INCREASING PRODUCT YIELD AND/OR THROUGHPUT BY

SUPERSATURATION OF SUBSTRATE

CROSS-REFERENCE TO RELATED APPLICATION

[0001 ] This application claims the benefit of the United States Provisional Patent Application, Serial No. 61 /491 ,710, filed 3 1 May 201 1 , entitled INCREASING PRODUCT YIELD AND/OR THROUGHPUT BY SUPERSTATURATION OF SUBSTRATE, which is hereby incorporated by reference in its entirety,

REFERENCE TO A "SEQUENCE LISTING"

[0002] A Sequence Listing is being filed concurrently with the electronic filing of this application, The accompanying Sequence Listing, identified as "Cargill N00 I 25USP1 sequence listing.txt", is herein incorporated by reference.

FIELD

[0003] The present disclosure relates generally to increasing the yield and/or throughput of a product through supersaturation of a solubility-limited substrate. Aspects of the disclosure are particularly directed to increasing the yield and/or throughput of monatin produced in a multi-step equilibrium pathway by increasing the concentration of tryptophan in solution through supersaturation.

BACKGROUND

[0004] Monatin (2-hydroxy-2-(indol-3-ylmethyl)-4-aminogIutaric acid) is a naturally occurring, high intensity or high potency sweetener that was originally isolated from the plant Sclerochiton ilicifolius, found in the Transvaal Region of South Africa, Monatin has the chemical structure:

[0005] Because of various naming conventions, monatin is also known by a number of alternative chemical names, including: 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid; 4- amino-2-hydroxy-2-(l H-indol-3-y]methy!)-pentanedioic acid; 4-hydroxy-4-(3- indoIylmethyI)glutamic acid; and, 3-(l -amino- l ,3-dicarboxy-3-hydroxy-but-4-y!)indoie.

[0006] Monatin has two chiral centers thus leading to four potential stereo isomeric configurations; the R,R configuration (the "R,R stereoisomer" or "R,R monatin"); the S,S configuration (the "S,S stereoisomer" or "S,S monatin"); the R,S configuration (the "R,S stereoisomer" or "R,S monatin"); and the S,R configuration (the "S,R stereoisomer" or "S,R monatin").

[0007] Reference is made to WO 2003/091396 A2, which discloses, inter alia, polypeptides, pathways, and microorganisms for in vivo and in vitro production of monatin. WO 2003/091396 A2 (see, e.g., Figures 1 -3 and 1 1 - 13) and U.S. Patent Publication No. 2005/282260 describe the production of monatin from tryptophan through multi-step pathways involving biological conversions with polypeptides (proteins) or enzymes. One pathway described involves converting tryptophan to indole-3-pyruvate ("I-3-P") (reaction (1 )), converting indole-3-pyruvate to 2- hydroxy 2-(indol-3-ylmethy!)-4-keto glutaric acid (monatin precursor, "MP") (reaction (2)), and converting MP to monatin (reaction (3)). The three reactions can be performed biologically, for example, with enzymes.

[0008] The use of high intensity sweeteners allows for the formulation of sweetened beverages with zero or significantly fewer calories, an important health and wellness feature as many countries are attempting to address weight related public health concerns. In particular a naturally occurring high intensity sweetener with a pleasing, sugar-like taste profile, such as monatin is desirable. Since it is desirable to have an economic source of monatin, there is a continuing drive to increase the efficiency of monatin-producing pathways, including the biological multistep pathway described above.

SUMMARY

[0009] Provided herein are methods and systems for increasing the yield and/or throughput of a product by supersaturating a substrate used to make the product. Monatin may be produced biosynthetically via a mu!ti-step equilibrium pathway, and contacting the mixture with a membrane. This results in a retentate having an increased concentration of substrate in solution compared to a concentration of substrate in solution upstream of the membrane. The increased concentration of substrate in solution in the retentate is greater than the substrate's normal solubility limit at the same temperature. In some embodiments, the increased concentration of substrate in solution in the retentate is at least three times greater than the substrate's normal solubility limit at the same temperature. [0010] In some embodiments, the product is monatin and the limited-solubility substrate is tryptophan, and the multi-step equilibrium pathway includes conversion of tryptophan to indole-3- pyruvate (I3P), I3P to 2-hydroxy 2-(indol-3-yImethyl)-4-keto glutaric acid (MP) and MP to monatin. In some embodiments, the yield of monatin is increased by at least about five percent compared to when the tryptophan is at or below its normal solubility limit at the same temperature at the start of the equilibrium pathway. In some embodiments, the product is produced by a batch process. In some embodiments, the product is produced by a continuous process.

[001 1] In some embodiments, the method above further comprises adding supplemental substrate to the retentate after the concentration of substrate in the retentate falls below a predetermined concentration. Prior to adding the supplemental substrate to the retentate, the method may further comprise heating a slurry containing the supplemental substrate to a first temperature until the substrate is fully soluble in solution and then cooling the solution to a second temperature that is less than the first temperature. Prior to adding the supplemental substrate to the retentate, the method may further comprise increasing solubility of the supplemental substrate in solution such that the concentration in solution is higher than its normal solubility limit. In some embodiments, the method above further comprises adding supplemental substrate to the retentate to maintain the concentration of substrate in the retentate at a constant concentration. The substrate may be tryptophan and the concentration may be between about 100 and about 1 30 mM.

[0012] In one embodiment, a method of increasing production of monatin in a multi-step equilibrium pathway includes combining water, tryptophan, pyruvate, a first enzyme and a second enzyme to form a mixture, and increasing a solubility of tryptophan in the mixture beyond its normal solubility limit at the same temperature. In some embodiments, increasing the solubility of tryptophan beyond its normal solubility limit includes contacting the mixture with a membrane to retain a second mixture having an increased concentration of tryptophan in solution compared to the first mixture. The concentration of tryptophan in the second mixture may be at least three times greater than its normal solubility limit at the same temperature. The concentration of tryptophan in the second mixture may be at least about 1 15mM at about 1 5 degrees Celsius.

[0013] In some embodiments of the method above, increasing the solubility of tryptophan beyond its normal solubility limited is performed after the ractions to convert tryptophan to monatin have reached equilibrium. In some embodiments of the method above, increasing the solubility of tryptophan beyond its normal solubility limit is performed at least about 24 hours after the mixture comprising water, tryptophan , pyruvate, a first enzyme and a second enzyme is formed. [0034] In one embodiment, a method of using a membrane to produce monatin includes combining water, tryptophan, pyruvate, a first enzyme and a second enzyme to form a mixture in which the tryptophan in the mixture is at a first concentration, and contacting the mixture with a membrane, resulting in a retentate comprising tryptophan at a second concentration that is greater than the first concentration. The second concentration may be greater than tryptophan's normal solubility limit at the same temperature. In some embodiments, the second concentration is at least three times greater than the normal solubility limit at the same temperature.

[0015] In some embodiments of the method above, the monatin is produced by a continuous process after a given start-up time. The method may further comprise adding supplemental tryptophan to the retentate to maintain a constant concentration of tryptophan in the retentate. The method may further comprise adding supplemental tryptophan to the retentate after the concentration of tryptophan in the retentate falls below a predetermined concentration. In some embodiments, the supplemental tryptophan added to the retentate is fully soluble in solution and is at a concentration greater than its normal solubility limit at the same temperature. In some embodiments, the method further comprises removing a portion of the retentate.

