This application incorporates by reference the contents of a 505 kb text file created on December 18, 2020 and named “CP0188US00sequencelisting.txt,” which is the sequence listing for this application.

BACKGROUND OF THE INVENTION

Efforts are being made to use the abundant sugar glucose (also called dextrose) as a feedstock the production of a variety of useful industrial compounds including for example, furan dicarboxylic acid (FDCA) and furan dicarboxylic methyl ester (FDME). Glucose, as with other aldol hexoses, may be oxidized by chemical catalysis to form glucaric acid, which can be subsequently dehydrated in two steps by known methods, such as acid catalysis to form FDCA or dehydrated and esterified with methanol to form FDME. Catalytic oxidation of glucose to glucaric acid as described for example, in US patent number 9,156,766, unfortunately also produces a mixture of mono and di acids and other byproducts that must be separated from glucaric acid before conversion to FDCA. Significant effort is required in the processing of the oxidation reaction product to separate glucaric acid from the other byproducts.

Enzymatic dehydration of glucaric acid to 5-keto-4-deoxy glucaric acid (KDG) by the enzyme glucarate dehydratase provides an enzymatic route for producing KDG that in turn can be subject to further acid catalyzed dehydration to FDCA. The dehydration of glucaric acid to KDG by glucarate dehydratase is therefore a key enabling step for a combined chemo-enzymatic process for making FDCA/FDME from dextrose. Studies of the enzymatic conversion of glucaric acid to KDG have been primarily directed towards understanding the mechanism of the glucarate dehydratase enzyme and its role in degradation of glucarate by microorganisms. Most literature references state the optimal activity of known naturally occurring glucarate dehydratase enzymes is 30-40°C and that the activity significantly falls off at higher temperatures. Studies also show that glucarate dehydratase is subject to inhibition by other diacids typically produced in the chemical oxidation of glucose to glucaric acid, including for example tartaric acid. There is a need for methods that do not have the drawbacks and limitations of enzymatic methods for dehydrating glucarate of KDG using known glucarate dehydratase enzymes. In particular there is a need for thermostable glucarate dehydratase enzymes that have sustained activity at temperatures greater than 30-40°C. Ideally there is a need for thermostable enzymes that can operate at temperatures of greater than 50°C, more preferably at greater than 60 °C, still more preferably greater than 65 °C and most preferably greater than 70°C. Operating at higher temperature will increase the catalytic activity of the enzyme and can use the heat derived from exothermic process of oxidizing glucose to form glucaric acid. There is also a need for thermostable glucarate dehydratase enzymes that are resistant to inhibition by tartrate and other byproducts present in a crude glucose oxidation mixture containing glucaric acid thereby eliminating the need to purify the glucaric acid prior to dehydration.

BRIEF SUMMARY

Provided herein are naturally occurring or improved glucarate dehydratases having increased thermostability and or resistance to inhibition by tartrate, which may be used in methods of making 5-keto-4-deoxy glucaric acid (KDG), which in turn provides improved methods of making FDCA or FDME.

In one aspect there is provided method of making 5-keto-4-deoxy glucaric acid (KDG) comprising contacting glucaric acid with a glucarate dehydratase enzyme at a temperature of at least 50°C where the enzyme has at least one characteristic selected from the group consisting of: (i) exhibiting thermostability at a temperature of at least 50 °C and (i) exhibiting at least 50% of its enzymatic activity converting glucarate to KDG in the presence of 10 mM glucarate and 10 mM tartrate as it exhibits in the absence of tartrate.

In various embodiments, the glucarate dehydratase enzyme is selected from the group consisting of (i) a naturally occurring enzyme having a protein sequence according to any one of SEQ ID NOS 141 -148; (b) a variant sequence of any one of SEQ ID NOS 141 -148 made by substituting at least 3 amino acids of SEQ ID NOS: 141 -148 and that retains or has improved thermostability or resistance to tartrate.; (c) a protein sequence according to any one of SEQ ID NO 2-140; and (d) a variant of SEQ ID NO 2-140 made by substituting at least one amino acid present in any of SEQ ID NO 2-140 and hat retains or has improved thermostability or resistance to tartrate.

In certain exemplary embodiments, the glucarate dehydratase enzyme is from Th. carboxydivorans having a protein sequence according to SEQ ID NO: 147 or from Lactobacillus pentous having a protein sequence according to SEQ ID NO: 146 or a variant derived by alteration of at least one amino acid in SEQ ID NO 146 or 147 that retains or has improved thermostability or resistance to tartrate. The tartrate is at least one of D-tartrate, L-tartrate, and meso-tartrate.

In exemplary embodiments the glucarate dehydratase enzyme exhibits enzymatic activity at a pH range of 5.5-7.0. In certain embodiments the glucarate dehydratase exhibits enzymatic activity at greater than 60°C, greater than 65°C and in some embodiments greater than 70°C. In some particular embodiments, the glucarate dehydratases exhibits at least 72% of enzymatic activity at temperatures above 50°C as exhibited at 37-45°C and that is resistant to inhibition by D-tartrate and guluronate relative to the parent and other naturally occurring glucarate dehydratases.

The forging can be used of making furan dicarboxylic acid (FDCA) comprising obtaining KDG made by the forging methods and dehydrating the KDG to form FDCA. In a further embodiment, the FDCA is esterified with methanol to furan dicarboxylic methyl ester (FDME). In certain desired embodiments the glucarate dehydratase enzyme is immobilized on a column.

In another aspect there are provided new glucarate dehydratase enzymes with improved thermostability and resistance to tartrate and guluronate. In broad embodiments there is provided a glucarate dehydratase enzyme comprising the amino acid sequence according to SEQ ID NO 146 except having at least one mutation selected from the group of individual mutations present in SEQ ID NOS: 2-140 and that exhibit improved thermostability and/or resistance to in comparison to the glucarate dehydratase enzyme according to SEQ ID NO 146.

In typical embodiments the glucarate dehydratases have at least two mutations selected from the group of individual mutations present in SEQ ID NOS: 2-24. In certain embodiments the glucarate dehydratase has at least three mutations selected group of individual mutations present is SEQ ID NOS: 25-87. In further embodiments, the glucarate dehydratase enzyme has at least four mutations selected from the group of individual mutations present in SEQ ID NOS: 25-87. In still further embodiments, the glucarate dehydratase enzyme has at least five mutations selected from the group of individual mutations present in SEQ ID NOS: 88-140.

