# Patent

## Title
Arabinitol dehydrogenase from Neurospora crassa

## Abstract

Stable and active arabinitol dehydrogenases (LAD) from Neurospora crassa and mutants thereof are disclosed. Arabinitol dehydrogenases are useful in the production of xylitol and ethanol from an arabinose containing substrate. Recombinant and heterologously expressed arabinitol dehydrogenases are useful in converting biomass into biofuels and other industrial food products.

## Background
This application is a Divisional of co-pending U.S. patent application Ser. No. 12/235,253, filed Sep. 22, 2008, which claims priority to U.S. Provisional Application No. 60/975,023, filed Sep. 25, 2007, the contents of which applications are herein incorporated by reference in their entireties.


BACKGROUND
L-arabinitol 4-dehydrogenases (LAD) from Neurospora crassa and their uses in production of sugar alcohols including xylitol from arabinose containing media are disclosed.
Lignocellulosic biomass represents a renewable resource that is available in sufficient quantities from the corn wet-milling industry to serve as a low-cost feedstock. Some sources, particularly corn fiber, contain significant amounts of L-arabinose, an abundant pentose sugar second only to D-xylose in biomass composition. However, utilization of the L-arabinose content from hemicellulose hydrolysates for production of valued products has resulted in limited success. The inability of many yeasts and fungi to ferment L-arabinose appears to be a consequence of inefficient or incomplete assimilation pathways for this pentose sugar. It has also been suggested that the cofactor imbalance necessary for the catabolism of L-arabinose also plays a factor. Recently some progress has been made with the overexpression of either the bacterial utilization pathway or the fungal pathway for production of ethanol from L-arabinose. One benefit of utilizing the fungal pathway is that the intermediate xylitol is also formed, which is a five-carbon sugar alcohol that has attracted much attention because of its potential as a natural food sweetener, a dental caries reducer, and a sugar substitute for diabetics.
Xylitol is a pentitol and is used not only as a sweetener but also as a platform chemical for the production of industrially important chemicals. Studies have shown that among sugar substitutes, xylitol is one of the most promising candidates for application in a wide range of products due to several favorable properties. These include anti-cariogenicity, suitability for use by diabetic patients, and good gastrointestinal tolerance, in addition to possibly preventing osteoporosis and ear infections. In spite of its advantages, the use of xylitol is currently limited and falls well short of another, cheaper sugar alternative, sorbitol in the billion dollar polyol market. Other than its use as a sweetener, xylitol is also an industrially important chemical, and the US Department of Energy (DOE) has named it among one of their top 12 platform chemicals from agricultural sources.
L-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12), a common enzyme found in yeasts and filamentous fungi, catalyzes the second step of the recently elucidated fungal L-arabinose metabolic pathway by oxidizing L-arabinitol to L-xylulose with concomitant NAD+ reduction. LAD is purportedly a fungal orthologue of the eukaryotic sorbitol dehydrogenase (SDH) and belongs to the family of zinc-containing alcohol dehydrogenases. Several LADs have successfully been cloned and expressed. However, they are not optimal for in vitro enzymatic production of xylitol due to their poor stability and/or activity.
L-arabinose is a major constituent of some plant materials, up to 15% of materials such as wheat bran and corn cob hulls, so that L-arabinose processing is of relevance for micro-organisms using plant material as a carbon source. The bacterial pathway for L-arabinose catabolism is known. It includes an isomerase, a kinase, and an epimerase that sequentially convert L-arabinose to L-ribulose, L-ribulose 5-phosphate, and D-xylulose 5-phosphate. D-xylulose 5-phosphate is an intermediate of the pentose phosphate pathway. There is also a pathway for fungi that may include five enzymes, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, and xylulokinase. The intermediates are, in this order: L-arabinitol, L-xylulose, xylitol and D-xylulose.
SUMMARY
Cloning, heterologous expression, purification, and characterization of a new L-arabinitol 4-dehydrogenase (LAD) from Neurospora crassa are disclosed herein. This enzyme is very stable and active compared to other known arabinitol dehydrogenases.
An NAD+-dependent L-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) from Neurospora crassa was cloned and recombinantly expressed in Escherichia coli and purified to homogeneity. The enzyme was a homotetramer with a subunit molecular mass of 39,245 Da, and contains two Zn2+ ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases. Km values for substrates L-arabinitol, adonitol, and xylitol were 16 mM, 35 mM, and 290 mM, respectively. The enzyme showed strong preference for NAD+, with a Km of 174 μM, but also displayed very low yet detectable activity with NADP+. No activity was observed for D-mannitol, D-arabinitol, or D-sorbitol. The optimum activity was between 45-55° C. The pH optimum was approximately pH 9.5, and >60% of the activity remained in the pH span from 8.0 to 10.5. This enzyme is one of the most stable and active LADs ever reported, and is useful for in vivo and in vitro production of xylitol and ethanol from L-arabinose.
