Optimization of liquid-state fermentation conditions for the glyphosate degradation enzyme production of strain Aspergillus oryzae by ultraviolet mutagenesis

This study aimed to obtain strains with high glyphosate-degrading ability and improve the ability of glyphosate degradation enzyme by the optimization of fermentation conditions. Spore from Aspergillus oryzae A-F02 was subjected to ultraviolet mutagenesis. Single-factor experiment and response surface methodology were used to optimize glyphosate degradation enzyme production from mutant strain by liquid-state fermentation. Four mutant strains were obtained and named as FUJX 001, FUJX 002, FUJX 003, and FUJX 004, in which FUJX 001 gave the highest total enzyme activity. Starch concentration at 0.56%, GP concentration at 1,370?mg/l, initial pH at 6.8, and temperature at 30°C were the optimum conditions for the improved glyphosate degradation endoenzyme production of A. oryzae FUJX 001. Under these conditions, the experimental endoenzyme activity was 784.15?U/100?ml fermentation liquor. The result (784.15?U/100?ml fermentation liquor) was approximately 14-fold higher than that of the original strain. The result highlights the potential of glyphosate degradation enzyme to degrade glyphosate.     

Physiological aspects of glyphosate degradation in Alcaligenes spec. strain GL

Alcaligenes spec. strain GL (IMET 11314) is able to grow on glyphosate (N-[phosphonomethyl]glycine) and other phosphonates as sole source of phosphorus. Degradation of glyphosate to inorganic phosphate and sarcosine by this strain is subject to several regulatory principles. While uptake and dephosphonation of glyphosate are regulated by P_i starvation, the intensity of glyphosate degradation is also controlled by the cellular ability to utilize the C-skeleton derived from glyphosate. Depending on the external concentration of glyphosate, the liberated sarcosine is differentially metabolised. Utilization of the sarcosine moiety and complete incorporation of 3-[¹⁴C]-label of glyphosate into cellular material occur only in cultures adapted to higher concentrations (5 mM) of the herbicide. At low concentrations of glyphosate (1 mM) only the P_i required by the growing cultures is utilized but not the sarcosine. Initially high rates of glyphosate uptake obtained after P_i-starvation decrease in the presence of low glyphosate concentrations. It is suggested that uptake and metabolism of glyphosate are connected with the expression of the sarcosine metabolizing capacity of the Alcaligenes cells.Abbreviation AMPA aminomethylphosphonic acid     

Glyphosate utilization byPseudomonas sp. andAlcaligenes sp. isolated from environmental sources

A total of 21 bacterial cultures were isolated that could utilize glyphosate (N-phosphonomethyl glycine) as a sole source of phosphorus in a mineral salts medium. Sources of inocula for enrichment cultures included aerobic digester liquid, raw sewage, trickling filter effluent, pesticide disposal pit liquid, and soil. Eleven cultures were identified asPseudomonas sp., one asPseudomonas stutzeri, and nine asAlcaligenes sp. Aminomethylphosphonic acid, the major metabolic intermediate of glyphosate degradation in soil, could also serve as a sole phosphorus source for all 21 isolates. Neither glyphosate nor aminomethylphosphonic acid could serve as carbon sources in mineral salts media. Experiments withPseudomonas sp. SG-1 (isolated from aerobic digester liquid) suggested that enzymatic activity responsible for glyphosate degradation was intracellular, inducible, and required the cofactors pyruvate and pyridoxal phosphate. The degradation pathway for glyphosate in this culture may be similar to that previously reported for aminoethylphosphonic acid.     

Effect of glyphosate contaminated feed on rumen fermentation parameters and in sacco degradation of grass hay and corn grain

Four rumen fistulated wethers were used to investigate the effect of glyphosate contaminated feed on rumen fermentation. The rations were based on corn silage, urea and a vitamin-mineral premix, either in the absence or presence of 0.77?g glyphosate per kg DM. Furthermore, rations were fed either with or without aromatic amino acid supplementation. During four periods of 28 days, sheep received each of the four dietary treatments according to a Latin square. After 14 days of adaptation rumen fermentation parameters (pH, ammonia, volatile fatty acids) were measured on day 15 over a five-hour period after the morning feeding. The remaining 13 days served for in sacco degradation studies with grass hay and corn grain. Ammonia (NH₃) and pH of rumen fluid were within the normal range for all dietary treatments (NH₃: 9.1 – 32.3?mmol·l^???1, pH: 6.2 – 6.7). Neither rumen fermentation parameters nor in sacco DM and NDF degradation of incubated feedstuffs were significantly affected by glyphosate, with or without aromatic amino acid supplementation. Kinetic profiles of the in sacco dry matter and NDF degradation of grass hay were almost identical for the dietary treatments.     