In exemplary embodiments, the glucarate dehydratase has an amino acid sequence according any one of SEQ ID. NO 2-140.

In still another aspect, there is provided a method of obtaining a mutant glucarate dehydratase that exhibits at least one of thermostability and resistance to inhibition by tartrate and/or guluronic acid that includes: a) subjecting a parent glucarate dehydratase having a protein sequence according to any of SEQ ID NOs: 141-148 to a first round of mutagenesis to generate a first set of variants of the parent glucarate dehydratase that include at least three amino substitutions; b) determining the glucarate dehydratase activity of the first set of variants at a desired temperature of at least 50°C and/or determining activity of the variants in the presence of tartrate and/or guluronic acid; c) selecting a variant that has higher activity at the desired temperature and/or that has higher activity in the presence of tartrate and/or guluronic acid than the parent glucarate dehydratase, and optionally d) subjecting at least one member from the first set of selected variants to at least one subsequent round of mutagenesis and repeating steps b and c to select a second set of variants having higher activity at the desired temperature and/or that has higher activity in the presence of tartrate and/or guluronic acid than the variants from the first round of mutagenesis

In exemplary embodiments, the mutagenesis is conducted by synthesizing genes that encode a protein sequence with amino acid mutations of the parent or selected variant glucarate dehydratases, and the method of selecting includes expressing the synthesized genes from a host organism and assaying the expressed genes for glucarate dehydratase activity. The variants synthesized in subsequent rounds of mutagenesis retain at least one amino acid substitution present in the variants selected after the first round of mutagenesis.

The forgoing enzymes are used in another aspect of the present invention, which is a method of making 5-keto-4-deoxy glucaric acid (KDG) comprising contacting glucaric acid with any of the forgoing new glucarate dehydratase enzymes. In desirable embodiments, the glucarate dehydratase enzyme is immobilized on a column. The forging method can be used in a further method for making furan dicarboxylic acid (FDCA) comprising obtaining KDG made using the new glucarate dehydratase enzymes and dehydrating the KDG to form FDCA. In some practices, the method may further include is esterifying the FDCA with methanol to furan dicarboxylic methyl ester (FDME).

These and other aspects, embodiments, and associated advantages will become apparent from the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. IB, respectively, are graphs showing the effect of increasing L- tartrate concentration on glucarate activity between dehydratase variant 350378 (SEQ ID NO:2) (FIG. 1A) and wild-type dehydratase (336438, SEQ ID NO: 146) (FIG. IB). For each graph, the Y axis is KDG production rate (mM KDG/min), and the X axis is glucarate concentration (mM). The L-tartrate concentration (mM) of each curve is identified by the legend, and correspond to L-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM.

FIG. 2A and FIG. 2B, respectively, are graphs showing the effect of increasing D- tartrate concentration on glucarate activity between dehydratase variant 350378 (SEQ ID NO:2) (FIG. 2A) and wild-type dehydratase (336438, SEQ ID NO: 146) (FIG. 2B). For each graph, the Y axis is KDG production rate (mM KDG/min), and the X axis is glucarate concentration (mM). The D-tartrate concentration (mM) of each curve is identified by the legend, and correspond to D-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM.

FIG. 3 A and FIG. 3B, respectively, are graphs showing the effect of increasing meso-tartrate concentration on glucarate activity between dehydratase variant 350378 (SEQ ID NO:2) (FIG. 3A) and wild-type dehydratase (336438, SEQ ID NO: 146) (FIG. 3B). For each graph, the Y axis is KDG production rate (mM KDG/min), and the X axis is glucarate concentration (mM). The meso-tartrate concentration (mM) of each curve is identified by the legend, and correspond to meso-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM. FIG. 4 is a graph showing tartrate inhibition of variants 368238 (SEQ ID NO:90), 368265 (SEQ ID NO:94), 368298 (SEQ ID NO:98), and 357047 (SEQ ID NO:82). Left bar of each pair 40 mM D-tartrate, right bar of each pair 20 mM L-tartrate.

FIG. 5 A, 5B, and 5C, respectively, are graphs showing guluronate inhibition at 0 mM guluronate (FIG. 5A), 40 mM guluronate (FIG. 5B), and 80 mM guluronate (FIG. 5C) for variants 368238 (SEQ ID NO:90), 368265 (SEQ ID NO:94), 368298 (SEQ ID NO:98), and 357047 (SEQ ID NO:82). The R number in the prefix preceding the variant number indicates the round of mutation from which the variant was generated. Each of the R3 variants retained the mutations from the R2 variant 357047.

FIG. 6A, 6B, 6C, and 6D, respectively, are graphs showing glucarate dehydratase activity assays at 66° C (FIG. 6A), 68° C (FIG. 6B), 70° C (FIG. 6C), and 72° C (FIG. 6D) for variants 374373 (SEQ ID NO: 104), 374377 (SEQ ID NO: 107), 374388 (SEQ ID NO: 111), 374398 (SEQ ID NO: 116), 374413 (SEQ ID NO: 123), 374414 (SEQ ID NO: 124), 374423 (SEQ ID NO: 128), 368238 (SEQ ID NO:90), and 357047 (SEQ ID NO:82). The R number in the prefix preceding the variant number indicates the round of mutation from which the variant was generated.

FIG. 7 is a graph showing the results of forming KDG from glucarate using a glucarate dehydratase enzyme immobilized on a column for continuous production of the KDG reaction product from a glucaric acid feedstock passed over the column at a rate of

0.5 Bv/hr at 55°C.

FIG. 8 is a bar graph illustrating results of screening naturally occurring glucarate dehydratases present in crude extracts made from E. coli cultures engineered to heterolgously express candidate enzymes. The X axis shows assigned gene numbers and Y axis shows measured glucarate dehydratase activity. Notable candidate sources are identified.

