Method for improving yield of L-phosphinothricin

Abstract

The invention relates to a method for improving the yield of L-phosphinothricin. Compositions of L-phosphinothricin and methods for preparing L-phosphinothricin are provided. The process comprises the conversion of racemic glufosinate-ammonium to an L-glufosinate-ammonium enantiomer or the conversion of PPO to L-glufosinate-ammonium in an efficient manner. Accordingly, the methods described herein comprise converting or eliminating alpha keto acid or ketone byproducts to additional products to move the equilibrium to L-phosphinothricin.

Description

[0001] This application is a divisional application of PCT application PCT / US2019 / 049467, filed on September 4, 2019, with the invention name “Method for Improving L-phosphinothricin Yield”. The date on which the PCT application entered the Chinese national phase is March 4, 2021, and the application number is 201980058685.6.

[0002] Cross-reference to prior applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 727,322, filed on September 5, 2018, the entire contents of which are incorporated herein by reference.

[0004] field

[0005] Described herein is a process for preparing L-phosphinothricin (glufosinate) in high yield.

[0006] background

[0007] The herbicide phosphinothricin is a non-selective, foliar herbicide, and from the viewpoint of toxicology or environment, it is considered to be one of the safest herbicides. The commercial chemical synthesis method of phosphinothricin currently produces the racemic mixture of L- and D-phosphinothricin (Duke et al., 2010Toxin 2:1943-1962). However, L-phosphinothricin (also referred to as phosphinothricin or (S)-2-amino-4-(hydroxy (methyl) phosphinothricin) butyric acid) is more effective than D-phosphinothricin (Ruhland et al. (2002) Environ.Biosafety Res.1:29-37).

[0008] Therefore, there is a need for a method for preparing only or primarily the active L-phosphinothricin form. Although many methods for producing pure L-phosphinothricin or a mixture of D- and L-phosphinothricin enriched with L-phosphinothricin have been described, there is no cost-effective method for producing high yields of L-phosphinothricin from racemic phosphinothricin.

[0009] Overview

[0010] A new and cost-effective method for preparing L-phosphinothricin with high yield is described herein. The method includes converting racemic phosphinothricin to L-phosphinothricin enantiomers. In particular, the method includes using L-glutamate, racemic glutamate or another amine source as an amine donor to specifically aminate PPO (2-oxo-4-(hydroxy(methyl)phosphonyl)butyric acid) to L-phosphinothricin. PPO can be obtained by oxidative deamination of D-phosphinothricin to PPO or produced by chemical synthesis. PPO is then converted to L-phosphinothricin using a transaminase in the presence of an amine donor. When the amine donor provides amine to PPO, L-phosphinothricin and reaction byproducts are formed. For example, when the amine donor is a glutamate compound (including a racemic glutamate compound or an L-glutamate compound), L-phosphinothricin and reaction byproduct α-ketoglutarate (KG), also known as oxoglutarate, are produced.

[0011] The transamination reaction is an equilibrium reaction, which means that under certain conditions, when the reaction is in equilibrium, some PPO will remain. The remaining PPO represents the yield loss of L-phosphinothricin, so it is desirable to minimize the amount of PPO remaining in the reaction mixture. Degradation, other chemical modifications, extractions, separations, combinations or other methods that reduce the effective concentration of byproducts (i.e., α-keto acids or ketones corresponding to the selected amine donor) will move the reaction equilibrium to L-phosphinothricin, thereby reducing the amount of PPO and increasing the yield of L-phosphinothricin. Therefore, the methods described herein include converting α-keto acids or ketone byproducts into other products or eliminating them to move the equilibrium to L-phosphinothricin. α-keto acids or ketone byproducts can be converted by enzymes or chemical methods. α-keto acids or ketone byproducts can be reduced or eliminated by methods such as ion exchange, size exclusion or other resins. For example, when L-glutamic acid compounds are used as amine donors, by adding ketoglutarate decarboxylase (KGD) to the reaction, KG byproducts can be converted into succinic semialdehyde (SSA). The reduction of the amount of KG that can be used for being converted by transaminase can cause the higher productive rate of L-phosphinothricin.If product is produced by reducing the amount of by-product or eliminating by-product, it is referred to as conversion product.For example, if KG is by-product and KGD is used to reduce the amount of KG or eliminate KG, then conversion product is SSA.

[0012] The compositions described herein may include L-phosphinothricin and a conversion product. As described above, when a glutamic acid compound is used as an amine donor, KG is a byproduct and SSA is a conversion product. In such compositions, L-phosphinothricin is present in a molar amount of 0.5 to 1,000 times the molar amount of SSA or another conversion product.

[0013] Other compositions include L-phosphinothricin, PPO and SSA. In such compositions, PPO is present at less than 10% of the amount of L-phosphinothricin, and the conversion product is present in a molar amount of up to 2 times the amount of L-phosphinothricin.

[0014] Also described herein is a method for selectively controlling weeds. In one embodiment, the method can be used to control weeds in a field of a crop containing planted seeds or a crop optionally resistant to phosphinothricin. The method includes applying an effective amount of a composition comprising an L-phosphinothricin with an enantiomeric excess of greater than 90% relative to D-phosphinothricin to the field. Such methods of use include selectively controlling weeds in the field, controlling weeds in non-field areas, defoliating plants or crops, and / or drying crops before harvest, including applying an effective amount of a composition comprising an L-phosphinothricin with an enantiomeric excess of greater than 90% relative to D-phosphinothricin and greater than 0.01% but less than 10% PPO and less than about 10% ketone byproducts. In one embodiment, the composition comprises greater than 90% L-phosphinothricin, less than 10% PPO and less than 10% ketone byproduct conversion products (relative to the amount of L-phosphinothricin). In some embodiments, the ketone byproduct conversion product is SSA.

[0015] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram of an exemplary conversion of racemic phosphinothricin to L-phosphinothricin using an L-glutamate compound as an amine donor and ketoglutarate decarboxylase (KGD) as a method to reduce the amount of KG.

[0017] Figure 2 yes Figure 1 Schematic diagram of the reaction, if the selected aminotransferase (TA) is capable of converting L-glutamate and SSA into KG and γ-aminobutyric acid (GABA); if the TA is capable of performing this conversion at a high rate relative to the conversion of PPO and L-glutamate to KG and L-phosphinothricin, the method will not produce high levels of L-phosphinothricin.

[0018] Figure 3A -C shows the use of DAAO enzyme and TA enzyme, two starting concentrations of L-glutamate compound, no KGD enzyme (300mM L-glutamate compound ( Figure 3A )) and KGD enzyme (300mM L-glutamate compound ( Figure 3B ) or 150 mM L-glutamate compound ( Figure 3C)) Plot of the concentrations of L-phosphinothricin (black circles, black thick line), D-phosphinothricin (light gray circles, light gray solid line), PPO (dark gray diamonds, dark gray solid line), L-glutamic acid compounds (black triangles, black dashed line) and KG (dark gray squares, dark gray dashed line) during the racemization of racemic phosphinothricin. Detailed Description

[0019] Provided are compositions of L-phosphinothricin (also known as phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphono)butyric acid) and methods for preparing L-phosphinothricin. In particular, the methods described herein increase the yield of L-phosphinothricin or simplify the separation of L-phosphinothricin from a reaction mass. When the raw material for preparing substantially pure L-phosphinothricin is racemic phosphinothricin, the method comprises a three-step process, which can be carried out in a single container and almost simultaneously, in multiple containers and sequentially, or in multiple containers and almost simultaneously.

[0020] U.S. Pat. No. 9,834,802 (incorporated herein by reference) describes a two-step process for preparing L-phosphinothricin, comprising reacting D-phosphinothricin with D-amino acid oxidase (DAAO) to form PPO (2-oxo-4-(hydroxy(methyl)phosphonyl)butyric acid); then aminating PPO to L-phosphinothricin by transaminase (TA) using an amine group from one or more amine donors. The reaction of PPO with the transaminase results in the production of L-phosphinothricin and the corresponding alpha keto acid or ketone as a byproduct, depending on the selected amine donor. Suitable amine donors include L-aspartate or racemic aspartate, L-glutamate or racemic glutamate, L-alanine or racemic alanine, L-1-phenylethylamine or racemic 1-phenylethylamine, L-phenylalanine or racemic phenylalanine, phenethylamine, L-glycine or racemic glycine, L-lysine or racemic lysine, L-valine or racemic valine, L-serine or racemic serine, L-glutamine or racemic glutamine, isopropylamine, sec-butylamine, ethanolamine, 2-aminobutyric acid and diaminopropionic acid.

[0021] The present method adds a third step to the method described in the above-mentioned U.S. Pat. No. 9,834,802, which includes reducing the effective level of the alpha keto acid byproduct produced in the second step of the amination of PPO to L-phosphinothricin. The byproducts of the second step can be removed by enzyme or chemical conversion, by physical such as ion exchange, size exclusion, etc. Without being bound by theory, it is believed that by reducing the effective level of the byproducts of the second step, the second reaction can continue at a higher rate and / or reach an equilibrium concentration, which leads to a higher yield of L-phosphinothricin. By combining these reactions, the proportion of L-phosphinothricin can be significantly increased in a racemic phosphinothricin mixture. Therefore, a method for obtaining a composition substantially comprising L-phosphinothricin is provided herein. For the purposes of the methods and compositions described herein, the composition substantially comprising L-phosphinothricin contains less than 10% PPO, less than 5% PPO, less than 2% PPO, less than 1% PPO, contains at least 0.01% PPO or does not contain detectable PPO (relative to the amount of L-phosphinothricin). In addition, the composition contains a certain amount of alpha-keto acid or ketone byproducts (e.g., KG). For the purpose of the methods and compositions described herein, the composition comprising L-phosphinothricin substantially contains less than 50% by-products, less than 25% by-products, less than 10% by-products, less than 5% by-products, less than 2% by-products, less than 1% by-products, or does not contain detectable by-products (relative to the amount of L-phosphinothricin). The reduction of the amount of by-products causes the level of the conversion product to increase. Therefore, the composition can comprise at least 5% of the conversion product, at least 10% of the conversion product, at least 25% of the conversion product, at least 50% of the conversion product, at least 75% of the conversion product, at least 100% of the conversion product or at least 200% of the conversion product (relative to the amount of L-phosphinothricin). Since L-phosphinothricin is more effective than D-phosphinothricin, as a herbicide, a smaller amount of the composition is required to be effective.

