Methods And Systems For Increasing Production of Equilibrium Reactions

• US 20080015361A1
• Filed: 05/23/2007
• Published: 01/17/2008
• Est. Priority Date: 05/24/2006
• Status: Active Grant

• Current Assignee: Cargill, Inc. (Agricultural Services Division)
• Original Assignee: Cargill, Inc. (Agricultural Services Division)
• Inventors: Sanchez-Riera, Fernando, Hicks, Paula, Hilbert, Brent, Khare, Anil, Floy, Kent, Solheid, Christopher, McFarlan, Sara
• Granted Patent: US 7,888,081 B2
• Time in Patent Office: Days
• US Class Current: 548/491
• CPC Class Codes: B01D 15/34 Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• CPC Class Codes: B01D 15/362 Cation-exchange
• CPC Class Codes: B01D 15/363 Anion-exchange
• CPC Class Codes: B01D 15/3804 Affinity chromatography
• CPC Class Codes: C07D 209/20 substituted additionally by nitrogen atoms, e.g. tryptophane
• CPC Class Codes: C12P 17/10 Nitrogen as only ring hetero atom

Abstract

Methods and systems for increasing the production of equilibrium reactions are described. In some embodiments, a method comprises removing enzymes from a reaction mixture once equilibrium is achieved, selectively re-adding enzymes and reactants for driving the product-forming reaction to product, and optionally recycling stabilized intermediates, by-products and/or co-products back into the reaction mixture. In some embodiments, a method comprises purifying the desired product from a reaction mixture and selectively recycling one or more components of the reaction mixture, such as original reactants, intermediates, co-products or by-products back into the reaction mixture as desired to improve titer. Systems for implementing the methods are also provided. In some embodiments the methods and systems are implemented for increasing the production of monatin and monatin derivatives, produced in multi-step equilibrium pathways.

Claims

1. A method, comprising:

a) allowing a first composition comprising one or more reactants and one or more first facilitators to react to form an amount of monatin and an amount of one or more intermediates in a multi-step equilibrium pathway, wherein the pathway comprises a monatin-producing reaction and a limiting reaction that limits the amount of at least one of the one or more intermediates available to the monatin-producing reaction;

b) removing, inhibiting, inactivating, or degrading the one or more facilitators after a desired time period forming a second composition; and

,

c) producing an additional amount of monatin by;

adding a second facilitator to the second composition, wherein the second composition facilitator can be the same as at least one of the first facilitators;

or uninhibiting or reactivating one or more of the first facilitators involved in the monatin-producing reaction;

or combinations thereof; and

adding a component into the second composition for driving the monatin-producing reaction forward.

2. The method of claim 1, wherein the desired time period is the time required to reach equilibrium in (a).

3. The method of claim 1, wherein said component can be the same as at least one of the one or more reactants from a) or can be the same as a compound produced in step a).

4. The method of claim 1, wherein said method further comprises:

purifying said monatin from said second composition and forming a reaction purification mixture.

5. The method of claim 1, wherein said removing, inhibiting, inactivating, or degrading comprises removing all of the one or more facilitators.

6. The method of claim 1, wherein said removing is selected from one or more of filtration, chromatography, and extraction.

7. The method of claim 6, wherein said filtration is ultrafiltration.

8. The method of claim 6, wherein said chromatography is selected from size exclusion, ion exchange, and affinity chromatography.

9. The method of claim 1, wherein at least one of the one or more reactants is an amino acid and at least one of the one or more reactants is an amino group acceptor, and said one or more first facilitators comprises two or more facilitators.

10. The method of claim 9, wherein said amino acid is L-tryptophan, D-tryptophan, or mixtures thereof.

11. The method of claim 9, wherein one of the two or more facilitators is the second facilitator.

12. The method of claim 9, wherein said amino group acceptor is pyruvate, oxaloacetate, alpha-ketoglutarate, or mixtures thereof.

13. The method of claim 9, wherein said amino group acceptor reacts to produce an amino group donor chosen from alanine, aspartate, and glutamate.

14. The method of claim 1, wherein said one or more first facilitators is chosen from enzymes, inorganic catalysts, and RNA.

15. The method of claim 14, wherein said enzymes are chosen from aminotransferases, racemases, and aldolases.

16. The method of claim 4, wherein said amino group acceptor is pyruvate, the amino group acceptor reacts to produce alanine, the component in c) is also alanine, and the method further comprises recycling alanine from the reaction purification mixture to the second composition, and optionally to the first composition.

17. The method of claim 3, wherein the component is alanine.

18. The method of claim 4, further comprising separating at least a portion of the alanine from the second composition after a desired time period and recycling the separated alanine into the first composition.