DETAILED DESCRIPTION

In one aspect, the present disclosure provides the discovery a handful of naturally occurring glucarate dehydratase enzymes that may be employed in a method for making KDG from glucaric at higher temperatures, i.e, at a temperatures of least 50°C, at least 60°C, at least 65°C or even at least 70°C. Some of these naturally occurring enzymes also show better resistance to tartrate inhibition than previously described enzymes.

Glucarate dehydratase has been identified from E. coli and has been well characterized. The properties of the E. coli D-glucarate dehydratase were first described by H. J. Blumenthal in Method of enzymology, 1966, 9, 660. The authors conclude that glucarate dehydratase is inhibited by galactaric acid, D-idaric acid, and tartaric acid. Roger Jeffcoat Eur. J. Biochem. 25 (1972) 515-523 has further characterized the enzyme from Pseudomonas and has shown that (+)-tartrate is a competitive inhibitor of that enzyme at a concentration of 2.5 mM. The E. coli D-glucarate dehydratase was used as the base enzyme to compare to other enzymes discovered or developed in accordance with the present disclosure. The E.coli enzyme was purified by fast protein liquid chromatography (FPLC) and then assayed for enzyme activity in the presence of various inhibitors known to exist in a crude glucose oxidation mixture containing glucaric acid. Example 1 provided hereafter shows the inhibitory properties of byproducts present in a typical glucose oxidation mixture to the glucarate conversion activity of the E. coli dehydratase enzyme.

Genes encoding E. coli glucarate dehydratase and a library of 94 homologues (all previously uncharacterized) were synthesized with optimized codons and expressed in E. coli. Some members in the library of 94 homologues to the E. coli glucarate dehydratase have as little as 30% protein sequence homology to the E. coli sequence yet still exhibited glucarate dehydratase activity. Crude lysates were made from E. coli cultures expressing the library of gene and used to screen for specific activity, temperature tolerance, pH tolerance, and resistance to the competitive inhibitor tartrate.

This screening identified 8 naturally occurring enzymes with higher thermostability and in most cases with better resistance to inhibition by tartrate than the E.coli enzyme. As used herein, thermostability means the enzyme exhibits a specific activity ( e.g ., KDG made from glucarate per minute / mg protein) at a temperature of 50°C or greater that is at least 75% of the specific activity exhibited by the E. coli enzyme in converting glucarate to KDG at a 30°C. The naturally occurring enzymes exhibiting higher thermostability are from Enterococcus sp., Oribacterium sp., Elalomonas sp., Elalomonas sp., F. plautii, Lactobacillus pentous, Th. Carboxydivorans, and Strep sp. OK885 identified herein as SEQ ID NOs: 141-148, respectively. All of these enzymes were judged to have thermostability in that they exhibited enzymatic activity at 50°C for at least 2 hours and in several cases for as long as 19 h. See Figure 8. Accordingly, all of these naturally occurring enzymes are suitable for use in a method of converting glucarate to KDG at a temperature of at least 50°C, at least 60°C and/or at least 65°C. Several also exhibited better resistance to inhibition by tartrate than the E. coli enzyme.

Follow up experiments revealed that the enzyme from Th. Carboxydivorans (a.k.a, Acetonema ) was the most thermostable enzyme screened, being active at a temperature as high as 70°C with an optimal temperature closer to 60° C. By comparison, published results show the E. coli enzyme is most active at 25-30°C and it was found that the E. coli enzyme denatures above 50°C. In our hands, the E. coli enzymes in-fact exhibits only about 60% of activity at 50°C as it exhibits at 27.5°C in a 15-minute reaction which further declines to about 40% of activity after one hour. At 60°C the E. coli enzyme exhibits only about 5% or less of the activity it exhibits at 30°C. Another feature of the Acetonema enzyme disclosed herein is its resistance to the presence of tartaric acid. While most enzymes in the library that was screened were indeed very sensitive to D-tartrate, the Acetonema enzyme was discovered to be much less sensitive to D-tartrate than the E. coli enzyme. This is a substantial and surprising result. There are literature reports ( e.g http://onjinelibrary. wiley .com/doi/ 723.x/epdf) that document the sensitivity of this class of enzymes to the competitive inhibitor D-tartrate

In another aspect, the present disclosure provides for a method to generate a broad family of variant glucarate dehydratase enzymes derived from any of the forgoing parent natural enzymes that exhibit enzyme activity at least 50°C, at least 60°C, at least 65°C or in some embodiments at least 70°C. In certain embodiments the variant enzymes are resistant to inhibition by the competitive inhibitors tartaric acid and in some embodiments also resistant to inhibition by guluronic acid. The enzymes disclosed herein are catalytically active in a crude glucose oxidation mixture containing glucarate, tartrate and guluronic acid. The crude glucose oxidation mixture used herein for illustrative purposes is a mixture containing glucaric acid, tartaric acid and other diacids resulting from oxidation of glucose made according to the method described in US patent number 9,156,766. The enzymes disclosed herein would, however, be active with any crude glucose oxidation mixtures containing glucaric acid and inhibitors of the dehydratase such as tartaric and guluronic acid. Figures 5A through 5C illustrate exemplary variants that show increased resistance to guluronic acid, where the mutations present in a second-round variant were preserved in third round variants, all of which showed increased resistance to inhibition by guluronic acid.

The method of improving at least one of thermostability and resistance to inhibition by tartrate by glucarate dehydratase includes subjecting a parent glucarate dehydratase having a protein sequence according to any of SEQ ID NOs: 141-148 to a first round of mutagenesis to generate variant glucarate dehydratases including at least three amino substitutions, determining the glucarate dehydratase activity of the variants at a temperature of at least 50°C, or at least 60°C , or at last 65°C or at last 70°C and selecting a variant that has higher activity at the desired temperature than the parent enzyme. The mutagenesis is exemplified herein by synthesizing a gene that encodes selected random mutations that span the entire peptide sequence of the parent enzyme, expressing the mutated genes from a suitable host, and making crude lysates of cultures expressing the genes, but the method may also be practiced by making purely random mutations in the coding sequence and expressing the mutant genes from a suitable host.