[0022] The methods described herein provide an effective method for converting PPO into L-phosphinothricin, particularly when transaminases are used in the reaction. As discussed, transaminases produce L-phosphinothricin and alpha ketoacids or ketone byproducts. The methods described herein provide for reducing alpha ketoacids or ketone byproducts to drive the reaction toward L-phosphinothricin, reducing the amount of PPO. Many amine donors can be used in the reaction. If a glutamic acid compound or an L-glutamic acid compound is an amine donor, the byproduct will be alpha ketoacid KG. If isopropylamine is an amine donor, the byproduct will be ketoacetone. If alanine is an amine donor, the byproduct will be alpha ketoacid pyruvic acid compound (pyruvate). If sec-butylamine is an amine donor, the byproduct will be ketone 2-butanone. If phenylethylamine is an amine donor, the byproduct will be ketone acetophenone. If lysine is an amine donor, the byproduct will be alpha ketoacid caproic acid or semialdehyde 2-aminoadipic acid-6-semialdehyde. If the aspartate compound is the amine donor, the byproduct will be the alpha keto acid oxaloacetate compound. If glycine is the amine donor, the byproduct will be the alpha keto acid glyoxylate compound. If valine is the amine donor, the byproduct will be the alpha keto acid alpha ketoisovalerate. If serine is the amine donor, the byproduct will be the alpha keto acid 3-hydroxypyruvate compound. If glutamine is the amine donor, the byproduct will be the alpha keto acid 2-oxoglutaramate compound. If ethanolamine is the amine donor, the byproduct will be the aldehyde glycolaldehyde. If 2-aminobutyric acid is the amine donor, the byproduct will be the alpha keto acid 2-oxobutyric acid. If diaminopropionic acid is the amine donor, the byproduct will be the alpha keto acid 3-amino-2-oxopropionic acid or the semialdehyde 3-oxoalanine.

[0023] In one embodiment, the first step is catalyzed by D-amino acid oxidase (DAAO), and the second step is catalyzed by transaminase (TA), and the glutamate compound is utilized by the TA enzyme as an amine donor. The byproduct produced is an alpha-ketoglutarate compound (KG). In this embodiment, ketoglutarate decarboxylase (KGD) can be used, for example (EC 4.1.1.71). Without limitation, SEQ ID NO: 1 to SEQ ID NO: 5 are exemplary KGD enzymes. Ketoglutarate decarboxylase converts KG into carbon dioxide and succinic semialdehyde (SSA). Figure 1 A schematic diagram of this exemplary reaction is included.While this method can be used to prepare substantially purified L-phosphinothricin in a batch reaction, it will be appreciated that a continuous process can be used.

[0024] One embodiment described herein is a composition comprising a mixture of L-phosphinothricin and SSA. Initially, the amount of L-phosphinothricin will be much higher than the amount of SSA. As the reaction proceeds, the amount of SSA increases, and can reach the same high as the amount of L-phosphinothricin, or even higher. The composition of L-phosphinothricin can include L-phosphinothricin and SSA, wherein the molar amount of L-phosphinothricin is 0.5 times to 1,000 times the amount of SSA. These compositions can optionally exist as dry powder or be dissolved in an aqueous or non-aqueous carrier, and other chemicals can optionally be present. Optionally, the composition is prepared and used in an ex vivo environment.

[0025] It is also recognized that L-phosphinothricin can be further separated and used as a herbicide in formulations. In many reactions known in the art to produce L-phosphinothricin, the reaction mixture contains many other components, usually multiples of significant levels relative to L-phosphinothricin. The complexity of these mixtures makes it difficult and / or expensive to separate L-phosphinothricin. In the case of the reaction described in U.S. Patent No. 9,834,802, the reaction mixture generally contains L-phosphinothricin, KG, PPO and glutamic acid. Separating L-phosphinothricin in the reaction mixture generally requires multiple operations because the chemical structures and chemical properties of these components are very similar. L-Glutamic acid is a major challenge because it exists in high concentrations relative to L-phosphinothricin and is structurally similar to L-phosphinothricin. In International PCT Patent Application PCT / US2018 / 042503, "Methods for Purifying L-phosphinothricin" (incorporated herein by reference in its entirety), improved separation methods are described, but simpler separations would be preferred. It will be appreciated that the reactions performed herein may have lower levels of KG, PPO, and glutamate, and higher levels of L-phosphinothricin, than the reactions described in US Pat. No. 9,834,802, and that this may simplify the isolation of L-phosphinothricin.

[0026] I. Synthesis Method

[0027] Provided is a method for converting D-phosphinothricin to L-phosphinothricin in an ex vivo environment with high yields. The methods described herein provide a means to convert a low-cost feedstock of a racemic mixture of D- and L-phosphinothricin into a more valuable product that is already enriched in L-phosphinothricin.

[0028] Figure 1 Shown is an exemplary schematic diagram of an embodiment as described herein.In this example, DAAO enzyme, TA enzyme and ketoglutarate decarboxylase (KGD) are combined, optionally combined with catalase, to catalyze several reactions that cause enrichment of L-phosphinothricin from racemic phosphinothricin.These reactions can occur in one or more than one reaction vessel, including in batches or flow reaction mode.These enzymes can be added as needed simultaneously (including by being immobilized on a solid support) or at different times.

[0029] if Figure 1 The reaction shown does not include a KGD enzyme, then at the beginning of the reaction, about 3-4 times the moles of glutamate compounds are required to fully favor L-phosphinothricin, so that the yield loss to PPO is less than 10% (see U.S. Patent No. 9,834,802). When the effective level of KG available to TA is reduced, as in the method described herein, the balance is more biased towards L-phosphinothricin. In fact, if the KGD enzyme can completely and solely catalyze the decarboxylation of KG to SSA, and if SSA is not a substrate for the TA enzyme, then the complete conversion of PPO to L-phosphinothricin can be obtained with a mole of glutamate compounds equal to the mole of PPO (which is half the mole of racemic phosphinothricin before the oxidative deamination of D-phosphinothricin catalyzed by the DAAO enzyme). The reaction can completely consume the input glutamate compounds, i.e., the byproduct KG, and convert all the produced PPO into L-phosphinothricin. The final reaction material may contain 0.5 times the molar amount of SSA of L-phosphinothricin, and no other major organic non-enzymatic reaction components. This simplified reaction material will obviously provide advantages for the purification and isolation of L-phosphinothricin.

[0030] Ketoglutarate decarboxylase (KGD) (EC 4.1.1.71) catalyzes the conversion of KG to carbon dioxide and SSA. KGD enzymes are well known in the art and can be found in a wide range of organisms, including M. smegmatis, S. apiospermum, M. maritypicum, A. enclensis, and K. phytohabitans. Without limitation, SEQ ID NO: 1 to SEQ ID NO: 5 are exemplary KGD enzymes. In a preferred embodiment, the KGD enzyme is based on a KGD enzyme from A. enclensis. Since KG is an α-keto acid and PPO is an α-keto acid, and decarboxylation occurs on the same terminal carboxylic acid of these relatively similar molecules, it is preferred to utilize a KGD enzyme that can rapidly enzymatically decarboxylate KG but cannot efficiently catalyze the decarboxylation of PPO. Various enzyme improvement techniques well known in the art are used to improve the efficiency of KG decarboxylation catalysis, reduce the efficiency of PPO decarboxylation catalysis, or both. KGD enzymes are examples of a broader class of carboxyl lyases, as indicated by all EC numbers starting from 4.1.1, and any of those carboxyl lyases may represent enzymes capable of reducing the amount of byproducts and providing the advantages described herein.

[0031] In some embodiments, SSA is separated from the reaction mass. This can preferably avoid undesirable reactions of SSA with other reaction components, to increase the conversion of KG to SSA, or to start purification and separation of L-phosphinothricin. SSA can be removed by methods known in the art, such as being attached to a resin (e.g., an amine resin, an ion exchange resin, a size exclusion resin or other resin), converted into an oxime (e.g., with a hydroxylamine derivative) and optionally precipitated, reduced (e.g., with NaBH4), polymerized or oxidized. SSA prepared by the KGD enzyme can be further oxidized to succinic acid, which is an important chemical intermediate.

[0032] The purpose of decarboxylating KG to SSA is to reduce the effective concentration of KG available to TA, thereby shifting the reaction equilibrium from PPO and glutamate compounds to L-phosphinothricin and KG. However, it is possible that SSA can be utilized by enzymes other than KGD enzyme in the reaction mass. In fact, the reaction naturally catalyzed by the E. coli gabT TA enzyme commonly used in U.S. Patent 9,834,802 is to convert SSA and L-glutamate compounds into KG and gamma-aminobutyric acid (GABA). Figure 2 Shown is the schematic diagram of this reaction as a part of whole L-phosphinothricin racemization reaction.Not bound by theory, it is believed that when this type of reaction is combined with the reaction catalyzed by KGD enzyme, it will eventually lead to the consumption of glutamate compounds and the accumulation of GABA. This may lead to the increase of PPO level and the reduction of L-phosphinothricin level, which is contrary to purpose as described herein.

[0033] In a preferred embodiment, a TA enzyme or an enzyme mutant or variant that is ineffective in utilizing the conversion product produced by reducing or eliminating the byproduct produced when L-phosphinothricin is aminated is used. Mutant variants of gabT have been described that exhibit different affinities for substrates including SSA (Liu et al., Biochemistry 2005, 44, 2982-2992). In a preferred embodiment, a TA enzyme that is ineffective in catalyzing the conversion of SSA and glutamate compounds to KG and GABA, but is effective in catalyzing the conversion of PPO and glutamate compounds to KG and L-phosphinothricin is used. In a more preferred embodiment, the E211S mutation in the E. coli gabT TA enzyme is used (SEQ ID NO: 6 is a wild-type TA enzyme with a leader sequence, and SEQ ID NO: 7 is an E211S mutant TA enzyme with a leader sequence). Sequence homology can be used to identify enzymes or mutations that are expected to confer desired substrate specificity. For example, the top 100 sequences found by blastp searching the NCBI non-redundant sequence database on August 31, 2018, against the wild-type E. coli gabT TA enzyme sequence, excluding all hits from Enterobacteraceae, yielded sequences from six genera. Although the sequence identity among these hits was as low as 76% compared to the E. coli gabT TA enzyme sequence (GenBank accession numbers CUJ92856.1 was 100%, KKF68526.1 was 91%, WP_061553513.1 was 89%, WP_025801403.1 was 89%, WP_074941369.1 was 76%, and PRD29013.1 was 76%), when cobalt alignment was used, all contained glutamic acid at the position equivalent to position E211 in the E. coli sequence, and therefore would be expected to exhibit the desired activity if the glutamic acid was converted to serine.

[0034] Structural homology can also be used to identify amino acids in the active site of TA enzymes that play a similar coordinating role as that played by E211 in the E. coli gabT enzyme and are therefore expected to confer similar changes in substrate specificity when mutated. Phylogenetically diverse collections of TA enzymes can be screened to identify those that efficiently catalyze the desired reaction (conversion of PPO to L-phosphinothricin) and those that are less efficient in catalyzing undesirable reactions (accepting conversion products as substrates, such as SSA). Thus, TA enzymes having a serine at a position equivalent to E211 when aligned to E. coli gab T TA (SEQ ID NO: 6) can be used in the methods described herein. It is also useful to use a variety of enzyme improvement techniques well known in the art to further improve desired activities, reduce undesirable activities, or both.

[0035] Other methods of reducing KG levels include non-enzymatic oxidation by various methods, such as hydrogen peroxide, enzymatic or non-enzymatic reduction, and degradation catalyzed by ethylene synthase. In CN107119084A, a method of reducing KG using ethylene synthase is described, which catalyzes the conversion of 3KG, 3O2, and 1 arginine into 2 ethylene, 1 succinate, 1 guanidine, 1 pyrroline-5-carboxylate, 7 carbon dioxide, and 3H2O. Although the use of ethylene synthase can reduce the effective amount of KG, it requires the addition of several co-substrates and produces many products that must be removed from the reaction mass.