[0016] In some embodiments of the method above, the monatin is produced by a batch process. The method may further comprise leaving the mixture for a time sufficient to reach equilibrium prior to contacting the mixture with a membrane. The mixture may be left for at least about 24 hours; in other embodiments, the mixture may be left for at least about 36 hours. In some embodiments, the method above further comprises leaving the mixture for at least about 24 hours after contacting the mixture with the membrane. In some embodiments, the method above further comprises contacting the mixture with the membrane a second time.

[0017] In some embodiments, a method of supersaturating tryptophan is used in a batch process for the production of monatin. In some embodiments, a method of supersaturating tryptophan is used in a continuous or semi-continuous process for the production of monatin.

[001 8] In some aspects of some or all of the embodiments of the invention, monatin is produced in a multi-step equilibrium pathway in which tryptophan is converted to indole-3- pyruvate (I3P), I3P is converted to 2-hydroxy 2-(indoi-3-y!methyl)-4-keto glutaric acid (MP), and MP is converted to monatin. In some aspects of some or all of the embodiments of the invention, the tryptophan is D-tryptophan and the monatin is a steroisomericaliy-enriched R,R monatin. In some aspects of some or all of the embodiments of the invention, the first enzyme is an aminotransferase and the second enzyme is an aldolase. [0019] In some aspects of some or ai! of the embodiments of the invention, the membrane is a reverse osmosis membrane. In some aspects of some or all of the embodiments of the invention, the membrane is a nanofiltration membrane. In some aspect of some or ali of the embodiments of the invention, the mixture contacting the membrane is at a pressure between about 500 and about 900 psi.

[0020] In some aspects of some or all of the embodiments of the invention, after the mixture contacts the membrane, the retentate comprises at least about 90 percent of the tryptophan contained in the mixture; in other aspects, the retentate comprises at least about 95 percent of the tryptophan contained in the mixture. In some aspects of some or all of the embodiments of the invention, after the mixture contacts the membrane, a volume of the retentate is about 10 to about 40 percent of the volume of the mixture; in other aspects, the volume is about 30 to about 35 percent of the volume of the mixture.

[0021 ] The details of one or more non-limiting embodiments of the invention are set forth in the description below. Other embodiments of the invention should be apparent to those of ordinary skill in the art after consideration of the present disclosure.

DETAILED DESCRIPTION

[0022] Monatin has an excellent sweetness quality, and depending on a particular composition, monatin may be several hundred times sweeter than sucrose, and in some cases thousands of times sweeter than sucrose. Monatin has four stereoisomeric configurations which exhibit differing levels of sweetness. The S,S stereoisomer of monatin is about 50-200 times sweeter than sucrose by weight. The R,R stereoisomer of monatin is at least about 2000-2400 times sweeter than sucrose by weight.

[0023] As used herein, unless otherwise indicated, the term "monatin" is used to refer to compositions including any combination of the four stereoisomers of monatin (or any of the salts thereof), including a single isomeric form.

[0024] As used herein, the term "solubility-limited" in reference to a solubility-limited substrate or molecule means the substrate or molecule becomes insoluble in water at a particular temperature after a given concentration is reached.

[0025] As used herein, the term "normal solubility limit" means the maximum concentration at which the substrate or molecule is fully soluble in water at a particular temperature, and may also be known as the saturation concentration. [0026] As used herein, the term "fully soluble" means all of the substrate or molecule is dissolved in water, and no crystals or appreciable crystalline or precipitate structure are present.

[0027] As used herein, the term "supersaturated" or "supersaturation" means the substrate or molecule is fully soluble and metastab!e in water at a concentration that is greater than its normal solubility limit at a particular temperature, it is recognized that when reference is made herein to a 'stable' solution that is supersaturated, the solution is actually in a metastable state,

[0028] As used herein, the term "about" encompasses the range of experimental error that occurs in any measurement.

[0029] Monatin may be synthesized in whole or in part by one or more of a biosynthetic pathway, chemically synthesized, or isolated from a natural source. If a biosynthetic pathway is used, it may be carried out in vitro or in vivo and may include one or more reactions such as the equilibrium reactions provided below as reactions (I )-(3). In one embodiment is a biosynthetic production of monatin via enzymatic conversions starting from tryptophan and pyruvate and following the three equilibrium reactions below:

( I ) Tryptophan Indole-3-pyruvate (13 P)

+ Pyruvate ^■ ^ - ₊ Alanine

Aminotransferase

(2) I3P + Pyruvate Monatin precursor (MP)*

Aldolase

(3) MP + Alanine Monatin + Pyruvate

Aminotransferase

* Monatin precursor (MP) is 2-hydrox.y 2-(indoI-3-yimethyl)-4-keto glutaric acid.

[0030] The following side-reactions may also occur, resulting in production of hydroxymethyl-oxo-glutarate (HMO), hydroxymethylglutamate (HMG) or a combination thereof:

(4) Pyruvate + Pyruvate HMO

Aldolase

(5) HMO + Alanine HMG + Pyruvate

Aminotransferase

[0031 ] In the pathway shown above, in reaction ( 1 ), tryptophan and pyruvate are enzymatically converted to indole-3-pyruvate (13 P) and alanine in a reversible reaction. As exemplified above, an enzyme, here an aminotransferase, is used to facilitate (catalyze) this reaction. In reaction ( 1 ), tryptophan donates its amino group to pyruvate and becomes BP. In reaction (3 ), the amino group acceptor is pyruvate, which then becomes alanine as a result of the action of the aminotransferase. The amino group acceptor for reaction (1 ) is pyruvate; the amino group donor for reaction (3) is alanine. The formation of indole-3-pyruvate in reaction (1 ) can also be performed by an enzyme that utilizes other a-keto acids as amino group acceptors, such as oxaloacetic acid and a-keto-glutaric acid. Similarly, the formation of monatin from MP (reaction (3)) can be performed by an enzyme that utilizes amino acids other than alanine as the amino group donor. These include, but are not limited to, aspartic acid, glutamic acid, and tryptophan.

[0032] Some of the enzymes useful in connection with reaction (1 ) may also be useful in connection with reaction (3). For example, aminotransferase may be useful for both reactions (1 ) and (3). The equilibrium for reaction (2), the aldolase-mediated reaction of indole-3-pyruvate to form MP (i.e. the aldolase reaction), favors the cleavage reaction generating indo!e-3-pyruvate and pyruvate rather than the addition reaction that produces the alpha-keto acid precursor to monatin (i.e. MP). The equilibrium constants of the aminotransferase-mediated reactions of tryptophan to form indole-3-pyruvate (reaction ( 1 )) and of MP to form monatin (reaction (3)) are each thought to be approximately one. Methods may be used to drive reaction (3) from left to right and prevent or minimize the reverse reaction. For example, an increased concentration of alanine in the reaction mixture may help drive forward reaction (3). Reference is made to US Publication No. 2009/0198072 (Application Serial No. 12/315,685), which is also assigned to Cargill, the assignee of this application.