The method is illustrated herein using E. coli harboring the mutant genes in an expression vector, but other hosts with suitable expression sequences may be used. Separately, or in addition, the activity of the variants expressed may be determined in the presence of tartrate or guluronic acid at temperatures of at 50°C, or at least 60°C or at last

65 °C a leas 65 °C and variants are selected that have also greater activity than the parent enzyme in the presence of these inhibitors. The concentration of inhibitors used to test for resistance to inhibition should be 1/4* to four times the amount of glucaric acid used in the assay. In exemplary embodiments, assays are performed with about 10 mM glucaric acid in the presence of 5 mM to 40 mM of the inhibitors. Reactions containing 10 mM glucarate and 10 mM of tartrate or other inhibitors are suitable for use in screening for resistance to the inhibitors. In other exemplary embodiments, a crude glucose oxidation mixture is used as the substrate for enzymatic conversion of glucarate, and the amount of inhibitors present is as inherently exist in a crude glucose oxidation mixture, which is shown in Example 1 of this disclosure.

Preferably in the method, at least one mutation introduced to make variants in the first round of mutations is preserved and at least a second round of mutation is made in the parent sequence wherein the second round includes at least one preserved mutation from the first round of mutations that showed increased activity at the desired temperatures and optionally in the presence of tartrate or guluronic acid. In preferred embodiments at least two, or at least three mutations from the first round of mutations that show improved thermos tolerance and or inhibitor resistance are preserved. The second round introduces new mutations, and the thermostability and/or resistance to inhibitors are determined for the new variants at the desired temperature optionally in the presence of the inhibitors and those with increased thermostability and/or resistance to the inhibitors are again selected. In an optimal method, at least four rounds of mutation are conducted, each creating variants that preserve at least one beneficial mutation selected from earlier rounds. In most effective embodiments at least two, or most preferably at least three mutations from prior rounds of mutation are preserved in subsequent rounds.

The tables from Examples 5, and 7-9 illustrate examples of variants made in each of 4 rounds of mutation. The variants made in the first round of mutation contained 3 mutations altering up to three amino acids and those mutations exhibiting the same or higher thermostability and/or resistance to tartrate (SEQ ID NOS: 2-24) were retained for use in a second round. Each of the variants made in the second round retained at least two and often all three of the mutations made in the first round and those mutations that exhibited thermostability or resistance inhibition (SEQ ID NOS 25-87) were retained for use in a third round of mutation which new mutations therefore also included at least three mutations from the first and second round. Similarly, beneficial mutations identified in the third round were preserved for combination with new mutations made in the fourth round, and those that exhibited thermostability or resistance to inhibition in the fourth round (SEQ ID NOS: 88-140) therefore contained at least four of the mutations made in earlier rounds. Mutations could further be made in subsequent rounds that would preserve at least 5 of the individual mutations present in SEQ ID NOS: 88-140.

The forgoing method provides a third aspect of the present disclosure, which is a variant glucarate dehydratase derived from a parent glucarate dehydratase according to

SEQ ID NOS 141-148 that has improved thermostability at 50°C , 60°C, 65°C or 70°C and/or improved resistance to inhibition by tartaric acid or guluronic acid. The Examples that follow illustrate this aspect of the disclosure by providing variant glucarate dehydratases having peptides sequences according to SEQ ID NOS: 2-140, which were generated by the forgoing method after one, two, three or four rounds of mutation of the glucarate dehydratase from Lactobacillus pentous where the parent protein had the peptide sequence according to SEQ ID NO: 146. Variants made in each subsequent round of mutation preserved at least one, two, three or more mutations discovered to be beneficial for thermostability or resistance to inhibitors from at least one previous round of mutation. Often, at least two and most often at least 3 mutations from prior rounds were preserved in subsequent rounds of invention. Production of these variants is illustrated in Examples 5 through 9 of the present disclosure. All of the variants should retain at least 94%, more preferably at least 96% and most preferably at least 98% sequence identity to the parent enzyme. The variants exemplified herein retain at least 97.5% sequence identity to the parent sequence.

The glucarate dehydratase from L. pentous having the peptide sequence according to SEQ ID NO: 146 was selected to illustrate this aspect of the invention because although the naturally occurring enzyme from Acetoma having the protein sequence according to SEQ ID NO: 147 was judged to have the best thermostability and good resistance to inhibition by tartrate using purified substrate and inhibitors, the Acetoma enzyme was also judged to be less resistance to inhibition using as substrate a crude glucose oxidation mixture than the enzyme from L. pentous , as shown in Table 2 in Example 2. Nonetheless, the present invention can be practiced using any of the naturally occurring sequences according to SEQ ID NOS: 141-148 as the starting parent enzyme for making subsequent rounds of mutation. Figures 6A through 6D shows the activity of exemplary variants that are both resistant to guluronic acid and continue to exhibit thermostability at 66°C, at 68°C, at 70°C and at 72°C. These illustrative enzymes exhibit enzymatic activity at 72°C that is at least 72% of the enzymatic activity at reference temperatures of 37-45 °C. Not all variants from SEQ ID NOS 2-140 were assayed at these higher temperatures, but most if not, all are expected to also show both guluronic acid and increased thermostability. Minimally, the variants made in round 3 and round 4 of mutations (SEQ ID NOS: 87-140) should show similar thermostability and resistance to guluronic acid because the variants preserved the beneficial mutations from round 2 variants that exhibited these features.

The naturally occurring and variant glucarate dehydratase provided according to the present disclosure may be used in a fourth aspect of the present invention, which is, a method of making furan dicarboxylic acid (FDCA) that comprises contacting glucaric acid with a glucarate dehydratase enzyme having protein sequences according to SEQ ID NOS 141-148 or a variant thereof made according to the methods described herein at a temperature of above 50°C to form 5-keto-4-deoxy glucaric acid (KDG) and further dehydrating the KDG to form FDCA, The variants retain at least 94%, at least 96% and more preferably at least 98% sequence identity with the parent. The dehydration to KDG may be performed at a pH of 5.5-7.5 wherein the glucarate dehydratase enzyme is thermostable at a temperature of at least 50°C and /or is resistant to inhibition by tartrate. Particular embodiments of variant glucarate dehydratase are illustrated by SEQ ID NOS 2- 140. In illustrative embodiments, the glucaric acid is in a salt form selected from the group consisting of a potassium salt form, a sodium salt form, and an ammonium salt form. All of the embodiments of the method may be practiced using as the glucarate substrate, a crude glucose oxidation mixture, exemplified by a mixture made according to the method described in US Pat. No. 9,156,766. During the catalytic oxidation of glucose to glucaric acid, other oxidized compounds are produced. The following is a list of compounds other than glucaric acid present in a crude oxidation mixture: gluconate, glycerate, gycolate, formate, 2-keto-gluconate, glucoronate, chloride, 2-furonate, 5-keto-gluconate, malate, maleate, tartrate, tartronate, oxalate, glucose, fructose, and arabinaric acid. Example 1