A purified arabinitol dehydrogenase includes an amino acid sequence of SEQ ID NO: 1. An arabinitol dehydrogenase may be recombinant and may be heterologously expressed. In an aspect, purified arabinitol dehydrogenase includes a fusion protein. The purity of purified arabinitol dehydrogenases disclosed herein may range from about 85% to 90% and from about 90% to about 95% or 99%.
Arabinitol dehydrogenases disclosed herein include an amino acid sequence that is equal to or greater than or at least about 85% or 90% or 95% or 99% similar to SEQ ID NO: 1, which include spontaneous mutations or random variations. Arabinitol dehydrogenases disclosed herein include a nucleic acid sequence that is equal to or greater than or at least about 80% or 85% or 90% or 95% or 99% similar to SEQ ID NO: 2, which include spontaneous mutations or random variations.
A suitable heterologous host for expressing and purifying arabinitol dehydrogenases disclosed herein include for example, bacteria, yeast, and plants.
An isolated nucleic acid sequence encoding an arabinitol dehydrogenase includes a nucleic acid sequence designated by SEQ ID NO: 2. The nucleic acid of arabinitol dehydrogenase may be directly isolated from Neurospora crassa or may also be directly synthesized or may also be recombinantly generated.
Arabinitol dehydrogenases disclosed herein are useful in producing xylitol, ethanol and any suitable sugar alcohol. For example, an arabinitol dehydrogenase is useful in producing ethanol from a plant material, such as corn. Production of ethanol may be by fermentation.
In an aspect, the production of xylitol or ethanol utilizes a phosphite dehydrogenase-based NADP regeneration system. Purified arabinitol dehydrogenases are useful to metabolically enhance an organism used for fermentation of a plant biomass to produce ethanol.
A method of producing ethanol includes:
(a) obtaining a purified arabinitol dehydrogenase that includes an amino acid sequence of SEQ ID NO: 1; and
(b) providing conditions to produce ethanol from an arabinose containing medium.
A method of producing xylitol includes:
(a) obtaining a purified arabinitol dehydrogenase that includes an amino acid sequence of SEQ ID NO: 1; and
(b) providing conditions to produce xylitol from an arabinose containing medium.
A heterologous host expressing an arabinitol dehydrogenase includes for example, Escherichia coli, Saccharomyces cerevisiae, and a plant cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows overexpression and purification of recombinant N. crassa LAD. Lane 1, the molecular weight marker proteins (size in kDa is shown); lane 2, cell-free crude extract; lane 3, purified LAD enzyme with N-terminal GST-tag; lane 4, purified LAD enzyme with GST-tag removed by thrombin cleavage.
FIG. 2 demonstrates: A) kcat dependence on temperature. N. crassa LAD was assayed at different temperatures from 12 to 65° C. at saturating concentrations of 200 mM L-arabinitol and 2 mM NAD+. B) Thermal inactivation of LAD at 50° C. The heat inactivation at 50° C. was irreversible and followed first-order kinetics with a half-life of 45 min. C) pH rate profile. Saturating concentrations of 200 mM L-arabinitol and 2 mM NAD+ were used to measure the activity in a universal buffer at various pH values from 7.0 to 11.0.
FIG. 3 shows: A) Crystal structure of human SDH with bound NAD+ and catalytic zinc ion (1PL8). B) Homology model of N. crassa LAD with bound NAD+ and catalytic zinc ion, built using the Insight II (Accelrys Software Inc., San Diego, Calif.) and molecular operating environment (MOE) programs. The catalytic zinc ion (Zn), four catalytic zinc binding residues, and NAD+ cofactor are colored by atom type.
FIG. 4 shows HPLC size exclusion chromatography. A size exclusion standard was used to calibrate a Bio-Sil SEC-250, 300×7.8 mm column with a mobile phase of 0.1 M Na2HPO4, 0.15 M NaCl, 0.01 M NaN3, pH 6.8 at a flow rate of 1 ml/min. The standard proteins are represented by closed squares. LAD samples (represented by open squares) with and without 15% SDS were injected separately and fitted to the standard curve.
FIG. 5 shows an RT-PCR product of the N. crassa LAD gene. Lane 1 shows the DNA base pair ladder. Lane 2 is the control in which there was no reverse transcription. Lane 3 is the RT-PCR product amplified from N. crassa total RNA. The approximately 1.1 kb product in Lane 3 was the expected size for the 1080 bp N. crassa LAD encoding gene plus the extra primer length for cloning. This product was subsequently sequenced and determined to be the desired gene.
FIG. 6 shows amino acid sequence alignment of N. crassa LAD (SEQ ID NO: 1) with four other closely related LAD sequences (SEQ ID NOS 5-8, respectively, in order of appearance) from filamentous fungi and yeast. Residues highlighted in gray represent the four conserved residues that make up the catalytic zinc binding tetrad. Residues indicated by arrows represent the four conserved cysteine residues that make up the proposed structural zinc binding tetrad.
FIG. 7 shows HPLC analysis of the reaction products of N. crassa LAD. A) Cofactor stocks, 260 nm; B) Cofactor stocks, 340 nm; C) NAD+ reaction mix products, 340 nm; D) NADP+ reaction mix before (gray) and after (black) LAD enzyme addition, 340 nm (magnified).