Bioremediation of glyphosate-contaminated soils

Based on the results of laboratory and field experiments, we performed a comprehensive assessment of the bioremediation efficiency of glyphosate-contaminated soddy-podzol soil. The selected bacterial strains Achromobacter sp. Kg 16 (VKM B-2534D) and Ochrobactrum anthropi GPK 3 (VKM B-2554D) were used for the aerobic degradation of glyphosate. They demonstrated high viability in soil with the tenfold higher content of glyphosate than the recommended dose for the single in situ treatment of weeds. The strains provided a two- to threefold higher rate of glyphosate degradation as compared to indigenous soil microbial community. Within 1–2 weeks after the strain introduction, the glyphosate content of the treated soil decreased and integral toxicity and phytotoxicity diminished to values of non-contaminated soil. The decrease in the glyphosate content restored soil biological activity, as is evident from a more than twofold increase in the dehydrogenase activity of indigenous soil microorganisms and their biomass (1.2-fold and 1.6-fold for saprotrophic bacteria and fungi, respectively). The glyphosate-degrading strains used in this study are not pathogenic for mammals and do not exhibit integral toxicity and phytotoxicity. Therefore, these strains are suitable for the efficient, ecologically safe, and rapid bioremediation of glyphosate-contaminated soils.     

Evidence for two distinct phosphonate-degrading enzymes (C-P lyases) in Arthrobacter sp. GLP-1

Arthrobacter sp. GLP-1 can utilize a wide range of organophosphonates as its sole source of phosphorus. The in-situ formation of sarcosine and methane from glyphosate and methanephosphonic acid respectively was studied. These two processes are differentially induced during phosphorus-deprivation. Methanephosphonic acid strongly inhibits glyphosate degradation (I₅₀ 10 M), but glyphosate has very little effect on methane generation (I₅₀ 150 mM). The pattern of inhibition by other organophosphonates and organophosphonate analogues is also very different for the two systems. Degradation of glyphosate and methanephosphonic acid therefore represent distinct processes.Abbreviations f.wt. fresh weight - MP-lyase methanephosphonate lyase     

Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide

Endophytic bacteria are ubiquitous in most plant species influencing the host fitness by disease suppression, contaminant degradation, and plant growth promotion. This endophytic bacterial community may be affected by crop management such as the use of chemical compounds. For instance, application of glyphosate herbicide is common mainly due to the use of glyphosate-resistant transgenic plants. In this case, the bacterial equilibrium in plant–endophyte interaction could be shifted because some microbial groups are able to use glyphosate as a source of energy and nutrients, whereas this herbicide may be toxic to other groups. Therefore, the aim of this work was to study cultivable and noncultivable endophytic bacterial populations from soybean (Glycine max) plants cultivated in soil with and without glyphosate application (pre-planting). The cultivable endophytic bacterial community recovered from soybean leaves, stems, and roots included Acinetobacter calcoaceticus, A. junii, Burkholderiasp., B. gladioli, Enterobacter sakazaki, Klebsiella pneumoniae, Pseudomonas oryzihabitans, P. straminea, Ralstonia pickettii,and Sphingomonassp. The DGGE (Denaturing Gradient Gel Electrophoresis) analysis from soybean roots revealed some groups not observed by isolation that were exclusive for plants cultivated in soil with pre-planting glyphosate application, such as Herbaspirillum sp., and other groups in plants that were cultivated in soil without glyphosate, such as Xanthomonas sp. and Stenotrophomonas maltophilia. Furthermore, only two bacterial species were recovered from soybean plants by glyphosate enrichment isolation. They were Pseudomonas oryzihabitans and Burkholderia gladioliwhich showed different sensibility profiles to the glyphosate. These results suggest that the application at pre-planting of the glyphosate herbicide may interfere with the endophytic bacterial communitys equilibrium. This community is composed of different species with the capacity for plant growth promotion and biological control that may be affected. However, the evaluation of this treatment in plant production should be carried out by long-term experiments in field conditions.     

Microorganisms associated with the retting of flax treated with the herbicide glyphosate

Greater numbers of fungi were isolated from stems of flax following treatment with the herbicide glyphosate than from untreated stems although there was also a general increase in numbers with time. The fungal genera isolated were the same from both treated and untreated plants, the most common being Cladosporium, Aureobasidium, Epicoccum and Botrytis with Cladosporium being predominant. Populations of bacteria were generally not enhanced after glyphosate treatment. Partial dew-retting of crops resulted in the reduction in numbers of Cladosporium compared with conventionally-treated flax. The increase in numbers of fungi was associated with an increase in retting. Overretting also occurred, particularly in discrete pale areas on the stem. These were associated with colonisation by Botrytis cinerea and the tensile strength of fibres from these areas was 15 times weaker than from surrounding darker areas of the stem. Light microscopy showed death of cells after glyphosate application, followed by invasion and degradation of the epidermis and cortex by fungi and other microorganisms. Fibre bundles were partially dissociated but the individual fibres remained largely intact as did the xylem and medulla.     

