induce this condition (BOLEN et al., 2001). Therefore, there is obtained an efficient production control for the correct folding of limonene synthase, being expressed only by the condition of osmotic stress under the regulation of PuspA promoter. Once activated, it accurately coordinates the expression of limonene synthase.

Coupled to the previous process, we also seek to express a group of proteic chaperones, natural from Escherichia coli, to help the correct folding of limonene synthase. To avoid the use of antibiotics, the genetic circuit will be further integrated to the bacterial genome by the Tn7 system using Flip-Flop transposases.

UspA promoter

In order for the protein expression occurs only at favorable conditions, we will use the UspA promoter (universal stress global response regulator). The PuspA comprises a stress responsive promoter region; this response is related to the expression and translation of universal stress protein A, which characterizes an important element on cellular growth control (FAREWELL et al., 1996).

The promoter was selected using neural network prediction systems, such as Neural Network Promoter Prediction, and heuristics systems, like SCOPE and PePPER. Due the requirements of this project, it was not cloned completely promoting elements downstream.

Plasmolysis

The process of cellular volume retraction caused by the loss of water in a hypertonic medium is known as plasmolysis. In bacteria, the conformational change, eventually, is not noticed externally, in a way that the cellular membrane can lose total contact with the cell wall by the reduction of the cytoplasmic volume.

Figure 4: Plasmolysis. Extracted from the link.

The process of plasmolysis alters the cellular metabolism. In Escherichia coli the processes of transcription and translation are inhibited severely, leading to the reduction of other activities regarding the genetic material of the bacteria, and phospholipids synthesis, also inhibited, but in a lesser rate (RUBENSTEIN et al., 1970).

Polyethylene glycol 6000

Many osmolytes can be used to induce plasmolysis, whereas, depending on their characteristics, they can interact with in a greater or lesser extent with cellular components. One of the most used osmolyte to induce osmotic stress is the polyethylene glycol (PEG). The PEG has features based on its chemical structure that offers many forms of applications, in diverse areas such as: pharmacy, industry and alimentary (FRENCH et al., 2009). Its chemical composition and tridimensional arrangement gives hydrophilicity and low melting point, both variables according to the chain length. The solubility in water, methanol, ethanol and benzene makes it useful in many methodologies. The PEG exhibited to be efficient in inducing osmotic stress, mainly because its flexible and hydrophilic features creates high osmotic pressures, and its chemical arrangement make it less likely to interact with other biologic compounds present at manipulated organisms (MONEY et al., 1989).

PEG is a polymer of modifiable size. With increasing molecular weights it is observed an increasing viscosity. We have chosen PEG6000 because its lower viscosity allied to its cost effective nature, and keeping hability of plasmolysis induction.

Figure 5: Polyethylene glycol strucutre.

References

IBRAHIM M. A., PIRJO K., AFLATUNI A., TIILIKKALA K., HOLOPAINEN J. K. Insectidal, repellent, antimicrobial activity and phytotoxicity of essential oils: With special reference to limonene and its suitability for control of insect pests. Agricultural and Food Science in Finland, v. 10, p. 243 – 259, 2001;

CROTEAU R. Biosynthesis and catabolism of monoterpenoids. Chemical Reviews 87, 1987;

WEIDENHAMER, J. D., MACIAS, F. A., FISCHER, N. H., WILLIAMSON, G. B. Jus how insoluble are monoterpenes? Journal of Chemical Ecology 19: 1799-1807, 1993;

SCHUTTE H. R. Secondary plant substances. Monoterpenes. Progress in botany 46: 119-139, 1984;

FERRARINI S. R., DUARTE M. O., ROSA R. G., ROLIM V., EIFLER-LIMA V.L., POSER V., RIBEIRO G. V. L. Acaricidal activity of limonene, limonene oxide and amino alcohol derivatives on Rhipicephalus (Boophilus) microplus. Veterinay Parasitology, v.157, p.149-153, 2008;