[0036] Higher yields of L-phosphinothricin can also be achieved by enzymatic decarboxylation of α-ketoglutarate compounds to succinate compounds (succinic acid) by α-ketoglutarate-dependent dioxygenases. These iron-containing enzymes catalyze a wide range of oxidation reactions using oxygen and α-ketoglutarate compounds as co-substrates. Many of these enzymes also require ascorbic acid (vitamin C) as a reducing agent.

[0037] Taurine dioxygenase (EC 1.14.11.17) is an enzyme that converts α-ketoglutarate to succinate and carbon dioxide, while also converting taurine and oxygen to sulfite and aminoacetaldehyde. Xanthine dioxygenase (EC 1.14.11.48) is another enzyme that converts α-ketoglutarate to succinate and carbon dioxide, while also converting xanthine and oxygen to urate.

[0038] Using these dioxygenases, the α-ketoglutarate compound is converted to a succinate compound (succinate), which, unlike the succinate semialdehyde produced by KGD, is not a substrate for any other enzyme in the process.

[0039] If an amine donor other than a glutamate compound or an L-glutamate compound is used, a variety of other methods can be used to reduce the amount of by-products. Those known in the art include spontaneous decarboxylation of oxaloacetic acid compounds (if aspartic acid compounds are amine donors, then by-products) as described in U.S. Patent No. 6,936,444, and, although not used to prepare L-phosphinothricin, decarboxylation of pyruvic acid compounds (if alanine is an amine donor, then by-products) as described in U.S. Patent No. 9,074,228. If certain amine donors other than a glutamate compound or an L-glutamate compound are used, decarboxylases can also be used to reduce by-products. For example, branched-chain keto acid decarboxylase can be used to decarboxylate 2-oxobutyrate (a byproduct if 2-aminobutyrate is the amine donor) and alpha-ketoisovalerate (a byproduct if valine is the amine donor) (Gocke, D., Nguyen, CL, Pohl, M., Stillger, T., Walter, L. & Müller, M., Advanced Synthesis & Catalysis, 2007, 349, 1425-1435). Other methods for reducing the amount of byproducts include using chemical or enzymatic methods to reduce or oxidize carboxylic acids, aldehydes or ketones, such as glycolaldehyde dehydrogenase can be used to catalyze the reduction of glycolaldehyde (a byproduct if ethanolamine is the amine donor).

[0040] The effective level of KG exposed to the TA enzyme can also be reduced by isolating the KG from the TA enzyme and, optionally, from the rest of the reaction mass. In one embodiment, a solid substrate having an affinity for KG is present in or added to the reaction vessel. This can include ion exchange resins, affinity resins, size exclusion resins, or other resins or substrates. In another embodiment, the reaction mass is removed from the presence of the TA enzyme and exposed to the solid substrate. For example, the reaction mass can be filtered from the main reaction vessel and applied to a resin that preferentially binds KG. Then, the reaction mass containing a reduced amount of KG can be exposed to the TA enzyme again. Single, repeated, or continuous removal of KG from the reaction mass can shift the equilibrium of the reaction catalyzed by the TA enzyme toward L-phosphinothricin.

[0041] Several DAAO enzymes are known in the art and can be used in the methods described herein, as long as they can accept D-phosphinothricin as a substrate and provide activity at a level sufficient to drive the reaction. The DAAO enzymes that can be used in the methods described herein have an activity equal to or greater than about 3 μmol / min*mg, greater than about 4 μmol / min*mg or higher. Wild-type enzymes can be used in the methods described herein, as long as the enzyme has an activity level as described above. Such DAAO enzymes that can be used in the method include those modified to increase activity from Rhodosporidium toruloides, Trigonopsis variabilis, Fusarium sp, Candida sp, Schizosaccharomyces sp, Verticillium sp, Neolentinus lepideus, Trichoderma reesei, Trichosporonoleaginosus, etc. Any DAAO enzyme can be used as a starting enzyme, including those having sequences corresponding to Swissprot accession numbers P80324, Q99042, P00371, and P24552 or SPTREMBL numbers Q9HGY3 and Q9Y7N4 or GenBank numbers KZT28066.1, XP_006968548.1, and KLT40252.1. The DNA sequence encoding DAAO can be selected from the sequences listed in EMBL accession numbers A56901, RGU60066, Z50019, SSDA04, D00809, AB042032, RCDAAOX, A81420, and SPCC1450, or can be codon optimized from the above protein sequences for optimal expression in a selected expression host. U.S. Patent No. 8,227,228 describes a DAAO enzyme from Candida intermedia. Such sequences are incorporated herein by reference. These enzymes can be modified to increase activity and used in the methods described herein.

[0042] Other DAAO enzymes can be identified in a variety of ways, including sequence similarity and functional screening. The DAAO enzyme can be a mutant DAAO enzyme that can accept D-phosphinothricin as a substrate. In Hawkes et al. (see above), a mutant DAAO (composed of F58K and M213S mutations) based on a sequence from Rhodosporidium toruloides has been shown to accept D-phosphinothricin as a substrate (Hawkes et al. (2011), Plant Biotechnol J.9 (3): 301-14). Other DAAO enzymes can be similarly modified to accept D-phosphinothricin and have greater activity, i.e., the activity required to drive the methods described herein. In the same manner, known DAAO enzymes can be improved by mutagenesis, and / or new DAAO enzymes can be identified.

[0043] In some embodiments, mutant enzymes can be prepared and tested in the methods described herein. Compared with the wild-type sequence, mutant DAAO enzymes (e.g., from Rhodotorula gracilis) can include one mutation, two mutations, three mutations, or more than three mutations (e.g., four mutations, five mutations, six mutations, seven mutations, eight mutations, nine mutations, or ten mutations or more) at the position of the mutant sequence. Mutant DAAO can optionally be included in mutations at positions 54, 56, 58, 213, and / or 238. In some embodiments, when compared with the wild-type sequence, such mutants can be included in amino acid replacements at positions 54 and 56. In other embodiments, when compared with the wild-type sequence, such mutants can be included in amino acid replacements at positions 54 and 58. In other embodiments, when compared with the wild-type sequence, such mutants can include amino acid replacements at positions 54, 213, and 238. In other embodiments, when compared with the wild-type sequence, such mutants can include amino acid replacements at positions 54, 56, 58, and 213.

[0044] Optionally, the wild-type asparagine may be replaced by Ala, Cys, Gly, Ile, Ser, Leu, or more preferably Thr or Val at position 54. For example, the mutant DAAO may comprise one of the following mutations at position 54: N54C, N54L, N54T or N54V.

[0045] Optionally, at position 56, the wild-type threonine can be replaced by Ala, Cys, Gly, Ile, Asn, Arg, Ser, Thr, Met or Val. See U.S. Pat. No. 7,939,709, which is incorporated herein by reference. For example, the mutant DAAO can comprise a T56M or T56N mutation.

[0046] In addition, wild-type Phe may be replaced by Lys, Arg, Gln, Thr, Gly, Ser, Ala, Arg, Asn or His at position 58. The mutant DAAO may optionally comprise one of the following mutations at position 58: F58A, F58G, F58H, F58K, F58N, F58Q, F58R, F58S or F58T. In some embodiments, the mutant DAAO does not comprise a mutation at position 58.

[0047] Optionally, at position 213, the wild-type methionine is replaced by Arg, Lys, Ser, Cys, Asn or Ala. In some examples, the mutant DAAO may comprise the mutation M213S.

[0048] Optionally, at position 238, the wild-type tyrosine is replaced by His, Ser, Cys, Asn or Ala.

[0049] In some embodiments, mutant DAAO may comprise one or more of the following mutation combinations: F58K and M213S; N54T and T56M; N54V and F58Q; N54C and F58H; N54T and F58T; N54T and F58G; N54T and F58Q; N54T and F58A; N54L and F58R; N54V and F58R; N54V and F58N; N54V, T56N, F58H and M213S; and / or N54V, F58Q and M213S.

[0050] In one embodiment, the mutant DAAO comprises a mutation in the other DAAO enzyme at a position corresponding to position 54, 56, 58, 213 and / or 238 of Rhodosporidium toruloides DAAO or Trigonopsis variabilis DAAO.

[0051] Other suitable D-amino acid oxidases can preferably be obtained from fungal sources. Such DAAO enzymes can be identified and tested for use in the methods described herein. In order to determine whether the enzyme accepts D-phosphinothricin as a substrate, oxygen electrode assay (Hawkes, 2011, the same), colorimetric assay (Berneman A, Alves-Ferreira M, Coatnoan N, Chamond N, Minoprio P (2010) Medium / High Throughput D-Amino Acid Oxidase Colorimetric Method for Determination of D-Amino Acids. Application for Amino Acid Racemases. J Microbial Biochem Technol 2: 139-146) and / or direct determination of product formation (by high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC-MS) or similar methods) can be used.

[0052] The reaction catalyzed by the DAAO enzyme requires oxygen. Therefore, in one embodiment, ventilation is an integral part of the DAAO reaction step. In some embodiments, oxygen, oxygen-enriched air, oxygen-enriched gas stream or air is introduced into the reaction, in the head space or by bubbling the gas intermittently or continuously through the reaction vessel to increase the reaction rate. In addition, in other embodiments, optionally in combination with bubbling gas through the reaction vessel, a pressurized reactor can be used. That is, the reactor can be sealed and allow consumption of O2. The use of a sealed chamber will limit steam emissions.

[0053] When the DAAO enzyme catalyzes the conversion of D-phosphinothricin to PPO, hydrogen peroxide (H2O2) is released. This may damage the enzyme and other components of the bioconversion (e.g., products and / or substrates). Therefore, in one embodiment, in addition to the DAAO enzyme, an enzyme such as catalase may also be used to catalyze the elimination of hydrogen peroxide.

[0054] In some embodiments, catalytic and non-catalytic decomposition reactions can be used to eliminate hydrogen peroxide. For example, hydrogen peroxide can be eliminated by using a non-catalytic decomposition reaction of increased heat and / or pH. Hydrogen peroxide can also be eliminated by using a catalytic decomposition reaction such as potassium iodide, for example, of a transition metal and other reagents. In addition to eliminating hydrogen peroxide, the use of catalase also produces oxygen (O 2). Since DAAO needs oxygen to play a role, the production of oxygen by catalase can help use the DAAO enzyme to promote the conversion of D-phosphinothricin to PPO.

[0055] Other enzymes can be used to catalyze the conversion of D-phosphinothricin to PPO. For example, a DAAD enzyme that accepts D-phosphinothricin as a substrate can be used. It is recognized that in the method using DAAD, the reaction catalyzed by DAAD can include redox cofactor recycling. This includes oxidation of the reduced acceptor so that it can accept more electrons from D-phosphinothricin.

[0056] In one embodiment, chemical oxidative deamination in which an intermediate α-keto acid is produced from the parent amino acid can be used in the methods described herein to convert D-phosphinothricin to L-phosphinothricin. Chemical oxidative deamination involves converting an amine group to a keto group with the release of ammonia gas, typically using metal ions, such as copper or cobalt ions, in aqueous solution at a temperature between room temperature and the boiling point of the solution, and a pH in the range of about 4 to about 10. See, for example, Ikawa and Snell (1954) J. Am. Chem. Soc. 76 (19): 4900-4902, incorporated herein by reference.

[0057] Substantially complete (greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95%) conversion of D-phosphinothricin to PPO can occur within 24 hours, within 18 hours, within 12 hours, within 8 hours, or less.