Aldo‐keto reductase enzymes detoxify glyphosate and improve herbicide resistance in plants

In recent years, concerns about the use of glyphosate‐resistant crops have increased because of glyphosate residual levels in plants and development of herbicide‐resistant weeds. In spite of identifying glyphosate‐detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo‐keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate‐mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1‐ or OsAKRI‐expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta.     

Glyphosate and Previous Crop Residue Effect on Deleterious and Beneficial Soil-borne Fungi from a Peanut–Corn–Soybean Rotations

Bean seedlings (Phaseolus vulgaris L.) were transplanted to soil with corn previous crop residue, peanut previous crop residue and no agricultural soil, and treated with a range of glyphosate concentrations. Trichoderma, Gliocladium, Fusarium and Pythium soil‐borne fungi populations were monitored during 24 days after glyphosate treatment to study the glyphosate and previous crop residue effects on these populations. In addition, those genera of soil‐borne fungi were tested to study in vitro toxicity to glyphosate. Independently of glyphosate concentration, the highest population of Trichoderma spp. and Gliocladium spp. were registered on soil with previous corn residue. Fusarium and Pythium populations increased proportionally to the increment of glyphosate concentration. No effect of glyphosate was founded on Trichoderma and Gliocladium populations. The in vitro study results indicated an inhibitory effect of glyphosate on mycelial grown of the most studied soil‐borne fungi.     

Degradation of glyphosate and other pesticides by ligninolytic enzymes

The ability of pure manganese peroxidase (MnP), laccase, lignin peroxidase (LiP) and horseradish peroxidase (HRP) to degrade the widely used herbicide glyphosate and other pesticides was studied in separate in vitro assays with addition of different mediators. Complete degradation of glyphosate was obtained with MnP, MnSO₄ and Tween 80, with or without H₂O₂. In the presence of MnSO₄, with or without H₂O₂, MnP also transformed the herbicide, but to a lower rate. Laccase degraded glyphosate in the presence of (a) 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), (b) MnSO₄ and Tween 80 and (c) ABTS, MnSO₄ and Tween 80. The metabolite AMPA was detected in all cases where degradation of glyphosate occurred and was not degraded. The LiP was tested alone or with MnSO₄, Tween 80, veratryl alcohol or H₂O₂ and in the HRP assay the enzyme was added alone or with H₂O₂ in the reaction mixture. However, these enzymes did not degrade glyphosate. Further experiments using MnP together with MnSO₄ and Tween 80 showed that the enzyme was also able to degrade glyphosate in its commercial formulation Roundup^® Bio. The same enzyme mixture was tested for degradation of 22 other pesticides and degradation products present in a mixture and all the compounds were transformed, with degradation percentages ranging between 20 and 100%. Our results highlight the potential of ligninolytic enzymes to degrade pesticides. Moreover, they suggest that the formation of AMPA, the main metabolite of glyphosate degradation found in soils, can be a result of the activity of lignin-degrading enzymes.     

Biodegradation of glyphosate by soil bacteria: Optimization of cultivation and the method for active biomass storage

Conditions for obtaining the active biomass of Ochrobactrum anthropi GPK 3 and Achromobacter sp. Kg 16, bacteria which are able to degrade the herbicide glyphosate (N-phosphonomethylglycine), were investigated. In the batch culture, degradation was most effective in the medium with pH 6.0–7.0 and aeration at 10–60% of air saturation supplemented with glutamate and ammonium chloride as sources of carbon and nitrogen, respectively. Due to the adaptation of the cells and induction of the relevant enzymatic systems, the inoculum grown in the presence of glyphosate exhibited 1.5–2-fold higher efficiency of xenobiotic degradation than that grown with other sources of phosphorus (orthophosphate and methylphosphonic acid). The efficiency of the toxicant decomposition increased with an increase in a specific load of glyphosate, which the cells were subjected to during the initial stage of growth. The specific load was regulated both by the initial cell concentration and the concentration of the phosphorus source, and the effect was probably determined by its availability to microorganisms. Storage of the liquid biopreparation as a paste with stabilizers (ascorbate, thiourea, and glutamate) at room temperature for 50 days resulted in high level of bacteria viability and a degrading activity approximately equal to that obtained when the bacteria were maintained on the agar medium containing glyphosate at 4°C with monthly transfers to the fresh culture medium.     