Inhibitors of the E. coli Glucarate Dehydratase

Glucarate dehydratase activity was quantified using a continuous enzyme-coupled spectroscopic assay. 100 pL of reaction mixture contained 100 mM phosphate buffer at pH 7.5, 10 mM MgCk, 0.25 mM to 50 mM D-glucarate, 0.1 mg glucarate dehydratase and byproducts/inhibitors (1-20 mM) incubated at 30°C for 10 minutes. Semicarbazide (0.1 M) in triacetate was added to the reaction mass and incubated at room temperature for 10 minutes. The amount of ketoacid was quantitated by detection of its semicarbazone at 250 nm. Values of kcat and kcat/Km were determined by varying the concentration of the sugar acid substrate. Table 1 shows that for purified E. coli enzyme, DL-tartronic, tartaric and L- guluronic acid inhibit the D-glucarate dehydratase compared to other compounds typically present in a glucose oxidation reaction mixture to convert glucose to glucarate.

Table 1

Enzyme reactions were also carried out using the purified E. coli enzyme and a crude glucose oxidation mixture containing glucarate designated herein as Drum A material, which is the product of a glucose oxidation according to US patent number 9,156,766. The composition of Drum A material is shown below.

The reaction was carried at a concentration that formed 10% w/w dissolved solids from Drum A material as the substrate. The reactions did not go to completion likely due to inhibition by tartrate and the high amount of guluronic acid shown above to be competitive inhibitor of glucarate with the E. coli enzyme. Therefore, a need existed to discover glucarate dehydratase enzymes that were resistant to inhibition to by the byproducts present in the glucose oxidation reaction mixture.

Example 2

Identification of Natural Glucarate Dehydratase With Thermostability and Resistant to Tartrate Inhibition

Publicly available sequence databases were mined to discover candidate protein sequences with homology to the E.coli glucarate dehydratase or with annotations indicating likelihood of having a glucarate dehydratase activity. A total of ninety-five (95) naturally occurring gene sequences were identified as candidate glucarate dehydratase enzymes. Surprisingly, many shared less than 50% protein sequence identity with the E.coli enzyme. DNA sequences encoding these candidate enzymes with codons optimized for expression in E. coli were synthesized and introduced into an expression vector to express the candidate enzymes in E. coli.

Strains carrying the candidate genes were grown in rich media and crude lystates of these cultures were used to assay for glucarate dehydratase activity using as the substrate source the Durm A material described in Example 1., Drum A contains amongst other byproducts, tartaric acid. _, the previously known inhibitor of the E. coli glucarate dehydratase enzyme and also contains a relatively high concentration of guluronic acid shown in Example 1 to also be an inhibitor of the E. coli enzyme. Therefore, using Drum A material as a substrate for screening glucarate dehydratases for industrial use inherently screens for enzymes more likely be resistant to inhibition by these compounds. Thermostability also a desired characteristic for an industrial enzyme, therefore lysates containing the candidate enzymes were screened for activity at 50°C for a period of 2 hrs. and 19 hrs. Reactions were conducted at pH 6.5 with a 10% volume of crude lysate containing the expressed protein product with 20 % w/w dissolved solids from Drum A as the substrate. One result of such screening is shown in Fig. 8.

Similar screenings on the same candidates were done multiple time under different conditions of temperature (50- 70°C) different conditions of dissolved solids content (10-

50%) and for different amounts of time. Different temperatures and times were used to assess for thermostability and different amounts of dissolved solids content from Drum A material was used to assess for crude tolerance. Table 2 shows a summary comparison of activity ranking for 8 particularly useful naturally occurring glucarate dehydratases. The indicated performance values for each feature are expressed on a relative scale of 1 through 9, with the highest ranking being 1 and poorest being 9. For reference, on this relative scale the E. coli enzyme would be at least 10 for crude tolerance and thermo tolerance.

Table 2

All of these naturally occurring sequences are suitable for use in the methods of the present invention which is aimed at conversion of glucaric acid to KDG at a temperature of at least 50°C, most preferably in the presence of inhibitory byproducts of glucose oxidation. The sequence with the best combination of expressed activity and thermostability was Gene No. 333729, source Th. Carboxydivorans (a.k.a. Aectonema). Expressed activity and specific activity, however, are dependent at least in part on the E. coli host expression system and is not necessarily indicative of the best inherent activity of the enzyme itself. The sequence with the best combination of thermostability and crude resistance was gene No. 336438, source Lactobacillus pentous, which had the second-best crude tolerance with a medial level of thermostability. Example 3

Activity of Acetonema Glucarate Dehydratase in Converting Glucarate to KDG

Using pure glucaric acid

The dehydratase form Acetonema {i.e., Th. Carboxydivorans) was expressed in E. coli and cultured in a shake flask conditions LB medium with a IPTG inducer. Crude extracts were prepared by lysing the cell pellet with Bugbuster® and the activity in the crude extract was used to evaluate the conversion of potassium glucarate in a 100 uL reaction mixture (10% glucarate, 50 mM Tris pH 7.5, 2 mM Magnesium sulfate) at lysate to substrate dosages of 1:1. The reaction mixture was incubated at 50 C for 4 hr and the product mixture was analyzed by GC-MS. Each sample was diluted in dimethylformamide and trimethylsilylated with N,0-bis(trimethylsilyl)trifluoroacetamide containing 1 % trimethylchloro silane. Derivatized samples were analyzed by GC-MS with components separated through a 5 % phenyl-modified polydimethyl arylene siloxane capillary column.

After 4 hrs. the reaction was completed and with no glucarate detected.