[0058] The second step of the method described herein involves converting PPO to L-phosphinothricin using a transaminase (TA). TA, which has the desired stereospecificity to accept PPO as a substrate, catalyzes the amination of PPO to L-phosphinothricin.

[0059] If the reaction is carried out in a two-step process, wherein D-phosphinothricin is substantially converted to PPO in the absence of an amine donor and / or a transaminase, the starting amount of PPO in the second step typically ranges from 10 g / L to 140 g / L; 20 g / L to 140 g / L; or 30 g / L-140 g / L. If the reaction is carried out in a one-step process, the starting amount of PPO is typically less than 1 g / L, and the highest level of PPO during the reaction is typically less than 25 g / L. The amine donor is initially present in a 1-50-fold molar excess relative to the starting amount of racemic phosphinothricin.

[0060] TAs that can be used in the methods described herein include the gabT aminotransferase from Escherichia coli (UniProt P22256), which has been shown to catalyze the desired reaction with PPO as a substrate (Bartsch et al. (1990), Appl Environ Microbiol. 56(1): 7-12). Additional enzymes have been developed that catalyze the desired reaction at a higher rate using isopropylamine as an amine donor (Bhatia et al. (2004), Peptide Revolution: Genomics, Proteomics & Therapeutics, Proceedings of the Eighteenth American Peptide Symposium, edited by Michael Chorev and Tomi K. Sawyer, July 19-23, 2003, pages 47-48). In addition, TA enzymes from a variety of microorganisms such as Streptomyces hygroscopicus, Streptomyces viridochromogenes, Candida albicans, and others can be used to implement the methods described herein. In particular, see, for example, EP0249188 and U.S. Pat. No. 5,162,212, incorporated herein by reference. If desired, enzymes can be evolved by mutagenesis to increase their activity. Mutant TA enzymes having the desired activity can be selected by the assays outlined in Schulz et al., Appl Environ Microbiol (1990) Jan 56(1): 1-6 and / or by direct product measurement by HPLC, LC-MS or the like.

[0061] Additional TA enzymes for use in the methods can be identified by screening collections of TAs for the desired activity, such as those sold by Prozomix Limited (Northumberland, United Kingdom), SyncoZymes (Shanghai, China), Eavolatal (Monheim Am Rhein, Germany), Codexis (Redwood City, CA), or Abcam (Cambridge, United Kingdom). Alternatively, sequence similarity can be used to identify new TA enzymes. Finally, TA enzymes can also be identified from organisms that catalyze the desired reaction.

[0062] Selecting suitable amine donors is important for the economic conversion of D-phosphinothricin to L-phosphinothricin. Can consider multiple problems, comprise the cost of donor, equilibrium thermodynamics, potential recovery of donor, ketoacid product and separation of required L-phosphinothricin etc.Therefore, can use the TA enzyme that accepts several different amine donors, comprise low-cost amine donor, for example L-aspartic acid compound or racemic aspartic acid compound, L-glutamic acid compound or racemic glutamic acid compound, L-alanine or racemic alanine, L-phenylethylamine or racemic phenylalanine, L-glycine or racemic glycine, L-lysine or racemic lysine, L-valine or racemic valine, L-serine or racemic serine, L-glutamine or racemic glutamine, isopropylamine, sec-butylamine, ethanolamine, 2-aminobutyric acid and diaminopropionic acid. In some embodiments, the amine donor is not an aspartate compound or aspartic acid (eg, L-aspartic acid, D-aspartic acid, or racemic D,L-aspartic acid).

[0063] A wild-type TA that accepts the desired amine donor can be identified, or a TA that does not normally accept the desired amine donor can be evolved to accept the desired substrate. Optionally, the transaminase is not an aspartate transaminase. Optionally, the transaminase is not a 4-amino-butyrate:2-oxoglutarate transaminase. In some embodiments, the transaminase is not a combined enzyme system comprising a PPT-specific transaminase and a glutamate:oxalacetate transaminase.

[0064] As shown in the third step of the method for producing L-phosphinothricin, the generated alpha ketone byproduct is reduced, driving the reaction to completely or substantially completely convert PPO to L-phosphinothricin. As described above, the method of reducing byproducts may differ depending on the amine donor.

[0065] Using the methods described herein, substantially complete conversion of PPO to L-phosphinothricin can occur within 48 hours, within 24 hours, within 12 hours, within 8 hours, or within 4 hours. In this article, substantially complete means that the conversion of PPO to L-phosphinothricin is greater than about 90%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or all detectable PPO is converted.

[0066] If the reaction is conducted in a single container or vessel, the TA enzyme may be added together with the DAAO enzyme or added later, such as after the DAAO enzyme has been allowed to catalyze some or substantially all of the oxidative deamination.

[0067] Enzyme can be added to the reaction by a variety of methods. One method is to express enzyme in microorganisms such as E. coli, S. cerevisiae, P. pastoris, etc., and full cells are added to the reaction as full cell biocatalysts. Another method is to express enzymes, crack microorganisms, and add cell lysates. Also having another method is to purify or partially purify enzymes from lysates, and to add pure or partially pure enzymes in the reaction. If the reaction requires multiple enzymes, the enzyme can be expressed in one or more microorganisms, including expressing all enzymes in a single microorganism.

[0068] Another approach that can be combined with the above methods is to immobilize the enzyme on a support (an exemplary strategy is described in Datta et al. (2013) 3Biotech. Feb; 3(1): 1-9). Without intending to be limiting, the enzyme, either alone or in combination, can, for example, be adsorbed onto, or covalently or non-covalently linked to, or embedded in a natural or synthetic polymer or inorganic support, including aggregates of the enzyme itself. Once immobilized, the enzyme and support can be dispersed into a bulk solution or filled into a bed, column, or any number of similar methods to allow the reaction solution to interact with the enzyme. Since aeration is important for the DAAO reaction contemplated herein, bubble columns or the like can be used for enzyme immobilization. For example, the reaction mixture can be flowed through an immobilized enzyme column (flow reaction), added to a fixed bed or immobilized enzyme column, reacted, and removed from the bottom or top of the reaction vessel (plug flow), or added to a dispersed immobilized enzyme and reacted, and then the immobilized enzyme is removed by filtration, centrifugation, or the like (batch). Thus, any method for immobilizing the enzyme can be used in the methods described herein.

[0069] DAAO, TA and / or other reactions can occur in a buffer. Exemplary buffers commonly used in biotransformation reactions include Tris, phosphate or any Good buffer, such as 2-(N-morpholino)ethanesulfonic acid (MES); N-(2-acetamido)iminodiacetic acid (ADA); piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); N-(2-acetamido)-2-aminoethanesulfonic acid (ACES); β-hydroxy-4-morpholinopropanesulfonic acid (MOPSO); chloroethanolamine; 3-(N-morpholino)propanesulfonic acid (MOPS); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES); 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropanesulfonic acid (BES); 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES); 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); 1-dihydroxy-2-(hydroxymethyl)propane-1-sulfonic acid (DIPSO); acetylaminoglycine, 3-(N-tris(hydroxymethyl)methylamino(-2-hydroxypropanesulfonic acid (TAPSO); piperazine-N,N'-di(2-hydroxypropanesulfonic acid) (POPSO); 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) (HEPPSO); 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS); tris(hydroxymethyl)methylglycine (tricine); glycinamide; N,N-dihydroxyethylglycine (bicine); or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid (TAPS). Additional exemplary buffer formulations can be found in Whittall, J. and Sutton, PW (eds.) (2012) Front. Matter, Practical Methods for Biocatalysis and Biotransformations 2, John Wiley & Sons, Ltd, Chichester, UK. In some embodiments, ammonium can be used as a buffer. One or more organic solvents can also be added to the reaction.

[0070] Surprisingly, DAAO, TA and / or other reactions can occur without or with low levels (less than 1 mM) of added buffer (other than the ammonium optionally present due to the addition of racemic glufosinate ammonium). In particular, immobilized DAAO, TA and KGD enzymes can be stable and active in the presence of less than 1 mM phosphate buffer and in the absence of any ammonium present due to the addition of racemic glufosinate ammonium.

[0071] The racemic phosphinothricin starting material can be provided in various forms. A variety of salts of racemic phosphinothricin can be used, such as ammonium salts and hydrochlorides, or zwitterions. Racemic phosphinothricin can be in the form of a solid powder (e.g., a powder having a purity greater than 80%, 85%, 90% or 95%) or an aqueous solution (e.g., a solution of about 50% of racemic phosphinothricin).

[0072] In some embodiments, the reaction occurs within a defined pH range, which may be between pH 4 and pH 10 (eg, between pH 6 and pH 9, such as about pH 7.5 to pH 8).

[0073] In some embodiments, the reaction occurs at a defined temperature. The temperature may be maintained at a point between room temperature and the boiling point of the solvent, most typically between room temperature and 50°C.

[0074] Additional steps for further purification of L-phosphinothricin may be added. Such further purification and separation methods include ion exchange, extraction, salification, crystallization and filtration; each may be used multiple times or in appropriate combinations. If supported, the enzyme may be removed by simple filtration, or if free in solution, by using ultrafiltration, using absorbents such as diatomaceous earth, cellulose or carbon, or by various technical denaturation known to those skilled in the art.

[0075] The ion exchange process achieves separation by selectively adsorbing solutes onto a resin selected for this purpose. Because the product and impurities must be dissolved in a single solution before adsorption, it is usually necessary to concentrate the purified product stream by evaporation or distillation before separation. Examples of purification using ion exchange are described in EP0249188 (A2) by Schultz et al., U.S. Pat. No. 9,834,802, and International PCT Patent Application PCT / US2018 / 042503.

[0076] Purification can be achieved by adding suitable acid, including hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, etc., to form an insoluble salt of L-phosphinothricin. Similarly, purification can be achieved by adding a suitable alkali to form an insoluble salt. Available alkalis include hydroxides, carbonates, sulfates and phosphates of alkali metals, or hydroxides, carbonates, sulfates and phosphates of alkaline earth metals. Other nitrogenous bases can be used, including ammonia, hydroxylamine, isopropylamine, triethylamine, tributylamine, pyridine, 2-picoline, 3-picoline, 4-picoline, 2,4-lutidine, 2,6-lutidine, morpholine, N-methylmorpholine, 1,8-diazabicyclo [5.4.0] 11 carbon -7-ene and dimethylethanolamine. It may be advantageous to concentrate the mixture or add a solvent (or both) to maximize the yield and optimize the purity of the desired salt. Suitable solvents for this purpose include those in which the solubility of the desired salt is very low (such solvents are generally referred to as "anti-solvents"). Salts of L-phosphinothricin can be converted to a phosphinothricin form suitable for formulation by standard methods known to those skilled in the art. Alternatively, L-phosphinothricin can be isolated as a zwitterion.

[0077] U.S. Pat. No. 9,255,115 B2 describes how to convert the hydrochloride of L-phosphinothricin into a zwitterionic form with a base such as sodium hydroxide or sodium methoxide and then crystallize from an aqueous alcohol solvent to obtain relatively high purity L-phosphinothricin. This method has the advantage of producing crystalline L-phosphinothricin that is not hygroscopic, and therefore retains a higher purity than amorphous L-phosphinothricin after being exposed to moisture for a period of time.