[0033] The overall production of monatin from tryptophan and pyruvate is referred to herein as a multi-step pathway or a multi-step equilibrium pathway. A multi-step pathway refers to a series of reactions that are linked to each other such that subsequent reactions utilize at least one product of an earlier reaction. In such a pathway, the substrate (for example, tryptophan) of the first reaction is converted into one or more products, and at least one of those products (for example, indoIe-3-pyruvate) can be utilized as a substrate for the second reaction. The three reactions above are equilibrium reactions such that the reactions are reversible. As used herein, a multi-step equilibrium pathway is a multi-step pathway in which at least one of the reactions in the pathway is an equilibrium or reversible reaction.

[0034] Because the R.R stereoisomer of monatin is the sweetest of the four stereoisomers, it may be preferable to selectively produce R,R monatin. For purposes of this disclosure, the focus is on the production of R.R monatin. However, it is recognized that the present disclosure is applicable to the production of any of the stereoisomeric forms of monatin (R,R; S,S; S,R; and R,S), alone or in combination.

[0035] In some embodiments, the monatin consists essentially of one stereoisomer - for example, consists essentially of S,S monatin or consists essentially of R,R monatin. In other embodiments, the monatin is predominately one stereoisomer - for example, predominately S,S monatin or predominately R,R monatin. "Predominantly" means that of the monatin stereoisomers present in the monatin, the monatin contains greater than 90% of a particular stereoisomer. In some embodiments, the monatin is substantially free of one stereoisomer - for example, substantially free of S,S monatin. "Substantially free" means that of the monatin stereoisomers present in the monatin, the monatin contains less than 2% of a particular stereoisomer. In some embodiments, the monatin is a stereoisomericaily-enriched monatin mixture. "Stereoisomericaily-enriched monatin mixture" means that the monatin may contain more than one stereoisomer and at least 60% of the monatin stereoisomers in the mixture is a particular stereoisomer. In other embodiments, the monatin contains greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of a particular monatin stereoisomer. In another embodiment, a monatin composition comprises a stereoisomericaily-enriched R,R-monatin, which means that the monatin comprises at least 60% R,R monatin. In other embodiments, stereoisomericaily-enriched R.R-monatin comprises greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of R,R monatin.

[0036] For example, to produce R,R monatin using the three-step pathway shown above (reactions (l)-(3)), the starting material may be D-tryptophan, and the enzymes may be a D- aminotranferase and an R-specific aldolase. The three reactions, which are shown be!ow, may be carried out in a single reactor or a multiple-reactor system.

(6) D-Tryptophan lndole-3-pyruvate (I3P)

+ Pyruvate - ^{» ^} "~ + D-Alanine

D- Am inotran sferase

(7) I3P + Pyruvate -* ^{^ ^} R-MP

R-specific Aldolase

(8) R-MP + D-Aianine - ^» R.R-Monatin + Pyruvate

D- A m i notransfera se

[0037] In an embodiment in which a single reactor is used, the two enzymes (i.e. the D- aminotransferase and the R-specific aldolase) may be added at the same time and the three reactions may run simultaneously. The same enzyme may be used to catalyze reactions (6) and (8). A D-aminotransferase is an enzyme with aminotransferase activity that selectively produces, in the reactions shown above, D-alanine and R,R-monatin. An R-specific aldolase is an enzyme with aldolase activity that selectively produces R-MP, as shown in reaction (7) above. Although a focus in the present disclosure is on R,R monatin, it is recognized that the method and system of increasing monatin yield and/or throughput by supersaturation of tryptophan is applicable to any of the stereoisomeric forms of monatin.

[0038] There are multiple alternatives to the above pathway (i.e. reactions (6)-(8)) for producing R,R-monatin. For example, L-tryptophan may be used as a starting material instead of D-tryptophan. In that case, an L-aminotransferase may be used to produce indole-3-pyruvate and L-alanine from L-tryptophan, Because L-alanine is produced, this pathway may require the use of an alanine racemase to convert the L-alanine to D-alanine, thus adding a fourth reaction to the monatin production pathway. (D-alanine is required to produce R,R monatin from the R- stereoisomer of monatin precursor (R-MP)). In addition to requiring another enzyme (alanine racemase), undesired side reactions may also occur in this pathway. For example, L-alanine may react with the L-aminotransferase to produce R,S-monatin, or D-alanine may react with 13 to form D-tryptophan, resulting in a racemate of L-tryptophan and D-tryptophan, which has poor solubility. Some disadvantages of this pathway may be avoided by using a two reactor system as opposed to a single reactor system. It is recognized that there are additional alternatives not specifically disclosed herein for performing the three-step equilibrium pathway to produce monatin. The method and system described herein for increasing monatin yield and/or throughput by supersaturation of tryptophan is applicable to monatin produced using alternative pathways to what is disclosed herein. For example, the focus herein is on the production of R,R monatin starting from D-tryptophan, however, it is recognized that the method and systems described herein are applicable to starting with L-tryptophan.

[0039] As described above, in some pathways, it may be preferable to perform the monatin producing reactions in two or more separate reactors, while in other pathways it may be preferable to use a single reactor system. The decision to use a one reactor or a multiple reactor system may depend, in part, on whether D-tryptophan or L-tryptophan is used as a starting material. A single reactor system is obviously simpler in design, eliminating the need for a second reactor, as well as eliminating, in some cases, a need for a separation step between the first and second reactors.

[0040] The resulting monatin produced using the method described above may be present in a mixture that contains other components, including starting materials, intermediates, side products of the monatin-producing reactions or combinations thereof. It is preferable to separate the monatin from these other components, which may include, for example, tryptophan, pyruvate, alanine, I3P, MP, HMG and HMO. It may be preferable to separate and purify monatin from this mixture in order to achieve a particular purity of monatin; such separation and purification methods are not the focus of the present disclosure.

[0041 ] As described above, monatin may be produced by the three step pathway disclosed above, and each of the three reactions are reversible reactions. As such, it may be challenging to produce a high yield of monatin, particularly given that some of the intermediates may be unstable and side reactions may also occur. Moreover, the solubility of tryptophan, one of the starting materials or substrates in the disclosed pathway, is a limiting factor in the conversion of tryptophan to monatin. Given the significant commercial need to maximize monatin yield, a method of increasing the solubility of tryptophan is desirable. The present disclosure focuses on a method and system for increasing the concentration of tryptophan available for the monatin-producing reactions through supersaturation. As shown below, supersaturation of tryptophan results in an increase in monatin yield. As described below, tryptophan supersaturation may be achieved by various methods and at different points in the process of producing monatin.

[0042] Monatin yield is determined herein using the following equation:

(9) ∑ m ^Q -SSmonatin created

mass _{rryptophan dosed} + mass _{Pyruvate dosed}

[0043] As described below, supersaturation of tryptophan as described herein may be used to increase product throughput. Monatin throughput is defined herein as the mass of monatin created over a particular period of time for a given set of equipment. As shown in the example below, the impact of supersaturation of tryptophan on monatin production may also be expressed in terms of the mass increase of monatin.