Example 4

Activity of Acetonema Glucarate Dehydratase in Converting Glucarate to KDG Using a Semi-Crude Glucose Oxidation Reaction Mixture

The concentration of glucaric acid obtained from a crude glucose oxidation reaction mixture was enriched by precipitating as mono potassium salt and re-dissolving in water. The precipitated glucaric acid sample included ~ 400-500 ppm of tartaric acid, -500 ppm of arabinaric acid, and - 250 ppm of gluconic acid.

The dehydratase form Acetonema {i.e., Th. Carboxydivorans) was expressed in E. coli and cultured under fed batch fermentation conditions with a lactose inducer. Crude extracts were prepared by homogenizing the culture and the activity in the crude extract was used to evaluate the conversion of glucarate in the semi-crude reaction mixture (10% dissolved solids content in assay) at varying enzyme dosages at pH 7.0 at 55°C, and the results are shown in Table 3. Table 3

In this experiment it was found that as the amount of the enzyme was increased more glucaric acid in the reaction mixture was converted to the KDG product, but not all the glucarate was converted. Even though the newly discovered enzyme was found to be efficient in converting enriched glucaric acid solution to desired product, as the ratio of glucarate to tartrate becomes 2:1 to 3:1 (w/w), it was found that the reaction becomes sluggish and does not go to completion. The remaining glucaric acid indicates inhibition of the enzyme with tartaric acid. Further enzyme evolution was pursued to address this issue.

Example 5

Immobilization of Glucarate Dehydratases on a Column

Batch production of KDG by dehydration of glucarate with glucarate dehydratase can be inefficient and economically challenging when scaled up. Immobilization of glucarate dehydratases onto inert supports, was there investigated as a tool to use commercial production. Providing such immobilization will allow for multiple uses of the same amount the enzyme than is possible in batch processes where the enzyme is solubilized in the reaction mixture. To evaluate this possibility, two of the naturally occurring enzymes and one variant( 368238)derived from wild type enzyme Lactobacillus pentous identified in Example 2 were immobilized on a column which was used to convert glucarate to KDG by merely passing the glucarate substrate over the column.

Crude lysates from heterlogously expressed glucarate dehydratase enzymes 333729 (SEQ ID NO: 147), 336431 (SEQ ID NO: 148), and 368238 (SEQ ID NO: 146) were immobilized onto a silica resin with by infusing the column with the crude lysate and incubation with polyethyleneimine and glutaraldehyde for a time sufficient to cross link protein to the silica resin. The resin was then washed with buffer and evaluated for use as a reactor for making KDG from glucarate using potassium glucarate as the substrate feed stock providing the results shown in Table 4 below.

Table 4

The immobilized enzyme on the column was used for continuous conversion of glucaric acid into the KDG reaction product. The potassium glucarate feedstock was passed over the column at 0.5 BV/hr at 55°C and the material eluted from the column was measures over _ 80 _ column volumes with the results shown in FIG. 7. It was found that immobilization of the enzyme retained the activity as shown in Table 4.

Example 6

Production of Round 1 Variants of the Lactobacillus pentous Glucarate Dehydratase The Lactobacillus pentous glucarate dehydratase (gene No. 336438, SEQ ID NO

146) was subjected to spanned sequence mutagenesis to determine regions of the protein sequence that could most likely be mutated to improve thermostability and and/or resistance to inhibition by tartarate and other byproducts in a crude glucose oxidation mixture present in the Drum A material. 96 variant genes were synthesized to contain three randomly determined mutations targeting regions of the protein in such a way to span the entire protein sequence. The variants were then expressed in E. coli using the same host and vector used to express the naturally occurring genes. Crude lysates were made and assayed for activity at 60°C with pure glucaric acid, pure glucaric acid in the presence tartrate and with Drum A material at 60°C and 65 °C. Thermostability with Drum A material was assayed by comparison of the activity with this material at 60 minutes and 180 minutes.

Table 5 shows a summary of properties of variants obtained after round 1 of genetic modification. Amino acid changes are shown as the one letter abbreviation for the naturally occurring amino acid followed by its position in the wild type gene ID 33643 (SEQ ID NO: 146) followed by the one letter abbreviation for the amino acid in the created in variant. The reaction conditions for the various assays are shown in the footnotes of Table 5. The performance of the variants from this first round of mutations is indicated on a relative scale sorted in descending order from highest activity with pure glucarate which was exhibited by variant gene ID number 350378 (SEQ ID NO:2).

Table 5

Summary of Properties of Variants Obtained After Round 1 of Genetic Modification

¹ mM KDG/min, 10 mM glucarate substrate, 5 min. reaction time. Enzyme load (lysate concentration = soluble expression level) 1%, pH 6.5, 60°C.

² Fraction activity relative to no inhibitor, 10 mM glucarate substrate, 10 mM L-tartrate inhibitor, 5 min. reaction time. Enzyme load (lysate concentration = soluble expression level) 1%, pH 6.5, 60°C.

³ mM KDG/min, Drum A substrate, 60 min. reaction time. Enzyme load (lysate concentration = soluble expression level) 10%, pH 6.5, 60°C.

⁴ mM KDG/min, Drum A substrate, 60 min. reaction time. Enzyme load (lysate concentration = soluble expression level) 10%, pH 6.5, 65°C.

⁵ mM KDG/min, Drum A substrate, 1020 min. reaction time. Enzyme load (lysate concentration = soluble expression level) 10%, pH 6.5, 65°C.

⁶ Fraction Activity (180 min. / 60 min. reaction time). Enzyme load (lysate concentration = soluble expression level) 10%, pH 6.5, temp 65/60°C.

⁷ Soluble expression pg/ml,

Example 7

Comparison of Round 1 Variant 350378 to the Naturally Occurring Parent

The kinetics and tartrate inhibition of variant 350378 (SEQ ID NO:2) as compared to its wild-type parent hydratase (336438, SEQ ID NO: 146) was investigated. Reaction conditions were as follows: 5 minutes, with pure glucarate at 60°C, 1% lysate, 4mM MgS0 ₄ , 100 mM MES pH 6.5. The results are shown in FIG. 1A, FIG. IB, FIG. 2A, FIG. 2B, FIG. 3A, and Fig. 3B. For each graph depicted in these figures, the Y axis is KDG production rate (mM KDG/min), and the X axis is glucarate concentration (mM). The tartrate concentration (mM) of each curve is identified by the legend, and correspond to tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM, respectively.