[0078] Other salts of L-phosphinothricin are known in the art. U.S. Patent No. 5,767,309 and U.S. Patent No. 5,869,668 teach the use of chiral alkaloids to form diastereoisomer salts with racemic phosphinothricin. Because the amount of the salt of L-phosphinothricin precipitated from the solution is much larger than the amount of the corresponding salt of D-phosphinothricin, purification is achieved. Therefore, if necessary, the method can be used together with the method described herein to obtain the L-phosphinothricin with high enantiomeric excess.

[0079] Optionally, purification can be achieved by first crystallizing one or more impurities, removing the impurities by filtering, and then further purifying L-phosphinothricin from the resulting filtrate by forming a salt as described above. It is advantageous if the unreacted amine donor can be partially or completely separated and used in subsequent reactions. Similarly, partially or completely separated unreacted PPO can be recycled for subsequent reactions.

[0080] Extraction can be used to purify product.DE 3920570 C2 has described a method, wherein by precipitating excessive glutamic acid (as amine donor) with sulfuric acid regulating solution pH to 3.7-4.2.After filtering glutamic acid, the pH of the filtrate is reduced to 1-2, so other impurities are extracted into the solvent.After extraction and concentration, ammonia is added to the aqueous solution, making pH is 5-7, so ammonium sulfate precipitation.Remove ammonium sulfate by filtration, and the gained filtrate is concentrated, obtain the ammonium salt of L-phosphinothricin.

[0081] For example, in order to transport solid to the place of preparation or use, it may be necessary to separate L-phosphinothricin or its salt. Typical industrial separation methods can be used, such as filtering, centrifuging, etc. The separated product usually needs to remove water, volatile impurities and solvent (if present), and typical industrial drying equipment can be used for this purpose. The example of such equipment includes baking oven, drum dryer, stirring dryer, etc. In some cases, it may be advantageous to use a spray dryer.

[0082] It is not necessary to produce a solid product after purification. If the preparation of L-phosphinothricin occurs at the same position for the preparation of L-phosphinothricin, this may be advantageous. L-phosphinothricin and many salts thereof are soluble in water, and water is a convenient liquid for preparing products. For example, the amine donor is separated by filtration, and the resulting filtrate is concentrated by distillation. The pH of the filtrate can be adjusted to a desired value, and the resulting solution can be used directly or mixed with the formulation ingredients. In another example, the slurry of L-phosphinothricin or a salt thereof can be prepared as described above and separated by filtration. By adding water or a suitable solvent, the solid can be dissolved directly on the filter to obtain a solution of L-phosphinothricin.

[0083] II. Composition

[0084] Also described herein is a composition comprising the above-mentioned reaction product. In some embodiments, the composition substantially comprises L-phosphinothricin and an acceptable cationic salt or anionic salt form, such as hydrochloride, ammonium salt or isopropylammonium salt. In some embodiments, the composition comprises a mixture of L-phosphinothricin, PPO and SSA. In some other embodiments, the composition comprises a mixture of L-phosphinothricin and SSA.

[0085] The compositions described herein can be used to prevent or control weeds in fields applied to crop plants. The composition can be formulated into a liquid for spraying on the field. L-phosphinothricin is provided in an effective amount in the composition. As used herein, an effective amount refers to about 10 grams of active ingredients per hectare to about 1,500 grams of active ingredients per hectare, such as about 50 grams to about 400 grams or about 100 grams to about 350 grams. In some embodiments, the active ingredient is L-phosphinothricin. For example, the amount of L-phosphinothricin in the composition can be about 10 grams, about 50 grams, about 100 grams, about 150 grams, about 200 grams, about 250 grams, about 300 grams, about 350 grams, about 400 grams, about 500 grams, about 550 grams, about 600 grams, about 650 grams, about 700 grams, about 750 grams, about 800 grams, about 850 grams, about 900 grams, about 950 grams, about 1,000 grams, about 1,050 grams, about 1,100 grams, about 1,150 grams, about 1,200 grams, about 1,250 grams, about 1,300 grams, about 1,350 grams, about 1,400 grams, about 1,450 grams, or about 1,500 grams of L-phosphinothricin per hectare.

[0086] The herbicidal compositions described herein (including concentrates that require dilution prior to application to plants) contain L-phosphinothricin (i.e., the active ingredient), optionally some residual D-phosphinothricin and / or PPO, and one or more adjuvant components in liquid or solid form.

[0087] The compositions are prepared by mixing the active ingredient with one or more adjuvants, such as diluents, extenders, carriers, surfactants, organic solvents, wetting agents or conditioners, to provide compositions in the form of finely divided granular solids, pellets, solutions, dispersions or emulsions. Therefore, the active ingredient can be used together with an adjuvant, such as a finely divided solid, a liquid of organic origin, water, a wetting agent, a dispersant, an emulsifier or any suitable combination of these. From an economic and convenient perspective, water is a preferred diluent. However, not all compounds are hydrolysis-resistant, and in some cases, this may determine the use of a non-aqueous solvent medium, as understood by those skilled in the art.

[0088] Optionally, one or more additional components can be added to the composition to prepare the prepared herbicidal composition. Such prepared compositions can include L-phosphinothricin, carriers (e.g., diluents and / or solvents) and other components. The prepared composition includes an effective amount of L-phosphinothricin. Optionally, L-phosphinothricin can be present in the form of L-phosphinothricin ammonium. L-phosphinothricin ammonium can be present in an amount range of 10% to 30% by weight of the prepared composition. For example, L-phosphinothricin ammonium can be present in an amount of 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or 30% by weight of the prepared composition. Optionally, L-phosphinothricin ammonium is present in an amount of 12.25% or 24.5% by weight of the prepared composition.

[0089] In some instances, the composition of preparation can include one or more surfactants. Suitable surfactants for the composition of preparation include sodium alkyl ether sulfate. The surfactant can be present in an amount of 10% to 40% by weight of the composition of preparation. For example, the surfactant can be present in an amount of 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38% or 40% by weight of the composition of preparation. Optionally, sodium alkyl ether sulfate is present in an amount of 11.05%, 15.8%, 22.1% or 31.6% by weight of the composition of preparation.

[0090] The composition of preparation can optionally include one or more solvents (e.g., organic solvents). Optionally, the solvent can be 1-methoxy-2-propanol, dipropylene glycol, ethylene glycol, propylene glycol, and mixtures thereof. One or more solvents can be present in an amount of 0.5% to 20% by weight of the composition of preparation. For example, the total amount of solvent in the composition can be present in an amount of 0.5% to 18%, 5% to 15%, or 7.5% to 10% by weight of the composition of preparation.

[0091] Optionally, the solvent includes a combination of two solvents. For example, the solvent in the preparation can include 1-methoxy-2-propanol and dipropylene glycol. 1-methoxy-2-propanol can be present in an amount of, for example, 0.5% to 2% by weight of the composition prepared. For example, 1-methoxy-2-propanol can be present in an amount of 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% by weight of the composition prepared. Optionally, 1-methoxy-2-propanol is present in an amount of 0.5% or 1.0% by weight of the composition prepared. Dipropylene glycol can be present in an amount of 4% to 18% by weight of the composition prepared. For example, dipropylene glycol may be present in an amount of 4%, 6%, 8%, 10%, 12%, 14%, 16% or 18% by weight of the formulated composition. Optionally, dipropylene glycol is present in an amount of 4.3% or 8.6% by weight of the formulated composition.

[0092] The composition of preparation can also include one or more polysaccharide wetting agents. Examples of suitable polysaccharide wetting agents include, for example, alkyl polysaccharides, pentose, high fructose corn syrup, sorbitol and molasses. Polysaccharide wetting agents such as alkyl polysaccharides can be present in the preparation composition in an amount of 4% to 20% by weight of the preparation composition. For example, the total amount of polysaccharide wetting agents in the composition can be present in an amount of 4% to 18%, 4.5% to 15% or 5% to 10% by weight of the preparation composition. In some instances, the total amount of polysaccharide wetting agents such as alkyl polysaccharides present in the preparation composition can be 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17% or 18%. Optionally, alkyl polysaccharides can be present in an amount of 3.2%, 4.9%, 6.2% or 9.8% by weight of the preparation composition.

[0093] Diluents may also be included in the formulated composition. Suitable diluents include water and other aqueous components. Optionally, the diluent is present in an amount necessary to prepare the composition ready for packaging or use.

[0094] In one example, the formulated composition includes L-ammonium glufosinate in an amount of 12.25% by weight of the formulation; sodium alkyl ether sulfate in an amount of 31.6% by weight of the formulation; 1-methoxy-2-propanol in an amount of 1% by weight of the formulation; dipropylene glycol in an amount of 8.6% by weight of the formulation; and an alkyl polysaccharide in an amount of 9.8% by weight of the formulation.

[0095] In another example, the formulated composition includes L-ammonium glufosinate in an amount of 24.5% by weight of the formulation; sodium alkyl ether sulfate in an amount of 31.6% by weight of the formulation; 1-methoxy-2-propanol in an amount of 1% by weight of the formulation; dipropylene glycol in an amount of 8.6% by weight of the formulation; and an alkyl polysaccharide in an amount of 9.8% by weight of the formulation.

[0096] In another example, the formulated composition includes L-ammonium glufosinate in an amount of 12.25% by weight of the formulation; sodium alkyl ether sulfate in an amount of 15.8% by weight of the formulation; 1-methoxy-2-propanol in an amount of 0.5% by weight of the formulation; dipropylene glycol in an amount of 4.3% by weight of the formulation; and an alkyl polysaccharide in an amount of 4.9% by weight of the formulation.

[0097] In another example, the formulated composition includes L-ammonium glufosinate in an amount of 24.5% by weight of the formulation; sodium alkyl ether sulfate in an amount of 22.1% by weight of the formulation; 1-methoxy-2-propanol in an amount of 1% by weight of the formulation; and alkyl polysaccharide in an amount of 6.2% by weight of the formulation.

[0098] In another example, the formulated composition includes L- glufosinate ammonium in an amount of 24.5% by weight of the formulation; sodium alkyl ether sulfate in an amount of 22.1% by weight of the formulation; propylene glycol in an amount of 15% by weight of the formulation; and alkyl polysaccharide in an amount of 6.2% by weight of the formulation.

[0099] In another example, the formulated composition includes L-ammonium glufosinate in an amount of 12.25% by weight of the formulation; sodium alkyl ether sulfate in an amount of 22.1% by weight of the formulation; 1-methoxy-2-propanol in an amount of 1% by weight of the formulation; and an alkyl polysaccharide in an amount of 6.2% by weight of the formulation.

[0100] In another example, the formulated composition includes L-ammonium glufosinate in an amount of 12.25% by weight of the formulation; sodium alkyl ether sulfate in an amount of 11.05% by weight of the formulation; 1-methoxy-2-propanol in an amount of 0.5% by weight of the formulation; and alkyl polysaccharide in an amount of 3.1% by weight of the formulation.

[0101] Other components suitable for use in the formulated compositions provided herein are described in US Pat. Nos. 4,692,181 and 5,258,358, both of which are incorporated herein by reference in their entirety.