[0044] The monatin-producing reactions described above are commonly performed at a temperature between about 1 0 degrees Celsius and about 25 degrees Celsius. In some embodiments, the reactions are performed at a temperature of about 15 degrees Celsius. The solubility of tryptophan varies, in part, as a function of the temperature of the tryptophan/solvent mixture. The normal solubility limit of L-tryptophan or D-tryptophan (separately) in water is approximately 35 mM ± 5 mM at 15 °C. A mixture of L-tryptophan and D-tryptophan has a different solubility limit. Thus, for purposes of this disclosure, when reference is made to the solubility of tryptophan, it is recognized that reference is made to either the L- enantiomer or the D- enantiomer of tryptophan, but not to a mixture of the enantiomers. It is recognized that the normal solubility limit of tryptophan at a given temperature will also depend on the presence of salts and other ions and components in the solvent mixture.

[0045] Methods may be used to supersaturate tryptophan or increase the solubility of tryptophan above its normal solubility limit. Supersaturation is a metastable state and may be used to increase monatin yield and/or throughput. A higher concentration of tryptophan in solution means there is more tryptophan available to be converted to indole-3-pyruvate (13P) and ultimately monatin. In other words, having more tryptophan in solution shifts the three equilibrium reactions in the forward direction, resulting in a higher production of monatin. Supersaturation, as exhibited below, allows for a significantly higher concentration of tryptophan available in the solution to react with the enzymes and perform the conversions outlined above. Through supersaturation, it is possible to achieve stable water solutions at 1 degrees Celsius having a tryptophan concentration up to about 120 or about 130 mM. In some embodiments, the maximum stable concentration of tryptophan in a solution may be more or less depending on the presence of other salts in the solution.

[0046] As described further below, tryptophan may be supersaturated thermally by increasing the temperature of a solution containing tryptophan. Alternatively, supersaturation may be achieved by removing water from a solution containing tryptophan (i.e. dewatering), for example, by using a membrane.

[0047] In some embodiments, tryptophan supersaturation is achieved by increasing the temperature of a slurry of tryptophan (a combination of soluble and insoluble tryptophan) in water until essentially all of the tryptophan is dissolved in solution. The solution is then cooled to a desired operating temperature for performing the monatin-producing reactions (for example, about 1 5 degrees Celsius). The soluble concentration of tryptophan achievable through supersaturation will depend in part on the temperature that the slurry is heated to. It is recognized that increasing the temperature to a temperature greater than the operating temperature will result in increased solubility of tryptophan. Thus an increased concentration of soluble tryptophan results from heating the slurry above about 20 degrees Celsius. In some embodiments, the tryptophan slurry may be heated to a temperature ranging between about 20 and about 120 degrees Celsius; in other embodiments, between about 80 and about 105 degrees Celsius. In some embodiments, the slurry is heated to about 100 degrees Celsius. As demonstrated in the Examples below, a stable solution of tryptophan at a concentration of 130 mM was achieved in water at 1 5 degrees Celsius, after the slurry was heated to about 100 degrees Celsius. As stated above, the normal solubility limit of tryptophan in water at 15 degrees Celsius is about 35 mM. Thus soluble concentrations of tryptophan ranging between 40 and 130 mM can be achieved through thermal supersaturation.

[0048] In some embodiments, the tryptophan slurry may also include pyruvate and the enzymes for the monatin-producing reactions. Supersaturation of tryptophan is achieved as described above - heating at a given temperature and then cooling. The temperature may be limited due to the thermal stability of the enzymes, thus the slurry may be heated to a temperature in which the enzymes are still thermally stable.

[0049] In some embodiments, tryptophan supersaturation may be achieved thermally by starting with a dilute solution of tryptophan (rather than a slurry). The dilute solution is heated at a temperature that causes evaporation of excess water in the solution until a target concentration of tryptophan is reached.

[0050] In some embodiments, a membrane is used to remove water from a mixture comprising at least tryptophan and water. The membrane is selected based on its ability to retain tryptophan, thus creating a retentate having a concentration of tryptophan that is higher than the concentration of tryptophan in the original mixture. In some cases, the membrane may be selected based on its molecular weight cut-off. In some embodiments, over 90% of the tryptophan is retained by the membrane; in other embodiments, over 95% of the tryptophan is retained by the membrane.

[00 1 ] In some embodiments, the mixture passing over the membrane not only comprises tryptophan and water, but also comprises other substrates, intermediates and enzymes found in the monatin-producing reactions. In that case, the membrane is selected based on its ability to retain all of the carbon-containing molecules, while letting water pass through the membrane. In some embodiments, over 90% of the carbon-containing molecules are retained by the membrane; in other embodiments, over 95% of the carbon-containing molecules are retained by the membrane.

[0052] In some embodiments, the membrane is a reverse osmosis (RO) membrane. More specifically, in one embodiment, the RO membrane is a tubular RO membrane, such as, for example, AFC 99 from PCI Membranes. In another embodiment, the RO membrane is a spiral wound RO membrane, such as, for example, SW30-4040 from DOW Filmtec. In some embodiments, the membrane is a nanofiltration membrane, such as, for example, DL I 812 from GE. One of skill in the art recognizes that the particular membrane selected depends, in part, on the composition of the mixture, the target composition of the retentate, and the volume and flow rate of the mixture. [0053] In those embodiments in which a membrane is used to achieve supersaturation of tryptophan, the membrane dewatering step may be performed at various points within the monatin- producing process described above. In one embodiment, a method of using a membrane includes creating a mixture that includes water, tryptophan and pyruvate, as well as the enzymes that facilitate the reactions in the multi-step equilibrium pathway. After creating the mixture, a sufficient amount of time passes such that equilibrium of the reactions is reached and the mixture is fully soluble. At that point, the mixture includes monatin, tryptophan, pyruvate, alanine, I3P, MP, HMO and HMG. In some embodiments, the time to reach equilibrium is at least 24 hours; in other embodiments, the time to reach equilibrium is at least 36 hours; and in yet other embodiments, at least 48 hours. Following equilibrium, the mixture is passed across or comes into contact with a membrane. As a result, the water permeates through the membrane while the tryptophan and other carbon-containing molecules, including monatin, are retained by the membrane (forming a retentate). Given the significant reduction in volume of the retentate, as compared to the mixture, there is a significantly higher concentration of tryptophan in the retentate than the original feed.

[0054] When the mass of the main components in the retentate are compared to the mass of those components in the mixture, the result is an increase in monatin and MP, and a decrease in I3P, tryptophan and pyruvate. Thus the membrane dewatering step concentrates the components in the retentate, thereby perturbing the previously reached equilibrium, resulting in a new equilibrium. As an example, about 40 hours after creating the mixture and thus initiating the reactions, but prior to being dewatered using a tubular RO membrane, the monatin yield of the mixture was 1 1 ,7%. After the mixture was dewatered using the RO membrane, the monatin yield of the retentate was 16.7%, resulting in a 5% yield increase. The retentate was then held for about five days and the monatin yield was 20.7%, resulting in an additional 4% increase. The mass increase of monatin as a result of the dewatering step and the five-day hold was 77%. As shown in the examples below, the hold step after the RO concentration or dewatering step allows time for the enzymes to perform the monatin-producing reactions. However, even while the dewatering step is taking place and the concentration of tryptophan is increasing, the monatin-producing reactions are occurring. It is recognized that the monatin yield increase will depend in part on how long it takes to complete the dewatering step and how long the retentate is held after completion of the dewatering step.