Pathway and rate-limiting step of glyphosate degradation by Aspergillus oryzae A-F02

Aspergillus oryzae A-F02, a glyphosate-degrading fungus, was isolated from an aeration tank in a pesticide factory. The pathway and rate-limiting step of glyphosate (GP) degradation were investigated through metabolite analysis. GP, aminomethylphosphonic acid (AMPA), and methylamine were detected in the fermentation liquid of A. oryzae A-F02, whereas sarcosine and glycine were not. The pathway of GP degradation in A. oryzae A-F02 was revealed: GP was first degraded into AMPA, which was then degraded into methylamine. Finally, methylamine was further degraded into other products. Investigating the effects of the exogenous addition of substrates and metabolites showed that the degradation of GP to AMPA is the rate-limiting step of GP degradation by A. oryzae A-F02. In addition, the accumulation of AMPA and methylamine did not cause feedback inhibition in GP degradation. Results showed that degrading GP to AMPA was a crucial step in the degradation of GP, which determines the degradation rate of GP by A. oryzae A-F02.     

Identification of the first glyphosate transporter by genomic adaptation

The soil bacterium Bacillus subtilis can get into contact with growth-inhibiting substances, which may be of anthropogenic origin. Glyphosate is such a substance serving as a nonselective herbicide. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which generates an essential precursor for de novo synthesis of aromatic amino acids in plants, fungi, bacteria and archaea. Inhibition of the EPSP synthase by glyphosate results in depletion of the cellular levels of aromatic amino acids unless the environment provides them. Here, we have assessed the potential of B. subtilis to adapt to glyphosate at the genome level. In contrast to Escherichia coli, which evolves glyphosate resistance by elevating the production and decreasing the glyphosate sensitivity of the EPSP synthase, B. subtilis primarily inactivates the gltT gene encoding the high-affinity glutamate/aspartate symporter GltT. Further adaptation of the gltT mutants to glyphosate led to the inactivation of the gltP gene encoding the glutamate transporter GltP. Metabolome analyses confirmed that GltT is the major entryway of glyphosate into B. subtilis. GltP, the GltT homologue of E. coli also transports glyphosate into B. subtilis. Finally, we found that GltT is involved in uptake of the herbicide glufosinate, which inhibits the glutamine synthetase.     

Degradation of the Phosphonate Herbicide Glyphosate by Arthrobacter atrocyaneus ATCC 13752

Of nine authentic Arthrobacter strains tested, only A. atrocyaneus ATCC 13752 was capable of using the herbicide glyphosate [N-(phosphonomethyl)glycine] as its sole source of phosphorus. Contrary to the previously isolated Arthrobacter sp. strain GLP-1, which degrades glyphosate via sarcosine, A. atrocyaneus metabolized glyphosate to aminomethylphosphonic acid. The carbon of aminomethylphosphonic acid was entirely converted to CO₂. This is the first report on glyphosate degradation by a bacterial strain without previous selection for glyphosate utilization as a source of phosphorus.     

The absorption, translocation and distribution of the herbicide glyphosate in maize expressing the CP-4 transgene

Maize (Zea mays L. var. Bonnie) transformed with a gene encoding a 5-enolpyruvylshikimate 3-phosphate synthase with altered sensitivity showed over 100-fold greater resistance to the herbicide glyphosate (N-[phosphonomethyl]glycine) in comparison with its non-transformed progenitor (parental control) at the third-leaf stage. Studies with [¹⁴C]-glyphosate at a dosage lethal to the parental control, but sublethal to the transgenic, revealed that a maximum of 45-65% of the applied dose was absorbed, with greater absorption occurring in transgenic plants. Translocation of glyphosate was closely related to its absorption (r value 0.956) with approximately 15% more of the applied dose being mobilized in transgenic plants than the parental controls. Analysis of electronic autoradiograms along the treated leaf lamina found discrete internal regions of glyphosate accumulation closely associated with the site of application. These regions contained lower amounts of glyphosate present in the treated leaf lamina was almost completely translocated in transgenic plants, while in the parental controls more remained and the leaf became necrotic. In both types of maize there was a small accumulation of herbicide in the tip region of the leaf which was not mobilized. Younger shoot tissues and roots were major sinks for translocated glyphosate accumulating approximately 25-40% of the applied dose depending upon treatment. In the parental control, equal amounts of glyphosate were found distributed between young shoot tissues and roots; while in transgenic plants, the young shoot tissue accumulated around three times more glyphosate than the roots. In both plant types, glyphosate was localized in the meristems and young, actively growing leaves. Specific glyphosate activity (the amount of glyphosate per unit dry weight of tissue) in the major sinks of the transgenic declined towards the end of the treatment period but remained relatively constant in the parental control. In conclusion, enhancing glyphosate resistance by genetic transformation influenced the absorption, translocation and distribution of this herbicide in whole plants.Keywords: Zea mays, glyphosate (N-[phosphonomethyl]-glycine), transgenic, absorption, translocation, source-sink.      