[0102] The herbicidal compositions described herein, particularly liquids and soluble powders, may contain one or more surfactants as other adjuvant components in an amount sufficient to make a given composition easily dispersible in water or oil. The incorporation of surfactants into the compositions greatly enhances their efficacy. As used herein, surfactants include wetting agents, dispersants, suspending agents, and, among others, emulsifiers. Anionic, cationic, and nonionic agents may be used with the same equipment.

[0103] Suitable wetting agents include alkylbenzene sulfonates and alkylnaphthalene sulfonates, sulfated fatty alcohols, amines or amides, long-chain acid esters of sodium isothiosulfate, esters of sodium sulfosuccinate, sulfated or sulfonated fatty acid ester petroleum sulfonates, sulfonated vegetable oils, ditertiary acetylenic diols, polyoxyethylene derivatives of alkylphenols (especially isooctylphenol and nonylphenol) and polyoxyethylene derivatives of mono-higher fatty acid esters of hexitol anhydrides (e.g., sorbitan). Exemplary dispersants include methylcellulose, polyvinyl alcohol, sodium lignin sulfonate, polyalkylnaphthalene sulfonate, sodium naphthalene sulfonate, polymethylene dinaphthalene sulfonate, and sodium N-methyl-N-(long-chain acid) laurate.

[0104] A water-dispersible powder composition containing one or more active ingredients, an inert solid extender and one or more wetting agents and dispersants can be prepared. Inert solid extenders are usually mineral-derived, such as natural clays, diatomaceous earth, and synthetic minerals derived from silicon dioxide, etc. Examples of such extenders include kaolinite, attapulgite clay, and synthetic magnesium silicate. The water-dispersible powder described herein may optionally include about 5 to about 95 parts by weight of active ingredients (e.g., about 15 to 30 parts by weight of active ingredients), about 0.25 to 25 parts by weight of wetting agents, about 0.25 to 25 parts by weight of dispersants, and 4.5 to about 94.5 parts by weight of inert solid extenders, all of which are by weight of the total composition. If desired, about 0.1-2.0 parts by weight of solid inert extenders can be replaced with corrosion inhibitors or defoamers or both.

[0105] Aqueous suspensions can be prepared by dissolving or by mixing together and grinding an aqueous slurry of the water-insoluble active ingredient in the presence of a dispersant to obtain a concentrated slurry of very finely divided particles. The resulting concentrated aqueous suspension is characterized by its extremely small particle size, so that when diluted and sprayed, the coverage is very uniform.

[0106] Emulsifiable oils are typically solutions of active ingredients in water-immiscible or partially water-immiscible solvents together with surfactants. Suitable solvents for active ingredients described herein include hydrocarbons and water-immiscible ethers, esters or ketones. Emulsifiable oil compositions typically contain about 5 to 95 parts of active ingredients, about 1 to 50 parts of surfactants and about 4 to 94 parts of solvents, all parts being by weight based on the gross weight of the emulsifiable oil.

[0107] The compositions described herein may also contain other additives, such as fertilizers, plant poisons and plant growth regulators, pesticides, etc., used as adjuvants or in combination with any of the above adjuvants. The compositions described herein may also be mixed with other materials such as fertilizers, other plant poisons, etc., and applied in a single application.

[0108] In each formulation type described herein, eg, liquid and solid formulations, the concentration of the active ingredient is the same.

[0109] It is recognized that the herbicidal compositions can be used in combination with other herbicides. The herbicidal compositions described herein are often applied in conjunction with one or more other herbicides to control a wider variety of undesirable vegetation. When used in conjunction with other herbicides, the compounds claimed herein can be formulated with the other herbicides, tank-mixed with the other herbicides, or applied sequentially with the other herbicides. Some herbicides that can be used in conjunction with the compounds described herein include amide herbicides such as allidochlor, beflubutamid, benzadox, benzipram, bromobutide, cafenstrole, CDEA, chlorthiamid, cyprazole, dimethenamid, dimethenamid-P, diphenamid, epronaz, etnipromid, fentrazamide, flupoxam, fomesafen, halosafen, isocarbamid, isocarbamid, isoxaben, naproxam, naptalam, pethoxamid, propyzamide, quinonamid and tebutam; aniline herbicides, for example, chloranocryl, cisanilide, clomeprop, cypromid, diflufenican, etobenzanid, fenasulam, flufenacet, flufenican, mefenacet, mefluidide,metamifop, monalide, naproanilide, pentanochlor, picolinafen and propanil; arylanilide herbicides such as benzoylprop, flamprop and flamprop-M; chloroacetanilide herbicides such as acetochlor, alachlor, butachlor, butenachlor, acetochlor, alachlor, butachlor, butenachlor, acetochlor, alachlor, butachlor, butenachlor, acetochlor, alachlor, butachlor, butachlor, butachlor, butachlor, acetochlor, ala ...acetochlor, alachlor, butachlor, acetochlor, acetochlor, alachlor, butachlor, acetochlor, acetochlor, alachlor, butachlor, acetochlor, acetochlor, acetochlor, alachlor, butachlor, chlor), delachlor, diethatyl, dimethachlor, metazachlor, metolachlor, S-metolachlor, pretilachlor, propachlor, propisochlor, prynachlor, terbuchlor, thenylchlor and xylazine sulfonanilide herbicides such as benzofluor, perfluidone, pyrimisulfan, and profluazol; sulfonamide herbicides such as asulam, carbasulam, fenasulam, and oryzalin; antibiotic herbicides such as bilanfos; benzoic acid herbicides such as chloramben, dicamba, 2,3,6 -TBA and tricamba; pyrimidinyloxybenzoic acid herbicides, such as bispyribac and pyriminobac; pyrimidinylthiobenzoic acid herbicides, such as pyrithiobac; phthalic acid herbicides, such as chlorthal; 2-pyridinecarboxylic acid herbicides, such as aminopyralid, clopyralid and picloram; quinolinecarboxylic acid herbicides, such as quinclorac and quinmerac;Arsenic-containing herbicides, such as cacodylic acid, CMA, DSMA, potassium hexaflurate, MAA, MAMA, MSMA, potassium arsenite and sodium arsenite; benzoylcyclohexanedione herbicides, such as mesotrione, sulcotrione, tefuryltrione and tembotrione; benzofuranyl alkyl sulfonate herbicides, such as benfuresate and ethofumesate; carbamate herbicides, such as asulam, tefuryltrione; carboxazole, chlorprocarb, dichlormate, fenasulam, karbutilate and terbucarb; phenylcarbamate herbicides, such as barban, BCPC, carbasulam, carbetamide, CEPC, chlorbufam, chlorpropham, CPPC, desmedipham, pheniso cyclohexene oxime herbicides, such as alloxydim, butroxydim, clethodim, cloproxydim, cycloxydim, profoxydim, sethoxydim, tepraloxydim and tralkoxydim; cyclopropylisothiazide, ... Azole herbicides, such as isoflurane Isoxachlortole and isoxachlortole isoxaflutole; imide herbicides such as benzfendizone, cinidon-ethyl, trifluoroacetic acid dinitroaniline herbicides, such as benfluralin, butralin, diinitramine, ethalfluralin, fluchloralin, isopropalin, methalpropalin, nitralin, oryzalin, and oxazolidinone; ), pendimethalin, prodiamine, profluralin and trifluralin; dinitrophenol herbicides such as dinofenate, dinoprop, dinosam, dinoseb, dinoterb, DNOC, etinofen and medinoterb; diphenyl ether herbicides such as ethoxyfen; nitrophenyl ether herbicides such as acifluorfen; ifluorfen), aclonifen, bifenox, chlomethoxyfen, chlomitrofen, etnipromid, fluorodifen, fluoroglycofen, fluoronitrofen, fomesafen, furyloxyfen, halosafen, lactofen, nitrobenzene, fluorfen, nitrofluorfen, and oxyfluorfen; dithiocarbamate herbicides such as dazomet and metam; halogenated aliphatic herbicides such as alorac, chloropon, dalapon, flupropanate, hexachloroacetone, iodomethane, methylbromide, monochloroacetic acid, SMA, and TCA;imidazolinone herbicides, such as imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, and imazethapyr; inorganic herbicides, such as ammonium sulfamate, borax, calcium chloride, copper sulfate, ferrous sulfate, potassium azide, potassium cyanate, sodium azide, sodium chlorate, and sulfuric acid; acid; nitrile herbicides, such as bromobonil, bromoxynil, chloroxynil, dichlobenil, iodobonil, ioxynil, and pyraclonil; organophosphorus herbicides, such as amiprofos-methyl, anilofos, bensulide, bilanfos, butamifos, 2,4-DEP, DMPA, EBEP, fosamine, glyphosate, and piperophos; phenoxy herbicides, such as bromofenoxim, clomeprop, 2,4-DEB, 2, 4-DEP, difenopenten, disul, erbon, etnipromid, fenteracol and trifopsime; phenoxyacetic acid herbicides such as 4-CPA, 2,4-D, 3,4-DA, MCPA, MCPA-thioethyl and 2,4,5-T; phenoxybutyric acid herbicides such as 4-CPB, 2,4-DB, 3,4-DB, MCPB and 2,4,5-TB; phenoxypropionic acid herbicides such as cloprop, 4-CPP, dichlorprop, dichlorprop-P, 3,4-DP, fenoprop, mecoprop and mecoprop-P;Aryloxyphenoxypropionic acid herbicides, such as chlorazifop, clodinafop, clofop, cyhalofop, diclofop, ; Fenoxaprop, Fenoxaprop-P, fenthiaprop, fluazifop, fluazifop-P, haloxyfop, haloxyfop-P, isoprop isoxapyrifop, metamifop, propaquizafop, quizalofop, quizalofop-P and trifop; phenylenediamine herbicides such as diinitramine and prodiamine; pyrazolyl herbicides such as benzofenap, pyrazolynate, pyrasulfotole, pyrazoxane, pyrimidine ... pyrazolylphenyl herbicides such as fluazolate and pyraflufen; pyridazine herbicides such as credazine, pyridafol and pyridate; pyridazinone herbicides such as brompyrazon, chloridazon, dimidazon, flupyridazin-butyl and fluazifop-butyl; herbicides such as aminopyralid, cliodinate, clopyralid, dithiopyr, fluroxypyr, haloxydine, picloram, fluroxypyr, pyralid, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, pyridinium chloride, picolinafen, pyriclor, thiazopyr and triclopyr; pyrimidinediamine herbicides, such as iprymidam and tioclorim; quaternary ammonium herbicides, such as cyperquat, diethamquat, difenzoquat, diquat, morfamquat and paraquat;Thiocarbamate herbicides, such as butylate, cycloate, di-allate, EPTC, esprocarb, ethiolate, isopolinate, methiobencarb, molinate, orbencarb, pebulate, prosulfocarb, pyributicarb, sulfalate, thiocarb, bencarb, tiocarbazil, tri-allate and vemolate; thiocarbonate herbicides such as dimexano, EXD and proxan; thiourea herbicides such as methiuron; triazine herbicides such as dipropetryn, triaziflam and trihydroxytriazine; chlorotriazine herbicides such as atrazine, chlorazi ne), cyanazine, cyprazine, eglinazine, ipazine, mesoprazine, procyazine, proglinazine, propazine, sebuthylazine, simazine, terbuthylazine and trietazine; methoxytriazine herbicides such as atraton, etherthiocarbamide, methometon), prometon, secbumeton, simeton and terbumeton; methylthiotriazine herbicides, for example ametryn, aziprotryne, cyanatryn, desmetryn, dimethametryn, methoprene, prometryn, simetryn and terbutryn;triazone herbicides, such as amethidione, amibuzin, hexazinone, isomethiozin, metamitron and metribuzin; triazole herbicides, such as amitrole, cafenstrole, epronaz and flupoxam; triadimenes herbicides, such as amicarbazone, bencarbazone, carfentrazone, flucarbazone, propoxycarbazone, sulfentrazone and thiencarbazone-methyl; triazolopyrimidine herbicides, such as chlorpyrifos, ... cloransulam, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam and pyroxsulam; uracil herbicides, such as butafenacil, bromacil, flupropacil, isocil, lenacil and terbacil; 3-phenyluracil; urea herbicides, such as benzthiazuron, cumyluron, cycluron, dichloralurea, diflufenzopyr, isonoruron, iso; isouron, methabenzthiazuron, monisouron and noruron; phenylurea herbicides, such as anisuron, buturon, chlorbromuron, chloreturon, chlorotoluron, chloroxuron, daimuron, difenoxuron, buturon, chloram ... dimefuron, diuron, fenuron, fluometuron, fluothiuron, isoproturon, linuron, methiuron, methyldymron, metobenzuron, metobromuron, metoxuron, monolinuron, monuron, neburon, parafluron, acetyl chlorpyrifos phenobenzuron, siduron, tetrafluron and thidiazuron; pyrimidinylsulfonyl urea herbicides, for example amidosulfuron, azimsulfuron, bensulfuron, chlorimuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flup yrsulfuron), foramsulfuron, halosulfuron, imazosulfuron, mesosulfuron, nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron on and trifloxysulfuron; triazinesulfonyl urea herbicides, such as chlorsulfuron, cinosulfuron, ethametsulfuron, iodosulfuron, metsulfuron, prosulfuron, thifensulfuron, triasulfuron, tribenuron, triflusulfuron and tritosulfuron;Thiadiazolyl urea herbicides, such as buthiuron, ethidimuron, tebuthiuron, thiazafluron and thidiazuron; and unclassified herbicides, such as acrolein, allyl alcohol, aminocyclopyrachlor, azafenidin, benazolin, bentazone, benzobicyclon, buthidazole, calcium cyanamide, cambendichlor, chlorfenac, chlorfenprop, chlorflurazole, chlorflurenol, cinmethylin, isothiocyanate; Clomazone, CPMF, cresol, ortho-dichlorobenzene, dimepiperate, endothal, fluoromidine, fluridone, flurochloridone, flurtamone, fluthiacet, indanofan, methazole, methyl isothiocyanate, nipyraclofen, OCH, propyne Oxadiargyl, Oxadiazon, Oxaziclomefone, pentachlorophenol, cyclopentachlorophenol The herbicidal compositions described herein can also be used in combination with glyphosate or 2,4-D for glyphosate-tolerant or 2,4-D-tolerant crops. It is generally preferred that the compositions described herein be used in combination with herbicides that are selective for the crops being treated and that supplement the weed spectrum that these compositions control at the application rate used. It is also generally preferred to apply the compositions described herein and other supplemental herbicides simultaneously, either as a combination formulation or as a tank mix.