[0055] In another example, the monatin yield of a mixture including monatin, tryptophan, pyruvate, alanine, 13 P, MP, HMO and HMG measured about 36 hours after being created was 16.4%. After the mixture was dewatered using an RO membrane, the monatin yield of the retentate was 20.1 %. The mass increase of monatin as a result of the deatering step was 22%.

[0056] In some embodiments in which an RO membrane is used for dewatering, the volume of the retentate is equal to about 30 to about 35 percent of the volume of the original mixture. Thus, for those embodiments, the concentration of the components in the retentate, including tryptophan, immediately following the dewatering step is at least 3 times higher than the concentration in the mixture. In other embodiments, the retentate may be further concentrated, depending in part, on the composition of the original mixture. The extent of concentration also depends, in part, on the desired concentration of tryptophan in the retentate. In some embodiments, the volume of the retentate is between about 1 and about 20 percent of the volume of the original mixture; in other embodiments, between about 10 and about 40 percent of the volume of the original mixture.

[0057] In one embodiment, the membrane dewatering step is performed before the mixture, containing water, tryptophan, pyruvate, and the monatin-producing enzymes, has reached equilibrium. After the enzymes are added to the mixture, the membrane system is activated and continues until a desired concentration of tryptophan (for example, between about 1 15 m and about 130 mM) is reached. The membrane system may then be shut off and in some embodiments, the retentate is left or held for a period of time, for example, 24 hours, during which the monatin- producing reactions are occurring.

[0058] In some embodiments, after the first dewatering step and hold, a second dewatering step may be performed to concentrate the tryptophan back up to the desired concentration. Even with a second dewatering step, in some cases, this method may be completed in a shorter period of time as compared to the methodology in which dewatering is not performed until after the mixture has reached equilibrium. A favorable monatin yield is observed using this method. In one example, when two dewatering steps are performed using a spiral RO membrane, with a 24-hour hold following each dewatering step, an additional 5.5% yield increase was observed between the first dewatering step and the second dewatering step. In other embodiments, additional dewatering steps may be performed. The number of dewatering steps and the hold time (if any) may be determined based in part on the desired monatin yield and optimizing the overall efficiency of the process for producing monatin.

[0059] A driving force of the membrane dewatering step is the pressure difference between the feed side of the membrane and the permeate side of the membrane. The inlet pressure of the mixture being fed into the membrane will depend, in part, on the feed flow rate, the type and size of the membrane, as well as the composition of the mixture. In some embodiments, the inlet pressure is between about 200 and about 1500 psi; in other embodiments, the inlet pressure is between about 500 and about 900 psi. In some embodiments, the mixture may contain a higher salt content, which may impact the operating pressure.

[0060] As described above, there are various ways to achieve supersaturation of tryptophan. To be able to supersaturate tryptophan through dewatering with a membrane, it is important to start with a soluble system. In contrast, tryptophan can be supersaturated thermally starting from a slurry. In one embodiment, a method of supersaturating tryptophan includes starting with a tryptophan-containing slurry, using heat to create a soluble system and then further increasing a concentration of tryptophan using a membrane.

[0061 ] In some embodiments, additional tryptophan or supplemental tryptophan is added to the mixture (containing water, tryptophan, pyruvate, intermediates, monatin and enzymes) as the tryptophan is consumed in the monatin-producing reactions and the tryptophan concentration decreases. Supplemental tryptophan may be added in order to maintain or return the tryptophan to a supersaturated level. This results in an increased monatin yield and/or throughput since maintaining or returning the tryptophan to a higher concentration continues to drive the monatin- producing reactions forward.

[0062] The supplemental tryptophan may be at a supersaturated concentration prior to being added to the mixture, and the supersaturation may be achieved thermally, by membrane dewatering, or a combination of the two. In some embodiments, all or a portion of the supplemental tryptophan may be recycled from downstream in the process (for example, following separation and/or purification steps not focused on herein). In some embodiments, the supplemental tryptophan may be continuously fed to the mixture to maintain tryptophan at a particular concentration. For example, it may be desired to maintain tryptophan at a concentration ranging between about 100 and about 130 mM. In some embodiments, supplemental tryptophan may be periodically added to the mixture, for example, when the concentration in the mixture falls be!ow a predetermined concentration level.

[0063] The examples below are directed to a batch process; however, it is recognized that the methods and systems described herein are applicable to a continuous or semi-continuous process. In some embodiments, the method includes continuously operating a membrane system combined with a feed/retentate tank containing the mixture with water, tryptophan, pyruvate, intermediates, monatin and enzymes. Because the membrane system is continuously running, the mixture is also the retentate. Once the enzymes are added to the feed tank, the monatin-producing reactions are initiated and thus the retentate also includes monatin. Water is removed from the system as permeate, resulting in an increased or supersaturated concentration of tryptophan. Moreover, an additional volume of the retentate/reaction mixture may be continuously removed from the retentate side of the membrane. The removed-retentate may then undergo additional processing to separate and/or purify monatin from the additional components in the removed- retentate. To maintain a continuous or semi-continuous process, supplemental tryptophan is added back to the feed tank after a given start-up time. Moreover, pyruvate and enzymes are also added to the feed tank.

[0064] The supplemental tryptophan may be a dilute solution (at or below the norma! solubility limit) or a supersaturated solution, which may be achieved thermally, by membrane dewatering, or a combination thereof. In some embodiments, the supplemental tryptophan may be added in order to maintain tryptophan at a predetermined concentration in the retentate, for example, between about 100 and about 130mM. All or a portion of the supplemental tryptophan may be recycled from downstream in the process. All or a portion of the pyruvate and enzymes added back to the feed tank may also be recycled from downstream in the process.

[0065] As similarly described above, by maintaining the tryptophan at a supersaturated concentration and operating under a continuous or semi-continuous process, it is possible to continue to push the equilibrium reactions forward and produce more monatin over a shorter period of time, resulting in increased monatin throughput. This is significant due, in part, to the length of time required to reach equilibrium once the monatin-producing reactions are initiated during start-up.

[0066] As stated above, the presence of salts and other ions in solution will impact the concentration of soluble tryptophan achievable through supersaturation. It is also recognized that other components, such as cofactors, substrates, etc., which may be present in a tryptophan- containing mixture described herein may also impact the supersaturation concentration of tryptophan. For example, the salts may create nucleation sites that initiate or propagate the formation of tryptophan crystals.

[0067] It is recognized that there are additional ways of achieving supersaturation of tryptophan, and other solubility-limited substrates, not specifically disclosed herein. The focus of the present disclosure is the method of increasing the product yield and/or throughput of an equilibrium pathway through supersaturation of a solubility-limited substrate. Although the disclosure herein and the examples below are focused on methods of supersaturating tryptophan for increasing the production of monatin, it is recognized that the methods disclosed are applicable to other solubility-limited molecules, and in particular to a substrate or starting material of limited solubility which is used in a multi-step equilibrium pathway.