FIG. 1A and IB, respectively, show the effect of increasing F-tartrate concentration on glucarate activity between hydratase variant 350378 (SEQ ID NO:2) (FIG. 1A) and wild-type hydratase (336438, SEQ ID NO: 146) (FIG. IB). The F-tartrate concentration (mM) of each curve is identified by the legend, and correspond to F-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM, respectively. A comparison of FIG. 1A to FIG. IB shows that Variant 350378 (SEQ ID NO:2) has much greater resistance to F-tartrate than wild-type hydratase (336438, SEQ ID NO: 146). At 10 mM F-tartrate the variant retains greater than 50% the activity exhibited in the absence of F-tartrate.

FIG. 2A and FIG. 2B, respectively, are graphs showing the effect of increasing D- tartrate concentration on glucarate activity between hydratase variant 350378 (SEQ ID NO:2) (FIG. 2A) and wild-type hydratase (336438, SEQ ID NO: 146) (FIG. 2B). The D- tartrate concentration (mM) of each curve is identified by the legend, and correspond to D- tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM, respectively. A comparison of FIG. 2A to FIG. 2B shows that Variant 350378 (SEQ ID NO:2) has much greater resistance to D-tartrate than wild-type hydratase (336438, SEQ ID NO: 146). At 10 mM D-tartrate the variant retains greater than 70% the activity exhibited in the absence of D-tartrate.

FIG. 3 A and FIG. 3B, respectively, are graphs showing the effect of increasing meso-tartrate concentration on glucarate activity between hydratase variant 350378 (SEQ ID NO:2) (FIG. 3A) and wild-type hydratase (336438, SEQ ID NO: 146) (FIG. 3B). The meso-tartrate concentration (mM) of each curve is identified by the legend, and correspond to meso-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM, respectively. A comparison of FIG. 3A to FIG. 3B shows that variant 350378 (SEQ ID NO:2) has much greater resistance to meso-tartrate than wild-type hydratase (336438, SEQ ID NO: 146). At 20 mM L-tartrate the variant retains greater than 50% the activity exhibited in the absence of L-tartrate. Example 8

Production of Round 2 Variants of the Lactobacillus pentous Glucarate Dehydratase

Variant 350378 produced in in first round of mutation contained mutations A183S, T282V, and D412E in comparison wild-type hydratase 336438. A second set 96 variants were synthesized constituting a second round of spanning mutations, each or which preserved at least two and most often all three of these first-round mutations and further introduced three other mutations spanning the protein sequence. These second-round mutations expressed and assayed as with the first round of mutations at 60°C or 65°C under different conditions of pH i.e, at pH 5.5 or 6.0

Table 6 shows a summary of properties of variants obtained after this second genetic modification sorted in descending order by tartrate resistance. The conditions of temperature and pH for the various assays are shown in the footnotes to the table. All amino acid changes including those preserved from the first round are again shown relative to the wild-type amino acid sequence (gene ID 336438, SEQ ID NO: 146).

Table 6.

Summary of Properties of Variants Obtained After Round 2 of Genetic Modification

¹ mM KDG/min, 10 mM glucarate substrate, 5 min reaction time, enzyme load (lysate concentration = soluble expression level) 1% lysate, pH 5.5, 60°C

² fraction activity (1/2), Drum A substrate, 960 min reaction time, enzyme load 10% lysate, pH (1) 5.5 and (2) 6, 60°C

³ mM KDG/min, 960 min reaction time, enzyme load 10% lysate, pH 5.5, 60°C

⁴ mM KDG/min, 960 min reaction time, enzyme load 10% lysate, pH 6, 60°C

⁵ fraction activity relative to no inhibitor, 10 mM glucarate substrate, 20 mM L-tartrate inhibitor, 5 min reaction time, enzyme load 1% lysate, pH 5.5, 60°C

⁶ mM KDG/min, Drum A substrate, 60 min reaction time, enzyme load 10% lysate, pH 6, 65°C

7 fraction activity (1/2), Drum A substrate, reaction time (1) 180 min, (2) 60 min, enzyme load 10% lysate, pH 6, temperatures (1) 65°C / (2) 60°C

Example 9

Production of Round 3 Variants of the Lactobacillus pentous Glucarate Dehydratase

A third-round genetic variants produced were produced including at least one mutation selected from A183S, T282V and D412E shown to be beneficial for glucarate conversion activity in the first round of mutations and including at least one mutation that showed increased tartrate resistance from the second round of mutations.

Table 7 A is a summary of properties of variants obtained after round 3 of genetic modification. The assays for this round of mutation were conducted at 66°C or 69°C at pH

6.0 as indicated in the footnotes. The first row in Table 7 A provides properties for variant 335047 made during the second round of mutations for sake of comparison. Table 7B shows amino acid changes of these variants again relative to the wild-type amino acid sequence (gene ID 336438, SEQ ID NO: 146).

Table 7A

Summary of Properties of Variants Obtained After Round 3 of Genetic Modification

¹ mM KDG/min, 10mM Glucarate substrate, 3 min 1% lysate, pH 6, 66°C

² mM KDG/min, Drum A substrate, 180 min, 10% lysate, pH 6, 69°C fraction Activity relative to no inhibitor, 10 mM glucarate substrate, 20mM L-tartrate inhibitor, 3 min, 1% lysate, pH 6, 66°C

⁴ mM KDG/min, Drum A substrate, 30 min, 10% lysate, pH 6

⁵ Fraction Activity (1/2), (1)180/(2)30, 10% lysate, pH 6, temp (1)69 °C /(2)66°C

⁶ Fraction Activity (1/2), Drum A substrate, 180 min, 10% lysate, pH (1)6/(2)6.5, 69 °C

⁷ mM KDG/min, 10% lysate ⁸ mM KDG/min, 180 min, 10% lysate

Table 7B

Amino Acid Substitutions, Round 3

Example 10

Production of Round 4 Variants of the Lactobacillus pentous Glucarate Dehydratase

A fourth round of genetic mutations were made preserving at least one beneficial mutation discovered from each of the previous rounds and introducing further mutations. Tables 8 A is a summary of properties of variants obtained after round 4 of genetic modification. All assays were at 66 C at pH 6.0. Conditions are shown in the footnote to table 7A. Table 8B shows all amino acid changes of these variants relative to the wild- type amino acid sequence (gene ID 336438, SEQ ID NO: 146).