19. The method of claim 1, wherein said facilitator of step c) is an aminotransferase.

20. The method of claim 1, wherein said monatin is R,R monatin, S,S monatin, R,S monatin, S,R monatin, or mixtures thereof.

21. The method of claim 20, wherein said monatin is R,R monatin.

34. The method of claim 4, wherein monatin is purified using anion exchange chromotagraphy, SDVB column chromatography, or cation exchange chromatography.

22. A method of making monatin in a multi-step equilibrium pathway, said method comprising:

a) producing monatin in a reaction mixture via a pathway involving at least two reaction steps facilitated by enzymes, wherein at least one of the at least two steps is a monatin-producing step and at least one of the at least two steps has an unfavorable equilibrium;

b) compromising at least the reaction having the unfavorable equilibrium to form a compromised composition;

c) loading the compromised composition with at least one component to drive the monatin-producing step toward production of monatin.

23. The method of claim 22, wherein the pathway involves at least three reaction steps.

24. The method of claim 23, further comprising compromising all of the at least three steps by separating the facilitators or co-factors of the facilitors or a combination thereof from the reaction mixture, and wherein the at least one component is at least a first component and a second component, the first component being an enzyme that facilitates the monatin-producing step and the second component being a reactant involved in the monatin-producing step, and wherein the enzyme that facilitates the monatin-producing step also facilitates conversion of a less stable component in the pathway into a more stable component in the pathway.

25. The method of claim 24, wherein the less stable component is indole-3-pyruvate and the more stable component is tryptophan.

26. The method of claim 25, further comprising recycling the separated enzymes and tryptophan into the reaction mixture.

27. The method of claim 24, further comprising recycling byproducts, coproducts, or combinations thereof into the first composition, the compromised composition, or combinations thereof.

28. The method of claim 22, further comprising providing a set of initial components that produce 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid (“

MP”

) in the reaction mixture, wherein about 90% of the MP is converted into monatin.

29. The method of claim 28, wherein about 95% of the MP is converted into monatin.

30. The method of claim 28, wherein about 100% of the MP is converted into monatin.

31. The method of claim 28, wherein the concentration of monatin produced is about 1.5 times greater than would be produced in an undisturbed equilibrium pathway.

32. The method of claim 28, wherein the concentration of monatin produced is about 1.7 times greater than would be produced in an undisturbed equilibrium pathway.

33. The method of claim 28, wherein the concentration of monatin produced is about 2.0 times greater than would be produced in an undisturbed eqiulibrium pathway.

35. A method of making a product in a multi-step equilibrium pathway, said method comprising:

a) allowing a first composition comprising a set of reactants and a set of facilitators to react to form a second composition comprising said product and one or more intermediates in said pathway;

b) removing, inhibiting, inactivating, or degrading the facilitators to inactive or slow down the multi-step pathway;

c) reactivating or increasing the speed of at least a product-producing reaction, while maintaining reactions competitive to the product-producing step in an inactive or slowed state, and optionally adding at least one component involved in the product-producing step in an amount sufficient to drive the product-producing step toward production of product; and

,

d) purifying the product from the second composition.

36. A system for making a product in a multi-step equilibrium pathway, said system comprising:

a) a first receptacle for receiving and holding components of a first mixture comprising one or more reactants and one or more facilitators, which components can react to produce a desired product and intermediates;

b) a first apparatus in communication with the receptacle for separating the reactants, product, and intermediates, from the facilitators;

c) means for recycling the facilitators back into the receptacle;

d) a second receptacle in communication with said first apparatus to receive the reactants, the desired product, and intermediates from the first apparatus, the second receptacle also adapted for receiving facilitators useful for activating a reaction for producing the desired product involving at least one of the reactants and intermediates and adapted for receiving components useful for driving the product-producing reaction to form the desired product;

e) means for purifying the desired product in communication with the second receptacle;

f) means for recycling reactants and optionally intermediates from the means for purifying to the first receptacle; and

, optionally,

g) means for recycling components from the means for purifying to the second receptacle.

37. A method, comprising:

purifying monatin from an original reaction mixture leaving a new mixture, selectively recycling at least one component from the original reaction mixture into the new reaction mixture, and producing an amount of monatin from an original amount of reactants that is greater than an amount of monatin that would be produced by the equilibrium process alone, wherein purifying comprises at least one ultrafiltration step and at least one chromotagraphy step.

38. A method according to claim 37, wherein the at least one chromatography step comprises a cation exchange chromatography step and chromatography on a non-polar absorbent resin.

39. A method according to claim 37, wherein the method further comprises a desalting step, and the at least one chromatography step comprises a cation exchange chromatography step, an anion exchange chromatography step and a chromatography on a non-polar absorbent resin.

40. A method according to claim 37, wherein the at least one chromatography step comprises chromatography with a non-polar synthetic absorbent resin.