Methods for improving yields of L-glufosinate
Compositions and methods for the production of L-glufosinate are provided. The method involves converting racemic glufosinate to the L-glufosinate enantiomer or converting PPO to L-glufosinate in an efficient manner. In particular, the method involves the specific amination of PPO to L-glufosinate, using L-glutamate, racemic glutamate, or another amine source as an amine donor. PPO can be obtained by the oxidative deamination of D-glufosinate to PRO (2-oxo-4-(hydroxy (methyl) phosphinoyl) butyric acid) or generated via chemical synthesis. PPO is then converted to L-glufosinate using a transaminase in the presence of an amine donor. When the amine donor donates an amine to PPO. L-glufosinate and a reaction by product are formed. Because the PPO remaining represents a yield loss of L-glufosinate, it is desirable to minimize the amount of PPO remaining in the reaction mixture. Degradation, other chemical modification, extraction, sequestration, binding, or other methods to reduce the effective concentration of the by-product. i.e., the corresponding alpha ketoacid or ketone to the chosen amine donor will shift the reaction equilibrium toward L-glufosinate, thereby reducing the amount of PPO and increasing the yield of L-glufosinate. Therefore, the methods described herein involve the conversion or elimination of the alpha ketoacid or ketone by-product to another product to shift the equilibrium towards L-glufosinate.
1 . A method for making L-glufosinate, comprising:
reacting D-glufosinate with a D-amino acid oxidase (DAAO) enzyme in the presence of oxygen to form PPO (2-oxo-4-(hydroxy (methyl) phosphinoyl) butyric acid) in an oxidation step;
aminating the PPO by a transaminase (TA) enzyme, using an amine group from one or more amine donors to form L-glufosinate and an alpha ketoacid by-product in an amination step; and
reducing an amount of the alpha ketoacid by-product in a reduction step, wherein more than 90% of the PPO is converted to L-glufosinate,
wherein the DAAO enzyme is a mutant DAAO comprising mutations at positions 54, 56, 58, and 213 using SEQ ID NO:8 as a reference sequence,
wherein the mutations comprise N54V, T56N, F58H, and M213S,
wherein the TA enzyme has the amino acid sequence of SEQ ID NO:7,
wherein the alpha ketoacid by-product is reduced in amount by an addition of ketoglutarate decarboxylase (KGD), and
wherein the reduction of the amount of the alpha ketoacid by-product allows the amination step to proceed at a higher rate than without such reduction and/or reach an equilibrium concentration that results in a higher yield of L-glufosinate than without such reduction.
2 . The method of claim 1 , wherein more than 98% of the PPO is converted to L-glufosinate.
3 . The method of claim 1 , wherein the alpha ketoacid by-product is reduced in amount by enzymatic conversion.
4 . The method of claim 1 , wherein the TA enzyme is a GabT transaminase.
5 . The method of claim 1 , wherein the one or more amine donors comprises glutamate or L-glutamate.
6 . The method of claim 1 , wherein the oxidation, the amination, and the reduction steps are performed in a single container.
7 . The method of claim 1 , wherein all reagents are added at the start of the reaction.
8 . The method of claim 6 , wherein reagents for the oxidation, the amination, and the reduction steps are added to the single container at different times.
9 . The method of claim 1 , wherein the oxidation, the amination, and the reduction steps are performed in separate containers.
10 . The method of claim 1 , wherein the KGD comprises an amino acid sequence selected from SEQ ID NOS: 1, 2, 3, 4, or 5.