EXAMPLES

[0068] Aspects of certain embodiments in accordance with aspects of the invention are illustrated in the following non-limiting examples. The materials and methods described in these examples are illustrative and not intended to be limiting.

[0069] In all of the examples below, the monatin produced was predominantly R,R monatin.

[0070] In the examples below that included enzymes, a D-aminotransferase (see SEQ ID NO: 1 and SEQ ID NO:2) and an aldolase (see SEQ ID NO:3 and SEQ ID NO:4) were used.

Example 1

[0071 ] In this example, supersaturation of D-tryptophan was achieved thermally by heating a slurry of D-tryptophan in water.

[0072] A series of suspensions of D-tryptophan (>99% purity) in water from 6.12 g/L to 40.8 g/L (nominally 30 to 200 mM) in increments of 2.04 g/L (nominally 10 mM) were prepared in 20 mL tubes with threaded caps and heated to 100°C to fully dissolve the D-tryptophan. Weights and volumes of D-tryptophan and water are given in Table 1. After heating, the solutions were then placed in a water bath at about 15°C for about 72 hours.

Table 1. Composition of D-tryptophan solutions

[0073] After heating for 1 hour at 100°C, D-tryptophan was completely dissolved in all of the samples from Table 1. After cooling to 15°C and holding for about 72 hours, solutions having a concentration of D-tryptophan greater than 160 mM (samples N through R) had crystals present.

[0074] To determine if the clear solutions were supersaturated, a few seed crystals of D- tryptophan were added to each of the solutions ranging between concentrations of 30 through 1 50 mM (Samples A-M). Sixteen hours after adding the seed crystals, only Samples L and M at concentrations of 140 and 150 mM showed crystal growth. This indicates, for this example, a maximum stable supersaturation solubility of 130 mM for D-tryptophan in water at 15°C.

Example 2

[0075] In this example, supersaturation of L-tryptophan was achieved thermally by heating a slurry of L-tryptophan in water.

[0076] A series of solutions of L-tryptophan (>99% purity) in water from 6.12 to 30.6 g/L (nominally 30 to 150 mM) were prepared in 30 mL tubes with threaded caps and heated to 3 00°C to fully dissolve the L-tryptophan. Weights and volumes of L-tryptophan and water are given in Table 2. After heating, the solutions were then placed in a water bath at about 15°C for about 24 hours.

Table 2. Composition of L-tryptophan solutions

[0077] After heating for 1 hour at 100°C, all solutions were completely dissolved. After cooling to 1 °C for about 24 hours, Sample J ( 150 mM) had crystals present. A few seed crystals of L-tryptophan were added to each of the solutions to determine if the clear solutions were supersaturated. After about 48 hours, samples at concentrations of 120, 130 and 140 mM showed crystal growth. This indicates, for this example, a maximum stable superaturation solubility of 100 mM for L-tryptophan in water at 1 5°C.

[0078] There is no reason why D-tryptophan and L-tryptophan (separately) should have different supersaturation limits. Thus it is believed that the difference between 100 mM for L- tryptophan and 130 mM for D-tryptophan is within experimental error. The examples below further exemplify that stable supersaturation solubilities of about 120 or 130 mM for D-tryptophan were achieved. Possible explanations for the difference in solubility between D-tryptophan and L- tryptophan could be due, in part, to differences in the way the testing was done and/or differences in how the particular samples of D-tryptophan from Example 3 and L-tryptophan from this example were produced.

Example 3

[0079] In this example, supersaturation of D-tryptophan was achieved through membrane dewatering after the reactions in the monatin-producing pathway had reached equilibrium,

[0080] An initial 10 L solution was made in a bioreaction vessel, according to Table 3. The bioreaction was allowed to proceed at about 15 °C for about 40 hours after the enzymes were added. The headspace of the bioreaction was sparged with nitrogen to protect the mixture from oxygen exposure.

Table 3. Components in Initial Solution

[0081 ] After about 40 hours, the bioreaction mixture was transferred from the bioreaction vessel to a nitrogen blanketed tank. Prior to performing the dewatering or concentration step, the composition of the bioreaction mixture was sampled and the results are shown in Table 4. The total volume of the mixture in Table 4 remained at about 10 L. Tabie 4. Composition of Bioreaetion Mixture after 40 hours

[0082] The monatin yield of the mixture in Table 4 (using formula (9) from above) was 1 1.7%.

[0083] The mixture was then concentrated on a tubular RO membrane having 0.024 m ² surface area, AFC99 RO-type from PCI Membranes. The system consisted of the feed tank with a cooling coil, a feed pump with inline heat exchanger, a membrane housing, and a permeate collection tank. The system was operated at about 500 psi inlet pressure, and the feed tank was under a constant nitrogen blanket. The target operating temperature was 15°C, however, the temperature was not monitored.

[0084] Triplicate samples were taken of the retentate and permeate solutions during the concentration, and a final permeate and retentate sample was taken at the end of the concentration. The total volume of the permeate collected during the concentration was about 6.3 L. The total volume of the retentate was about 3.2 L. Table 5 shows the composition of the retentate essentially immediately after the dewatenng or concentration step, which, excluding shut downs and operating interruptions, operated for about 21 hours.

Table 5. Composition of Bioreaetion Mixture after Concentration Step

[0085] The monatin yield of the mixture in Table 5 was 16.7%. Thus, the dewatering or concentration step increased the monatin yield by 5%.

[0086] After the concentration step, the mixture was held for an additional five days to see the impact on yield if ample time was given for the equilibrium reactions to proceed. The results are shown in Table 6. Table 6. Composition of Bioreaction Mixture after a five day hoid

[0087] The monatin yield of the mixture in Table 6 was 20.7%. Thus an additional yield increase of 4% was observed by leaving the mixture for a significant time period following the dewatering step. A yield increase of 9% was observed through the combination of dewatering followed by a hold. The monatin mass after the hold, as compared to the monatin mass after 40 hours (see Table 4), resulted in a monatin mass increase of 77% ((93.90 g - 53,07 g)/53.07 g).

Example 4

[0088] In this example, supersaturation of tryptophan through dewatering was performed prior to adding the enzymes to the mixture containing tryptophan and pyruvate. More specifically, a solution of D-tryptophan and pyruvate was supersaturated using a tubular RO membrane. The solution was then dosed with the remaining materials for carrying out the monatin-producing reactions, including enzymes, and held for about 48 hours. The mixture was then concentrated on a tubular RO membrane again.

[0089] An initial solution of D-tryptophan and sodium pyruvate was made in a nitrogen blanketed tank according to Table 7.

