Team:UFRGS Brazil/Description
Project Inspiration and Description - GlyFloat - Team iGEM UFRGS 2019
It is undeniable that, as population increases - and consequential increase in food demand -, food production in large scale becomes essential. As there is a limited amount of resources on the planet, agriculture and livestock also become limited, due to the requirements of resources such as water and area destined to food production [1]. Especially in agriculture, many efforts have been made in the last years to overcome these challenges. Tools such as artificial selection, pest control using a variety of chemicals. Genetic engineering and the search for new resources are examples of such advances [1] - [5].
Glyphosate (N-(phosphonomethyl)glycine) is a chemical compound - a synthetic phosphonate - largely used as active ingredient for many different pesticides. The molecule was discovered in 1950, but only revealed itself as a powerful herbicide in 1970 [6]. It is incorporated by the plants on their surfaces, diffusing in the plant’s cuticle and transported through the phloem to the same tissues considered sucrose drains [7]. In high concentrations (phytotoxic levels) the molecule can reach the plant’s meristems, leaves and young roots, just as any other growing tissue or organ [8]. Its mechanism of action (MOA) consists in the inhibition of the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) involved in the shikimate pathway. This enzymatic inhibition leads to shikimate accumulation and decrease in the production of several essential compounds to the plant, such as aromatic amino acids and proteins, hence causing its death [7] - [11]. Due to its low specificity, glyphosate is able to be applied in a large spectrum of different species, becoming a very powerful and effective herbicide.
Genetic engineering has made it possible to develop different plant strains, capable of resisting the presence of glyphosate. These glyphosate resistant strains are able to withstand the presence of the molecule without experiencing its harmful effects, which enables the use of this herbicide to efficiently eliminate weeds, without harming the desired cultivar, in a relatively easy, cheap and adaptable way, leading to productivity increases [12] - [13]. Moreover, the use between plantations can yield up to 30% higher harvests, as reported in a study in Europe [14]. As it is a major agrochemical, much has been put up to debate about the possible harmful effects caused by recurrent use of this chemical in nature. One of the main advantages of glyphosate is its relative low environmental impact when compared to other agrochemicals [15], besides the high sorbent capacity of these molecules in the soil, which makes it less prone to contaminate groundwater [8]. On the other hand, in the presence of rain, glyphosate can be carried over to superficial water bodies - such as lakes and rivers - , where the damage can end up being more significant.
Even though it is considered to be a “probable carcinogen in humans” (category 2A by the International Agency for Research on Cancer), several evidences do not indicate high risks of glyphosate to human health [16]; nevertheless, much remains to be investigated [17]. The ecological disbalance caused by the chemical compound is, undoubtedly, the main concern in the present moment. Studies show that: (i) in water bodies, the chronic exposure of animals to glyphosate culminates in several damage caused by its toxicity, ranging from developmental problems to infertility [18]; beyond the fact that (ii) selective pressure resulting from the presence of glyphosate in water may end up selecting microorganisms capable not only of resisting the cytotoxic potential of the molecule, but also having the capacity to use it as phosphorus source [19], [20], resulting in an adaptive advantage to these organisms, which may disbalance ou compromise the ecosystem. One of the practical examples of such harmful effects was shown by a canadian research group, that even low concentrations of a glyphosate based herbicide in water are capable of altering both structurally and functionally phytoplankton communities that naturally occur in rivers, illustrating the dangers of the presence of glyphosate in these water bodies [21]. Another shocking report was from a paper published in 2005, where the application - in different environments - of another agrochemical containing glyphosate as active ingredient led to tadpole’s death rate between 96 and 100% [22].
Both chemical and photolytic degradation of glyphosate are slow and not always effective, mainly due to the molecule’s relatively high stability [23]. Moreover, the biological degradation of the compound is much more efficient, and is currently present in nature. Many microorganisms are capable of assimilating, degrading, or modifying glyphosate. Among many different routes of metabolization and degradation of the molecule, the most well known are (i) those involving the glyphosate oxirreduction in aminomethylphosphonic acid (AMPA) and glyoxylate; (ii) and those comprising the enzymatic breakdown of the carbon-phosphorus bond - an enzyme known as C-P lyase -, in a process that generates sarcosine and phosphate. The main metabolite from biological degradation of glyphosate, AMPA, has several properties similar to glyphosate - toxic and ecotoxic effects -, also being found in superficial waters and on soil, though AMPA has a higher mobility on ground than glyphosate (the affinity for soil particles is not as strong as glyphosate) [24]. As it is also a harmful compound, AMPA becomes a problem; however, the activity of a C-P lyase enzyme capable of breaking down the carbon-phosphorus bond can decompose this acid into methylamine and phosphate. Interestingly, studies indicate that in bacteria, C-P lyase that disrupts glyphosate C-P bondis apparently different from that responsible for the breakdown of AMPA C-P bond [25]. As it is an excellent model for studies involving molecular biology, many studies about the C-P lyase have been made using E.coli (due to its simple laboratorial manipulation), showing that in this bacteria, the enzyme is encoded by a series of coding sequences from an operon (phnCDEFGHIJKLMNOP), and acts in synergy with other products encoded by the same set of sequences, which aid in the transport and assimilation of phosphonates, regulated by the regulon Pho. According to previous studies, the operon phnCDEFGHIJKLMNOP works as follows: the sequences phnCDE encode a phosphonate transporting structure; the sequence phnF encodes a repressor protein; sequences phnGHIJKLM encode the holoenzyme; and the sequences phnNOP are regulatory or accessories of the enzymatic function of the C-P lyase protein [25], [26]. When acting together, these genes are capable of conferring, in bacteria, the ability to use phosphonates as a source of phosphorus.
In Brazil, the sales of the compound have reached over 173 thousand tons in 2017 [27], which shows the economical importance as regarding the use of this product. Intuitively, its water levels in Brazil may reach staggering values, indicating a potent threat to our rich national biodiversity. Furthermore, brazilian law permits a residual glyphosate concentration in drinking water of 500μg / L [28], which is 5000 times higher than in the European Union.
References
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[28] Anvisa
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