DUETZ W. A., BOUWMEESTER H., BEILEN V. J. B., WITHOLT B. Biotransformation of limonene by bacteria, fungi, yeasts, and plants. Microbiol Biotechnol, v.61, p. 269-277, 2003;

PRATES H. T., LEITE R. C., CRAVEIRO A. A. Identification of some chemical components of the essential oil from molasses grass (Melinis minutiflora Beauv.) and their activity against catle-tick (Boophilus microplus). J. Braz. Chem. Soc., v.9, p.193-197, 1998;

KOSTYUKOVSKY M., RAFAELI A., GILEADI C., DEMCHENKO N., SHAAYA E. Activation of octopaminergic receptors by essential oil constituents isolated from aromatic plants: possible mode of action against insect pests. Pest Manag Sci. Nov 2002;

TONELLI E. A. Experimental use of d-limonene in dips for treatment of flea and tick infestations in small animals. Vet. Argentina, 4: 931-937, 1987;

KASSIR J.T., MOHSEN Z. H. MEHDI N. Toxic effects of limonene against Culex quinquefasciatus say larvae and its interference with oviposition. Anzeiger fur Schadlingskunde, 62: 19-21, 1989;

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

CROWELL P. L., GOULD M. N. Chemoprevention and therapy of cancer by d-limonene. Crit Rev Oncog. 1994;

TSUDA H., OHSHIMA Y., NOMOTO H., FUJITA K., MATSUDA E., IIGO M., TAKASUKA N., MOORE M. A. Cancer prevention by natural compounds. Drug Metab Pharmacokinet. Aug 2004;

GOULD M. N. Cancer chemoprevention and therapy by monoterpenes. Environ Health Perspect. Jun 1997;

JUNIOR J. P. S. Ação de furocumarinas associadas à ultravioleta B em Staphylococcus aureus. In: 25º Congresso Brasileiro de Microbiologia, Porto de Galinhas, PE, 2009;

COLBY, S. M.; ALONSO, W. R.; KATAHIRA, E. J.; MCGARVEY, D. J.; CROTEAU, R. 4S-Limonene synthase from the oil glands of spearmint (Mentha spicata): cDNA isolation, characterization and bacterial expression of the catalytically active monoterpene cyclase. J Biol Chem 268:23016–23024.

RUBENSTEIN K. E., NASS M. M., COHEN S. S. Synthetic capabilities of plasmolyzed cells and spheroplasts of Escherichia coli. J Bacteriol. Oct 1970;

FRENCH A. C., THOMPSON A. L., DAVIS B. G. High-Purity Discrete PEG- Oligomer Crystals Allow Structural Insight. Angew. Chem. Int. Ed. 2009, 48, 1248 –1252, 2009;

SUSIN M. F., BALDINI R. L., GUEIROS-FILHO F., GOMES S. L. GroES/GroEL and DnaK/DnaJ Have Distinct Roles in Stress Responses and during Cell Cycle Progression in Caulobacter crescentus. J Bacteriol. Dec 2006;

MAGER W. H., DE BOER A. H., SIDERIUS M. H., VOSS H. Cellular responses to oxidative and osmotic stress. Cell Stress Chaperones. Apr 2000; 5(2): 73-75;

OGANESYAN N., ANKOUDINOVA I., KIM S., KIM R. Effect of Osmotic Stress and Heat Shock in Recombinant Protein Overexpression and Crystallization. Protein Expr. Purif. Apr 2007; 52(2): 280-285;

GUNASEKERA T. S., CSONKA L. N., PALIY O. Genome-Wide Transcriptional Responses of Escherichia coli K-12 to Continuous Osmotic and Heat Stresses. J. Bacteriol. May 2008;

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.

FAREWELL, A.; DIEZ, A. A.; DIRUSSO, C. C.; NYSTROM, T. Role of the Escherichia coli FadR regular in stasis survival and growth phase-dependent expression of the uspA, fad, and fab genes. J. Bacteriol. 1996.