Table 7. Components in Initial Solution

[0090] The D-tryptophan and pyruvate fully went into solution. The concentration of D- tryptophan in the solution was 28.4 inM, which is below the normal solubility limit. The test solution, was then concentrated on a tubular AFC99 RO membrane, having 0.88 m ² surface area, from PCI Membranes. The system consisted of a feed tank with a cooling coil, a feed pump with inline heat exchanger, a membrane housing, and two permeate collection vessels. The system was operated at 500 psi inlet pressure and an average temperature of about 18.5°C, and the feed tank was under a constant nitrogen blanket. After the concentration was completed, nitrogen was used to displace the solution remaining in the membrane housing back into the feed (retentate) tank. A membrane flush of 10 L was completed to recover the remaining tryptophan and pyruvate to determine how much stayed behind in the housing and on the membrane surface.

[0091 ] An initial sample of the feed was taken in triplicate before starting the concentration. During the concentration, samples were taken of the retentate and permeate solutions for every 5 kg of permeate removed. Triplicate samples were taken of the final permeate and retentate at the end of the concentration. The total permeate collected during the concentration was approximately 75 L.

[0092] The total volume of the retentate was about 24.0 L. The composition of the retentate is shown in Table 8. D-tryptophan was supersaturated to a concentration of 1 15.5 mM No tryptophan or pyruvate was measured in the permeate while the membrane flush sample had about 19.0 g of tryptophan and about 14.8 g of pyruvate.

Table 8. Composition of Solution after Concentration

[0093] The solution from Table 8 was then transferred from the plastic tank to a glass bioreactor vessel. Overnight the supersaturated solution was agitated in the reactor vessel at about 14- 15°C under a constant nitrogen blanket. This solution was initially stable, but D-tryptophan precipitated during an overnight hold. It was unclear whether this was due to a decrease in temperature and/or the addition of salts or some other factor. The following morning the bioreaction was initiated by adding the remaining reactants and enzymes from Table 9 to the tryptophan-pyruvate slurry to form the bioreaction mixture. The temperature of the mixture was maintained at about I 5°C.

Table 9. Additional Components Added to Solution from Table 8

Mass

(g) Component

17.7 Sodium Phosphate (dibasic) Anhydrous

5.1 Magnesium Chloride Hexahydrate

125.0 10 mM pyridoxai phosphate

2,7 Tween 80

20.33 1 H HCI

MON016J D-aminotransferase cell free

extract, containing 5.0 gra ms of active

1136 enzyme

MON013A Aldolase cell free extract,

50 containing 0.5 grams of active enzyme [0094] Five hours after the addition of the enzymes, the bioreaction mixture was transparent with almost no solids. Thus, although the initially supersaturated D-tryptophan came out of solution when left overnight, the monatin-producing reactions proceeded and D-tryptophan was pulled back into solution as it was used to create monatin. The bioreaction mixture was transferred from the bioreactor vessel to a clean nitrogen-blanketed tank. The mixture was then sampled about 48 hours after the addition of enzymes, and the composition is shown in Table 10. The volume of the mixture in Table 10 was about 25.0 L.

Table 10. Composition of Bioreaction Mixture after 48 hours

[0095] The monatin yield of the mixture in Table 10 was 1

[0096] The mixture was next concentrated on a tubular AFC99 RO membrane, having 0.88 m ² surface area, from PCI Membranes. The system consisted of a feed tank with a cooling coil, a feed pump with inline heat exchanger, a membrane housing, and a permeate collection tank. The system was operated at 500 psi inlet pressure and the feed tank was under a constant nitrogen blanket.

[0097] During the concentration, samples were taken of the retentate and permeate solutions and composite triplicate samples were taken of the final permeate and retentate at the end of the concentration. The total permeate collected during the concentration was approximately 17 L. Table 1 1 shows the composition of the retentate essentially immediately after concentration. The total volume of the retentate was about 7.8 L.

Table 11. Composition of Bioreaction Mixture after Concentration Step

[0098] The monatin yield of the mixture in Table l l was 16.7%. Thus performing the concentration step after the reactions had reached equilibrium resulted in a yield increase of 3.1 %. [0099] After the dewatering or concentration step was performed, the mixture was held for five days to see if there was an additional increase in monatin. The composition of the sample taken after a five day hold is shown in Table 12.

Table 12, Composition of Bioreaction Mixture after Five Day Hold

[00100] The monatin yield of the mixture in Table 12 was 18.8%. Thus, giving the enzymes additional time to facilitate the monatin-producing reactions after the tryptophan was supersaturated resulted in an additional 2, 1 % yield increase. A yield increase of 5.2% was observed through the combination of a second dewatering step followed by a hold. The monatin mass after the hold, as compared to the monatin mass after 48 hours (see Table 10), resulted in a monatin mass increase of 38%.

Example 5

[00101 ] This example was similar to Example 3 above to demonstrate supersaturation through dewatering, after the monatin-producing reactions had reached equilibrium.

[00102] An initial 25 L solution was made in a bioreaction vessel, according to Table 13. The bioreaction was allowed to proceed at about 35 °C for about 36 hours after the enzymes were added. The headspace of the bioreaction was sparged with nitrogen to protect the mixture from oxygen exposure.

Table 13. Components in Initial Solution

Mass

<g> Component

22500 Degassed deionized Water

628.0 D-Tryptophan

507.6 Sodium Pyruvate

17.7 Sodium Phosphate (dibasic) Anhydrous

5,1 Magnesium Chloride Hexahydrate

125.0 10 mM pyridoxal phosphate

2.7 Tween 80

4.5 10 M NaOH

MON016J D-amrnotransferase cell free

extract, containing 5.0 grams of active

1136 enzyme

ON018 Aldolase cell free extract,

80 containing 0.54 grams of active enzyme [00103] After about 36 hours, the bioreaction mixture was sampled and then transferred to a nitrogen blanketed tank. The sample results are shown in Table 14. The total volume of the mixture in Table 14 was 24.3 L. After the transfer, the bioreactor was rinsed with 5.03 kg of water to ensure that all carbon was transferred to the blanketed tank, to minimize transfer loss.

Table 14. Composition of the Bioreaction Mixture after 36 hours

[00104] The monatin yield of the mixture in Table 14 was 16.4%.

[00105] The mixture was then concentrated on a tubular RO membrane, having 0.88 m ² surface area, AFC99 RO-type from PCI Membranes. The system consisted of a feed tank with a cooling coil, a feed pump with inline heat exchanger, a membrane housing, and a permeate collection tank. The system was operated at about 500 psi inlet pressure, and the feed tank was under a nitrogen blanket. The target operating temperature was about 15°C _S however, over time the temperature increased to about 27°C.

[00106] Table 15 shows the composition of the retentate after the dewatering or concentration step. The total volume of the retentate was about 7.8 L. The mixture from Table 15 was evaluated about 48 hours after the enzymes were first added to the bioreaction vessel.

Table 15. Composition of the Bioreaction Mixture after Concentration Step

[00107] The monatin yield of the mixture in Table 1 was 20.1 %, resulting in a 3.7% yield increase. The monatin mass after the concentration or dewatering step, as compared to the monatin mass after 36 hours (see Table 14), resulted in a monatin mass increase of 22%.

Example 6

[00108] This example was very similar to the previous Example 5. [00109] An initial 25 L solution was made in a bioreaction vessel, according to Table 16, The bioreaction was allowed to proceed at about 15 °C for about 36 hours after the enzymes were added. The headspace of the bioreaction was sparged with nitrogen to protect the mixture from oxygen exposure.