[0110] III. Methods of using L-phosphinothricin compositions

[0111] Compositions as described herein can be used in methods for selectively controlling weeds in fields or any other regions, including, for example, railways, lawns, golf courses, and other regions where weeds need to be controlled. Optionally, fields or other regions can contain crops of planted seeds or crops that are resistant to phosphinothricin. The method can include applying an effective amount of a composition comprising L-phosphinothricin as described herein to the fields.

[0112] Compositions as described herein can be used to prevent or control weeds in fields applied to crop plants. Compositions can be formulated into liquids for spraying on fields. L-phosphinothricin is provided in an effective amount in the composition. As used herein, effective amount refers to about 10 grams of active ingredients per hectare to about 1,500 grams of active ingredients per hectare, such as about 50 grams to about 400 grams or about 100 grams to about 350 grams. In some embodiments, the active ingredient is L-phosphinothricin. For example, the amount of L-phosphinothricin in the composition can be about 10 grams, about 50 grams, about 100 grams, about 150 grams, about 200 grams, about 250 grams, about 300 grams, about 350 grams, about 400 grams, about 500 grams, about 550 grams, about 600 grams, about 650 grams, about 700 grams, about 750 grams, about 800 grams, about 850 grams, about 900 grams, about 950 grams, about 1,000 grams, about 1,050 grams, about 1,100 grams, about 1,150 grams, about 1,200 grams, about 1,250 grams, about 1,300 grams, about 1,350 grams, about 1,400 grams, about 1,450 grams, or about 1,500 grams of L-phosphinothricin per hectare.

[0113] IV. Exemplary Embodiments

[0114] Non-limiting embodiments include:

[0115] 1. A method for preparing L-phosphinothricin, comprising: in an oxidation step, reacting D-phosphinothricin with D-amino acid oxidase (DAAO) to form PPO (2-oxo-4-(hydroxy(methyl)phosphonyl)butyric acid) while aerating; in an amination step, aminating PPO by transaminase (TA) using amine groups from one or more amine donors to form L-phosphinothricin and an alpha-keto acid byproduct or a ketone byproduct; and in a reduction step, reducing the amount of the alpha-keto acid byproduct or the ketone byproduct, wherein greater than 90% of the PPO is converted to L-phosphinothricin.

[0116] 2. The method of embodiment 1, wherein greater than 91% of the PPO is converted to L-phosphinothricin.

[0117] 3. The method of embodiment 1, wherein greater than 92% of the PPO is converted to L-phosphinothricin.

[0118] 4. The method of embodiment 1, wherein greater than 93% of the PPO is converted to L-phosphinothricin.

[0119] 5. The method of embodiment 1, wherein greater than 94% of the PPO is converted to L-phosphinothricin.

[0120] 6. The method of embodiment 1, wherein greater than 95% of the PPO is converted to L-phosphinothricin.

[0121] 7. The method of embodiment 1, wherein greater than 96% of the PPO is converted to L-phosphinothricin.

[0122] 8. The method of embodiment 1, wherein greater than 97% of the PPO is converted to L-phosphinothricin.

[0123] 9. The method of embodiment 1, wherein greater than 98% of the PPO is converted to L-phosphinothricin.

[0124] 10. The method of embodiment 1, wherein greater than 99% of the PPO is converted to L-phosphinothricin.

[0125] 11. The method of embodiment 1, wherein the DAAO enzyme is a mutant DAAO comprising one or more mutations at positions 54, 56, 58, 213 and 238, using SEQ ID NO: 8 as a reference sequence, wherein the mutation at position 54 is selected from N54C, N54L, N54T and N54V; the mutation at position 56 is T56M or T56N; and the mutation at position 58 is selected from F58A, F58G, F58H, F58K, F58N, F58Q, F58R, F58S and F58T.

[0126] 12. The method of embodiment 11, wherein the mutations include N54V, T56N, F58H and M213S.

[0127] 13. The method of embodiment 1, wherein the alpha-keto acid byproduct is reduced by enzymatic conversion.

[0128] 14. The method according to any one of embodiments 1 to 13, wherein the TA enzyme is an enzyme encoded by SEQ ID NO: 6 or SEQ ID NO: 7.

[0129] 15. The method according to any one of embodiments 1 to 13, wherein the TA enzyme is a GabT aminotransferase having the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0130] 16. The method according to any one of embodiments 1 to 15, wherein the amine donor is a glutamic acid compound or an L-glutamic acid compound.

[0131] 17. The method of embodiment 16, wherein the amount of alpha-keto acid byproduct is reduced by adding ketoglutarate decarboxylase (KGD).

[0132] 18. The process of any one of embodiments 1 to 17, wherein the oxidation, amination and reduction steps are performed in a single vessel.

[0133] 19. The method according to any one of embodiments 1 to 17, wherein all reagents are added substantially at the beginning of the reaction.

[0134] 20. The method of embodiment 18, wherein the reagents for the oxidation, amination and reduction steps are added to a single container at different times.

[0135] 21. The process of any one of embodiments 1 to 17, wherein the oxidation, amination and reduction steps are performed in separate vessels.

[0136] 22. The method according to any one of embodiments 1 to 17, wherein one or more enzymes are immobilized.

[0137] 23. The process of any of embodiments 1 to 17, wherein the by-products are separated from the reaction by combining or further converting into additional products.

[0138] 24. A method for converting PPO to L-phosphinothricin, comprising aminating PPO by a transaminase (TA) using amine groups from one or more amine donors to form L-phosphinothricin and an alpha keto acid or ketone byproduct, and reducing the amount of the alpha keto acid or ketone byproduct, wherein greater than 90% of the PPO is converted to L-phosphinothricin.

[0139] 25. The method of embodiment 24, wherein the amount of alpha keto acid or ketone by-product is reduced by adding an enzyme that reacts with the by-product.

[0140] 26. The method of embodiment 25, wherein the amine donor is an L-glutamic acid compound.

[0141] 27. The method of embodiment 25, wherein the enzyme that reacts with the by-product is KGD.

[0142] 28. The method of embodiment 27, wherein KGD comprises one of the sequences shown in SEQ ID NOs: 1-5.

[0143] 29. The method of embodiment 24, wherein the TA enzyme is an enzyme encoded by SEQ ID NO: 6 or SEQ ID NO: 7.

[0144] 30. The method of embodiment 24, wherein the TA enzyme is a GabT aminotransferase having the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0145] 31. The method of embodiment 24, wherein the TA enzyme has a lower affinity for SSA than for PPO.

[0146] 32. A composition comprising L-phosphinothricin and succinic acid semialdehyde (SSA).

[0147] 33. A composition comprising L-ammonium glufosinate in an amount of 10-30% by weight of the composition; one or more additional components selected from sodium alkyl ether sulfate in an amount of 10-40% by weight of the composition; 1-methoxy-2-propanol in an amount of 0.5-2% by weight of the composition; dipropylene glycol in an amount of 4-18% by weight of the composition; and an alkyl polysaccharide in an amount of 4-20% by weight of the composition.

[0148] 34. A composition comprising L-ammonium glufosinate in an amount of 10-30% by weight of the composition; one or more additional components selected from sodium alkyl ether sulfate in an amount of 10-40% by weight of the composition; propylene glycol in an amount of 5-20% by weight of the composition; and an alkyl polysaccharide in an amount of 4-20% by weight of the composition.

[0149] 35. A method for selectively controlling weeds in an area, comprising: applying to the area an effective amount of a composition comprising L-phosphinothricin having an enantiomeric excess of greater than 99% relative to D-phosphinothricin.

[0150] 36. A composition comprising L-phosphinothricin, PPO and SSA, wherein PPO is present in an amount less than 10% of the amount of L-phosphinothricin, and SSA is present in a molar amount of up to 2 times the amount of L-phosphinothricin.

[0151] 37. A composition comprising greater than 90% L-phosphinothricin, less than 10% PPO, less than 10% alpha ketoacid or ketone byproducts, and at least 25% conversion products relative to the amount of L-phosphinothricin.

