Screening of halotolerant glyphosate oxidases native of salt flats from northern Argentina

ZERDA MOREIRA, ANDREA; LOPEZ, MARIA BELÉN; OTERINO, MARÍA BELÉN; GONZALEZ, JAVIER M.

Buenos Aires

Workshop; Tercer Encuentro y Primer Workshop de la Red Argentina de Tecnología Enzimática; 2021

Red Argentina de Tecnología Enzimática

Glyphosate (N-phosphonomethylglycine)is one of the most widely used organophosphonate herbicides in the world andhas been strongly questioned for its toxic effects on humans. It is often used excessivelydue to the emergence of resistant weeds, leading to its presence in varied foods(fruits, vegetables, honey, etc.), cotton textiles, and other derivatives.Thus, cost-effective technologies for detection and remediation of glyphosate in situ are needed. Weexplore salt flats in northern Argentina in search for natural variants of the enzymeglycine oxidase (GO) from halophilic strains of Bacillus sp., exhibiting glyphosate oxidase(GLO) activity. Indeed, this flavoenzyme belongs to the family of D-aminooxidases (Pfam PF01266), and bacteria producing GO with residual GLO activityare capable of degrading glyphosate into AMPA (amino methyl phosphonic acid)and glyoxylate, which are further assimilated as phosphorus and carbon sources,respectively.We typically collect samples of saline soil and incubate them in high-saltmedia specific for growth of halophilic bacteria, enriched with increasing concentrationsof glyphosate, followed by environmental DNA (eDNA) extraction for PCR analysis.In this work, we sampled the Ambargasta salt flat in southwestern Santiago delEstero, Argentina, a saline plain of more than 9000 square kilometers, with someseasonal salty lagoons and streams that harbor halophilic microorganisms potentiallyadapted to metabolize xenobiotics present in the aquifers. Indeed, Ambargastaconstitutes a dead-end salt flat for rivers Salí-Dulce that carry waters acrossTucumán and Santiago del Estero, likely accumulating diverse herbicides andchemical effluents from crop irrigation and industrial waste. We propose thatsuch environment promotes the growth of halophilic microbes with the ability toassimilate those chemicals. Thus, we incubated Ambargasta soil samples in theabovementioned conditions, which were collected during the dry winter season, andmonitored growth of microorganisms harboring genes related to GO enzymes byPCR, using primers specific for halophilic Bacillussp. GO enzymes, and aliquots of eDNA as templates. While the total eDNA yieldwas comparable between samples and control cultures without glyphosate, theexpected PCR products appeared only in samples incubated with added glyphosatefor several days, as compared with control cultures without the herbicide, stronglyindicating that we amplified the correct genes. However, we faced the technicaldifficulty of cloning PCR products of unknown sequence, without flankingrestriction sites. We decided to avoid restriction cloning because such procedurecould potentially digest some GO-like genes, and we would therefore lose themor clone fragmented versions. Instead, we are currently adapting a pET-typeexpression vector for in-phase cloning of PCR products through the TA-cloningmethod, which will enable inducible expression of intact GO-like enzymes from alibrary of putative GO-encoding genes. These plasmids will be transformed into E.coli BL21(DE3) cells in order to screen for oxidase activity in vivo as production of hydrogenperoxide, by colony PCR, or by differential growth in media with and without expressioninducer (IPTG), using glycine or glyphosate as carbon sources.Our research will eventually allow us to isolateoxidases with GLO activity, which will be purified and characterized biochemicallyand biophysically. Since halotolerant enzymes are often resistant to denaturationdue to extremes of salinity, pH and temperature, halotolerant GLO enzymes will exhibitdesirable biophysical properties that make them ideal for biotechnological applications,which is our long-term goal.