Table 16. Components in Initial Solution

[001 10] After about 36 hours, the bioreaction mixture was sampled and then transferred to a nitrogen blanketed tank. The sample results are shown in Table 17. The total volume of the mixture in Table 17 was 24.5 L. After the transfer, the bioreactor was rinsed with 4.82 kg of water to ensure that all carbon was transferred to the blanketed tank, to minimize transfer loss.

Table 17. Composition of the Bioreaction Mixture after 36 Hours

[001 1 1 ] The monatin yield of the mixture in Table 17 was 16.4%.

[001 12] The mixture was then concentrated using the same membrane system as in previous Example 5, A full membrane cleaning procedure was completed in between running of Examples 5 and 6, The system was operated at about 500 psi inlet pressure, and the feed tank was under a nitrogen blanket. The target operating temperature was about 1 5°C, however, over time the temperature increased to about 29°C. [001 13] Table 38 shows the composition of the retentate after the dewatering or concentration step. The total volume of the retentate was about 7.7 L. The mixture from Table 18 was evaluated about 44 hours after the enzymes were first added to the bioreaction vessel.

Table 18. Composition of the Bioreaction Mixture after Concentration Step

[001 14] The monatin yield of the mixture in Table 1 8 was 1 7.5%, resulting in a 1 .1 % yield increase. The monatin mass after the concentration or dewatering step, as compared to the monatin mass after 36 hours (see Table 17), resulted in a monatin mass increase of 7%.

Example 7

[001 15] in this example, supersaturation of D-tryptophan was achieved through membrane dewatering, essentially immediately after adding the enzymes (i.e. prior to equilibrium).

[001 16] An initial 100 L solution was prepared according to Table 19.

Table 19. Components in Initial Solution

[001 17] Essentially immediately after the enzymes were added, the concentration of the solution (i.e. dewatering) was initiated. The test solution was concentrated on a spiral wound SW30 4040 RO membrane, having a surface area of 7.4 m ² , from DOW Filmtec. The concentration system consisted of a feed tank with a cooling coil, a feed pump with an inline heat exchanger, a membrane housing, and a permeate collection tank. The system was operated at 700 psi inlet pressure and about 33°C, and the feed tank was under a nitrogen blanket.

[001 18] After a target concentration of 1 15 mM D-tryptophan was reached in the retentate, the concentration system was stopped and the retentate, containing the enzymes, was allowed to react for about 24 hours. During this time additional D-tryptophan was consumed. After 24 hours, a sample of the retentate was taken and the composition is shown in Table 20. The volume of the retentate was about 16.5 L.

Table 20. Composition of Bioreaction Mixture after Initial Concentration and 24 Hour Hold

[001 19] The monatin yield of the mixture in Table 20 was 19.7%.

[00120] The mixture/retentate from Table 20 was then concentrated a second time using the same RO membrane system from the initial concentration. The temperature was about 16.5 °C during this second concentration. The mixture/retentate was concentrated until the concentration of tryptophan in the new retentate reached 1 15 mM, The membrane system was shut off and a sample was taken. The composition of the sample is shown in Table 21 . The volume of the mixture was 10.9 L.

Table 21. Composition of Bioreaction Mixture after Second Concentration

[00121 ] The monatin yield of the mixture in Table 21 was 20.6%.

[00122] After the second concentration, an additional 0.5 grams of aldolase (active enzyme supplied as 75 grams of MONO 18 cell free extract) was added to the mixture. The mixture was held for about 24 hours to observe the impact on monatin yield. The composition of the sample after the 24 hour hold is shown in Table 22. Table 22. Composition of Bioreaction Mixture after Second Concentration and 24 Hour Hold

[00123] The monatin yield of the mixture in Table 22 was 25.2%, thus an additional 4.6% yield increase resulted from holding the mixture for 24 hours following the second concentration step.

Example 8

[00124] This example was very similar to the previous Example 7.

[00125] An initial 100 L solution was prepared according to Table 23.

Table 23. Components in Initial Solution

[00126] Essentially immediately after the enzymes were added, the concentration of the solution was initiated using the same membrane and operating system described in Example 7, as well as the same operating pressure. The temperature was about 14°C.

[00127] After a target concentration of 1 1 5 mM D-tryptophan was reached in the retentate, the concentration system was stopped and the retentate, containing the enzymes, was allowed to react for about 24 hours. During this time additional D-tryptophan was consumed. After 24 hours, a sample of the retentate was taken and the composition is shown in Table 24. The volume of the retentate was about 15.0 L.

Table 24. Composition of Bioreaction Mixture after Initial Concentration and 24 Hour Hold

[00128] The monatin yield of the mixture in Table 24 was 20.2%

[00129] The mixture/retentate from Table 24 was then concentrated a second time using the same RO membrane system from the initial concentration. The temperature was about 14°C during this second concentration. The mixture/retentate was concentrated until the concentration of D- tryptophan in the new retentate reached 1 15 mM The membrane system was shut off and a sample was taken. The composition of the sample is shown in Table 25. The volume of the mixture was

10.8 L.

Table 25. Composition of Bioreaction Mixture after Second Concentration

[00130] The monatin yield of the mixture in Table 25 was 20.0%.

[0013 1 ] After the second concentration, an additional 0.5 grams of aldolase (active enzyme supplied as 75 grams of MONO 18 cell free extract) was added to the mixture. The mixture was held for about 24 hours, and a sample was taken. The composition of the sample is shown in Table 26.

Table 26. Composition of Bioreaction Mixture after Second Concentration and 24 Hour Hoid

[00132] The monatin yield of the mixture in Table 26 was 23.7%. Thus holding the for 24 hours following the second concentration step resulted in a 3.7% yield increase. Example 9

[00133] This example explored supersaiuration of tryptophan through dewatering, using a nanofiltration (NF) membrane.

[00134] An initial solution was made using about 12,000 g of deionized water and about 73.5 g of D-tryptophan. The water was weighed into a tank at about 1 5 °C. The D-tryptophan was added and the pH was adjusted to 7.8 using sodium hydroxide (NaOH). The solution was then recirculated overnight in a chilled tank at about 15°C. Triplicate samples were then taken. The average concentration of D-tryptophan was 25.9 mM and the volume of the solution was about 12.0 L.

[00135] The solution was then concentrated using a DL1812 nanofiltration (NF) membrane from GE. The system was operated at an inlet pressure of about 500 psi and about 15 °C. The membrane system was operated until the volume of the retentate was about 2.6 L, which was about 29 minutes. The concentration of tryptophan in the retentate was 93.4 mM and the mass was 50.2 g. The solution was stable at this concentration for over four days at about 15 °C.

[00136] The mass balance is 82% for the permeate and the retentate. The membrane did allow some of the D-tryptophan to pass through the membrane which explains why the mass of D- tryptophan in the retentate was lower than the mass in the initial solution. It is assumed that the missing D-tryptophan was left on the surface of the membrane which could be recovered with a rinse step.

[00137] It is recognized that various modifications to the described invention may be made without departing from the spirit and scope of the disclosure. It is recognized that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Accordingly, other embodiments are within the scope of the following claims.