[0152] The following examples are offered by way of illustration and not limitation. Example

[0153] Example 1: Activity of selected KGD enzymes using KG as substrate

[0154] In this embodiment, the activity of KG degradation to SSA by various KGD enzymes was tested. The following reactants were added at the beginning of the 470 μL reaction: 15 mM KG, 107 μM thiamine pyrophosphate (TPP), 4.5 mM MgCl2-6H2O, 63-197 μg / mL KGD enzyme, 30 mM Kphos (pH 8). The reaction was incubated at 30 ℃ with 300 rpm shaking for 21 hours, then placed on the bench for 5 hours, and then HPLC analysis was performed. After the KG initial concentration was normalized, the KG degradation over time was shown in Table 1.

[0155] Table 1

[0156]

[0157]

[0158] Example 2: Activity of selected KGD enzymes using PPO as substrate

[0159] In this example, the activity of multiple KGD enzymes for decarboxylating PPO was tested. The following reactants were added at the beginning of a 500 μL reaction: 40 mM PPO, 40 mM L-phosphinothioate, 0.1 mM thiamine pyrophosphate (TPP), 4.2 mM MgCl2-6H2O, ~30-90 μg / mL KGD enzyme. The reaction was incubated at 30°C with 300 rpm shaking for 4 hours. The reactants were then placed on the bench for 71 hours, at which point an endpoint sample was taken. The normalized degradation of PPO over time is shown in Table 2.

[0160] Table 2

[0161]

[0162] Example 3: Activity of wild-type and mutant TA enzymes in L-phosphinothricin reaction

[0163] In this example, the activity of wild-type E. coli gabT TA enzyme and its E211S variant on the desired reaction of converting PPO and glutamate compounds to KG and L-phosphinothricin was tested. The following reactants were added at the beginning of a 500 μL reaction: 25 mM PPO, 25 mM L-phosphinothricin, 180 mM L-glutamate compound (3.6-fold molar excess of PPO + Glu-L), 0.2 mM PLP, 25 mM Kphos (pH 8.0) to pH ~ 7.5, 75 μg / mL gabT. The reaction was incubated at 30 ° C with shaking at 300 rpm and samples were taken for HPLC analysis. The normalized performance of KG over time is shown in Table 3.

[0164] Table 3

[0165] Time (hours) wild type E211S mutant 0.0 0.0 0.0 0.2 10.9 2.0 0.5 14.2 5.1 1.0 14.5 7.9 5.0 14.4 14.1 24.5 14.5 14.8

[0166] Example 4: Activity of wild-type and mutant TA enzymes in their native reactions

[0167] In this example, the activity of the wild-type E. coli gabT TA enzyme and its E211S variant on the natural reaction of SSA and glutamate compounds into KG and GABA was tested. The following reactants were added at the beginning of the 500 μL reaction: 250 mM L-glutamate compound, 50 mM SSA, 0.2 mM PLP, 50 mM Kphos (pH 8.0) and 30 mM NaOH to pH 7.5, 75 μg / mL gabT enzyme. The reaction was incubated at 30°C with 300 rpm shaking, and samples were taken for HPLC analysis. The normalized performance of KG over time is shown in Table 4.

[0168] Table 4

[0169] Time (minutes) wild type E211S mutant 0 0.0 0.0 10 12.4 0.0 30 13.2 0.0 60 13.5 0.5 1200 15.5 4.9

[0170] Example 5: Improved Yield of L-phosphinothricin by Adding KGD Enzyme

[0171] In this example, racemic glufosinate was deracemized using DAAO enzyme, TA enzyme, and with or without KGD enzyme. The following reactants were added at the beginning of a 500 μL reaction: 300 mM racemic glufosinate, 150 or 300 mM L-glutamate compound, 0.1 mM thiamine pyrophosphate (TPP), 4.2 mM MgCl2-6H2O, 0.2 mM PLP, 0.2 mg / mL catalase lysate, 1 mg / mL DAAO (pure enzyme), 0.4 mg / mL E211S gabT (2.35 mg / mL CFE), + / -0.5 mg / mL KGD from A. enclensis (7.15 mg / mL CFE), and ~35 mM Kphos (pH 8) and 5 mM NaOH to adjust the pH to ~7.5. Reactions were incubated open at 30°C with shaking at 320 rpm for the first 6-7 hours. The tubes were sealed and incubated overnight to minimize evaporation. FIG. 3 shows the progress of the reaction without (A) or with (B and C) addition of KGD enzyme, and with an initial L-glutamate concentration of 300 mM (A and B) or 150 mM (C). The concentrations of L-phosphinothricin (black circles, black thick line), D-phosphinothricin (light gray circles, light gray solid line), PPO (dark gray diamonds, dark gray solid line), L-glutamate (black triangles, black dashed line), and KG (dark gray squares, dark gray dashed line) are shown in the figure.

[0172] Example 6: Improved yield of L-phosphinothricin using mutant TA enzymes

[0173] In this example, a mutant TA enzyme having a reduced affinity for SSA as a substrate was used for racemization, and the yield of L-phosphinothricin was higher than that of the wild-type TA enzyme, although the initial production rate of the wild-type TA was higher. The following reactants were added at the beginning of the 500 μL reaction: 300 mM D, L-phosphinothioate, 150 mM L-glutamic acid compound (MSG), 0.1 mM thiamine pyrophosphate (TPP), 4.2 mM MgCl2-6H2O, 0.2 mM pyridoxal phosphate (PLP), 0.2 mg / mL catalase powder (Sigma), 1 mg / mL DAAO Ac302, 0.4 mg / mL TA (SEQ ID NO: 6 for wild type, SEQ ID NO: 7 for E211S mutant), 0.36 mg / mL A. enclensis KGD (SEQ ID NO: 4), ~30 mM Kpho (pH 8) and 10 mM NaOH to adjust pH to 7.7. The reaction was incubated open at 30°C with shaking at 320 rpm for 4 hours. The tubes were incubated overnight with sealing to minimize evaporation. The normalized amounts of L-phosphinothricin (mmol) over time are shown in Table 5.

[0174] Table 5

[0175] Time (hours) wild type E211S mutant 0 146 145 2 171 153 4 194 164 23 233 259

[0176] Example 7: Improved Yield of L-phosphinothricin by Adding Various KGD Enzymes

[0177] In this example, racemic phosphinothricin was deracemized using DAAO enzymes, TA enzymes, and with or without various KGD enzymes. The following reactants were added at the beginning of a 500 μL reaction: 300 mM D,L-phosphinothricin, 150 mM L-glutamic acid compound (MSG), 0.1 mM thiamine pyrophosphate (TPP), 4.2 mM MgCl2-6H2O, 0.2 mM pyridoxal phosphate (PLP), 0.2 mg / mL catalase powder (Sigma), 1 mg / mL DAAO Ac302, 0.4 mg / mL TA (E211S mutant), 0.5 mg / mL KGD (0.36 mg / mL for A. enclensis KGD, 0 mg / mL for no KGD), ~30 mM Kpho (pH 8), and 10 mM NaOH to adjust the pH to 7.7. The reaction was incubated open at 30°C with shaking at 320 rpm for the first 4 hours. The tubes were sealed and incubated overnight to minimize evaporation. The percentage of PPO converted to L-phosphinothricin over time is shown in Table 6.

[0178] Table 6

[0179]

[0180]

[0181] Example 8: Activity of KGD enzyme immobilized on HA403 resin

[0182] In this example, KG was decarboxylated using a variety of immobilized KGD enzymes. The following reactants were added at the start of a 2.5 mL reaction: 100 mM KG, 0.17 mM TPP, 4 mM MgCl2-6H2O, 2 mg / mL KGD immobilized on 0.25 g Relizyme HA403 resin (Resindion), 100 mM Kphos (pH 7), 177.5 mM NaOH to pH 6.47 at 23°C. The reactants were incubated in a closed 50 mL tube at 30°C with 275 rpm shaking. The concentration of KG over time is shown in Table 7.

[0183] Table 7

[0184]

[0185] Example 9: Activity of KGD enzyme immobilized on HA403 resin

[0186] In this example, KG was decarboxylated using a variety of immobilized KGD enzymes. The following reactants were added at the start of a 2.5 mL reaction: 100 mM KG, 0.1 mM TPP, 4 mM MgCl2-6H2O, 1 mg / mL KGD immobilized on 0.5 g Lifetech ECR8209F (Purolite), 100 mM Kphos (pH 7), 170 mM NaOH to pH 6.5 at 2°C. Reactants were incubated in a closed 50 mL tube at 30°C with shaking at 275 rpm. The concentration of KG over time is shown in Table 8.

[0187] Table 8

[0188]

[0189]

[0190] All publications and patent applications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated herein by reference.

[0191] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A method for preparing L-phosphinothricin, include: In the oxidation step, D-phosphinothricin is reacted with D-amino acid oxidase (DAAO) in the presence of oxygen to form PPO (2-oxo-4-(hydroxy(methyl)phosphono)butyric acid); In an amination step, PPO is aminated by a transaminase (TA) using amine groups from one or more amine donors to form L-phosphinothricin and an alpha keto acid byproduct or a ketone byproduct; and In the reduction step, the amount of alpha-keto acid byproduct or ketone byproduct is reduced, wherein greater than 90% of the PPO is converted to L-phosphinothricin; wherein the DAAO enzyme is a mutant DAAO comprising one or more mutations at positions 54, 56, 58, 213 and 238, using SEQ ID NO: 8 as a reference sequence; The mutations include N54V, T56N, F58H and M213S.

2. The method of claim 1, wherein greater than 98% of the PPO is converted to L-phosphinothricin.

3. The method of claim 1, wherein the amount of alpha-keto acid byproduct is reduced by enzymatic conversion.

4. The method of claim 1, wherein the TA enzyme is an enzyme encoded by SEQ ID NO: 6 or SEQ ID NO:

7.

5. The method of claim 1, wherein the TA enzyme is GabT aminotransferase having the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO:

7.

6. The method of claim 1, wherein the one or more amine donors comprise a glutamic acid compound or an L-glutamic acid compound.

7. The method of claim 6, wherein the amount of alpha-keto acid byproduct is reduced by adding ketoglutarate decarboxylase (KGD).

8. The method of claim 7, wherein KGD comprises the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO:

5.

9. The process of claim 1, wherein the oxidation, amination and reduction steps are performed in a single vessel.

10. The method of claim 1, wherein substantially all of the reagents are added at the beginning of the reaction.

11. The method of claim 9, wherein the reagents for the oxidation, amination and reduction steps are added to a single container at different times.

12. The process of claim 1, wherein the oxidation, amination and reduction steps are performed in separate vessels.

13. A composition comprising greater than 90% L-phosphinothricin, less than 10% PPO, less than 10% alpha ketoacid or ketone byproduct, and at least 25% alpha ketoacid or ketone byproduct conversion products relative to the amount of L-phosphinothricin.

14. The composition of claim 13, wherein the conversion product is SSA.

15. The composition of claim 14, wherein the SSA is present in a molar amount of up to 2 times the amount of L-phosphinothricin.

16. A mutant DAAO enzyme comprising one or more mutations at positions 54, 56, 58, 213 and 238, using SEQ ID NO: 8 as a reference sequence, The mutations include N54V, T56N, F58H and M213S.

17. Use of the mutant DAAO enzyme according to claim 16 for converting D-phosphinothricin into PPO.

18. A method for converting D-phosphinothricin into PPO using the mutant DAAO enzyme of claim 16.

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