Table 8A

Summary of Properties of Variants Obtained After Round 4 of Genetic Modification

'Fraction Activity (1/2), Drum A substrate, (1)180/(2)30, 10% lysate fraction Activity (1/2), (1)180 min /(2)30 min, 10% lysate, pH 6 ³ mM KDG/min, Drum A substrate, 10% lysate ⁴ mM KDG/min, 10% lysate

⁵ Fraction Activity (1/2), Drum A substrate, (1)180 min /( 2)30 min, 10% lysate

⁷ mM KDG/min, 10 mM glucarate substrate, 3 min, 1% lysate, pH 6, 66°C fraction Activity relative to no inhibitor, 20 mM L-tartrate inhibitor, 3 min, 1% lysate, pH 6, 66 °C

⁹ mM KDG/min, 10% lysate

'“Fraction Activity (1/2), Drum A substrate, 30 min, 10% lysate, (1)6 min /(2)6.5 min, 66°C

Table 8B

Amino Acid Substitutions, Round 4

Example 11

Resistance to Tartrate Inhibition by Selected Variants at 66°C pH 6.0

Certain variants were tested for fractional activity at 10 mM glucarate, pH 6.0, at 66° C, with 1% lysate, in the presence of 40 mM D-tartrate or 20 mM L-tartrate, and the results are shown in FIG. 4. FIG. 4 shows tartrate inhibition of variants 368238 (SEQ ID NO:90), 368265 (SEQ ID NO:94), 368298 (SEQ ID NO:98), and 357047 (SEQ ID NO:82). It was found that variant 368238 (SEQ ID NO:90) had overall similar qualities as variant 368265 (SEQ ID NO:94), even though it is a simpler variant, having fewer substitutions than variant 368265 (SEQ ID NO:94).

Example 12

Resistance to Guluronate Inhibition by Selected Variants at 66°C pH 6.0

Certain variants were tested for guluronate inhibition. Test conditions were 10 mM glucarate, pH 6.0, and 66° C, with 1% lysate, and the results are shown in FIG. 5A, 5B, and 5C. The variants that were tested the same as in Example 10, with at 0 mM guluronate (FIG. 5A), 40 mM guluronate (FIG. 5B), and 80 mM guluronate (FIG. 5C).

Example 13

Resistance to Guluronate Inhibition by Selected Variants at higher Temperatures

Certain variants were tested for guluronate inhibition at different temperatures, and the results are shown in FIG. 6A, 6B, 6C, and 6D. These figures, respectively, are graphs showing KDG production rates over time, at pH 6.0, with 10% lysate at 66° C (FIG. 6A), 68° C (FIG. 6B), 70° C (FIG. 6C), and 72° C (FIG. 6D) for variants 374373 (SEQ ID NO: 104), 374377 (SEQ ID NO: 107), 374388 (SEQ ID NO: 111), 374398 (SEQ ID

NO: 116), 374413 (SEQ ID NO: 123), 374414 (SEQ ID NO: 124), 374423 (SEQ ID

NO: 128), 368238 (SEQ ID NO:90), and 357047 (SEQ ID NO:82). For each graph, the Y axis KDG formed (mM/minute), and the X axis is time (minutes).

At 70° C (FIG. 6C) and 180 minutes, the variants of round 4, i.e., 374373 (SEQ ID NO: 104), 374377 (SEQ ID NO: 107), 374388 (SEQ ID NO: 111), 374398 (SEQ ID

NO: 116), 374413 (SEQ ID NO: 123), 374414 (SEQ ID NO: 124), 374423 (SEQ ID

NO: 128), and the variant of round 3, i.e., variant 368238 (SEQ ID NO:90), all out performed the variant of round 2, i.e., variant 357047 (SEQ ID NO:82). Table 9

Amino Acid Substitutions and SEQ ID NOs for All Useful Variants with Improved Thermostability and Resistance to Inhibion

Table 9 summarizes the amino acid substitutions of 139 variants made through various rounds of mutations of parent SEQ ID NO: 146 from Lactobacillis pentous, all of which exhibit thermostability in retaining at 60°C, at least 50% of the activity exhibited by the parent enzyme at 30-40°C in converting glucarate to KDG and which exhibit resistance to inhibition by tartrate by retaining at least 50% of the activity in converting glucarate to KDG in the presence of 10 mM tartrate in comparison to the activity exhibited in the absence of tartrate.

While the production of glucarate dehydratase variants with improved thermostability and tartrate resistance is exemplified above by making variants of the Lactobacillis pentous enzyme, a person of ordinary skill in the art will recognized from the present disclosure that similar improvements can be made starting with any of the naturally occurring sequences according SEQ ID NOS: 141-148 first disclosed herein as having greater thermostability and resistance to inhibition to tartrate than the E.coli enzyme.

Those skilled in the art having the benefit of the present disclosure will recognize that the compositions disclosed herein can be used in an integrated process with a particular crude feedstock. The integrated process may comprise a two-step process. The first step may comprise oxidizing glucose with a metal catalyst to glucaric acid. The second step may comprise converting glucaric acid to KDG using a thermostable glucarate dehydratase. In the process, due to the glucarate dehydratase enzymes disclosed in the present disclosure, inhibitors, e.g., tartaric acid, do not inhibit the enzymatic conversion of glucaric acid to KDG. The glucarate dehydratase enzymes disclosed in the present disclosure can be immobilized on a substrate in a column and the dehydration occurs by flowing the crude glucose oxidation mixture over the column and collecting KDG from the eluate.

Those having skill in the art, with the knowledge gained from the present disclosure, will recognize that various changes can be made to the disclosed processes in attaining these and other advantages, without departing from the scope of the present disclosure. As such, it should be understood that the features of the disclosure are susceptible to modifications and/or substitutions. The specific embodiments illustrated and described herein are for illustrative purposes only, and not limiting of the invention as set forth in the appended claims.