Prospects of in vivo 31P NMR method in glyphosate degradation studies in whole cell system

The degradation of the phosphonate herbicide glyphosate (N-phosphonomethylglycine) by four taxonomically distinct microorganisms was studied in vivo in whole cell system using phosphorus nuclear magnetic spectroscopy (³¹P NMR). The time-course of glyphosate metabolization in dense cell cultures was followed by means of ³¹P NMR up to 21 days after the addition. The results obtained by this non-invasive way confirmed that the cells of Spirulina platensis and Streptomyces lusitanus biodegrade herbicide. Moreover, phosphorus starvation influenced the rate of glyphosate degradation by S. platensis. On the other hand, the results of similar measurements in the cultures of green algae Chlorella vulgaris showed that this aquatic plant, however growing in the medium containing 1 mM of N-phosphonomethylglycine, did not seem to posses the ability of its biodegradation. Additionally, the use of this method allowed us to find the new fungal strain Fusarium dimerum, which is able to biodegrade and utilize the glyphosate as the sole source of phosphorus. The results of our studies on usefulness of in vivo ³¹P NMR for tracing glyphosate degradation in whole cell systems revealed that this non-invasive, one-step method, might be considered as a valuable tool in environmental biotechnology of organophosphonate xenobiotics.     

Bacterial glyphosate resistance conferred by overexpression of an <Emphasis Type="Italic">E. coli</Emphasis> membrane efflux transporter

Glyphosate herbicide-resistant crop plants, introduced commercially in 1994, now represent approximately 85% of the land area devoted to transgenic crops. Herbicide resistance in commercial glyphosate-resistant crops is due to expression of a variant form of a bacterial 5-enolpyruvylshikimate-3-phosphate synthase with a significantly decreased binding affinity for glyphosate at the target site of the enzyme. As a result of widespread and recurrent glyphosate use, often as the only herbicide used for weed management, increasing numbers of weedy species have evolved resistance to glyphosate. Weed resistance is most often due to changes in herbicide translocation patterns, presumed to be through the activity of an as yet unidentified membrane transporter in plants. To provide insight into glyphosate resistance mechanisms and identify a potential glyphosate transporter, we screened Escherichia coli genomic DNA for alternate sources of glyphosate resistance genes. Our search identified a single non-target gene that, when overexpressed in E. coli and Pseudomonas, confers high-level glyphosate resistance. The gene, yhhS, encodes a predicted membrane transporter of the major facilitator superfamily involved in drug efflux. We report here that an alternative mode of glyphosate resistance in E. coli is due to reduced accumulation of glyphosate in cells that overexpress this membrane transporter and discuss the implications for potential alternative resistance mechanisms in other organisms such as plants.     

Mutation by DNA shuffling of 5‐enolpyruvylshikimate‐3‐phosphate synthase from Malus domestica for improved glyphosate resistance

A new 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate‐tolerant plants. However, wild‐type MdEPSPS is not suitable for the development of transgenic glyphosate‐tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate‐resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site‐directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single‐site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate‐resistant crops.     

Degradation of the Herbicide Glyphosate by Members of the Family Rhizobiaceae

Several strains of the family Rhizobiaceae were tested for their ability to degrade the phosphonate herbicide glyphosate (isopropylamine salt of N-phosphonomethylglycine). All organisms tested (seven Rhizobium meliloti strains, Rhizobium leguminosarum, Rhizobium galega, Rhizobium trifolii, Agrobacterium rhizogenes, and Agrobacterium tumefaciens) were able to grow on glyphosate as the sole source of phosphorus in the presence of the aromatic amino acids, although growth on glyphosate was not as fast as on P_i. These results suggest that glyphosate degradation ability is widespread in the family Rhizobiaceae. Uptake and metabolism of glyphosate were studied by using R. meliloti 1021. Sarcosine was found to be the immediate breakdown product, indicating that the initial cleavage of glyphosate was at the C—P bond. Therefore, glyphosate breakdown in R. meliloti 1021 is achieved by a C—P lyase activity.