DETAILED DESCRIPTION
NCU00643.1 (EAA36547.1) (hypothetical) from N. crassa was found to encode an L-arabinitol 4-dehydrogenase of 363 amino acid residues with a calculated MW of 39,245. Sequence alignment with other reported LADs shows sequence similarity of about 70-80%, with conserved regions for Zn2+ binding, cofactor binding, and active site residues (FIG. 6). Comparison with several mammalian SDHs (mouse, rat, bovine, sheep, and human) showed  ̃40% similarity, whereas comparison with xylitol dehydrogenases (Hypocrea jecorina, Aspergillus oryzae, Candida tropicalis, Pichia stipitis, and Aspergillus fumigatus Af293) showed  ̃30% similarity.
Kinetic parameters of characterized LADs from Hypocrea jecorina, Aspergillus niger, and Aspergillus oryzae are displayed in Table 3. The Km value of N. crassa LAD was 16 mM for L-arabinitol, which when compared to LADs from H. jecorina and A. niger, is one of the lower values reported of characterized LADs. With a specific activity of the purified N. crassa LAD equal to about 31 U/mg, it is almost 20-fold greater than H. jecorina LAD purified from S. cerevisiae heterologous expression, and orders of magnitude higher than other LADs except for A. niger LAD, which shows about 3-fold greater specific activity than that of N. crassa LAD. However, it was also reported that the purified A. niger LAD was highly unstable, with rapidly diminishing activity at 4° C., and complete loss of activity after freeze-thawing of the enzyme. In contrast, N. crassa LAD is quite stable and does not markedly lose activity when frozen repeatedly.
There has been no in-depth study of the substrate binding residues for LAD, but the enzyme has been postulated to be a fungal orthologue of the eukaryotic D-sorbitol dehydrogenases which have been investigated more thoroughly. Based on these reports, the active site substrate binding residues are all strictly conserved in all LADs characterized to date (FIG. 6). When comparing these residues in N. crassa LAD to sorbitol dehydrogenases, F59 was not conserved, which instead was a tyrosine residue in all of the SDHs examined. Mutational studies of this position were examined for F59A, F59S, and F59Y, to determine what effects this residue has on substrate specificity alteration, and are shown in Table 4. Replacement of the native F59 residue with the homologous tyrosine found in SDH decreased the catalytic efficiency towards all active substrates. The ability of LAD to bind each substrate markedly decreased as the size of the amino acid at position 59 was decreased. Although these results suggest this residue is important for binding and catalysis for the active substrates of the wild type LAD, it did not confer the ability to accept D-sorbitol as a substrate. Amino acids flanking the active site cleft may be responsible for the activity and affinity patterns between LAD and SDH.
A gene from N. crassa encoding an LAD was cloned and purified. The enzyme is highly active and stable, acts on several five carbon sugar alcohol substrates, and operates over a wide pH range. This enzyme is useful in the production of xylitol and ethanol from L-arabinose derived from renewable resources.
In certain embodiments, purified LADs are about 95% similar to SEQ ID NO: 1 and may include naturally occurring variations in N. crassa arabinitol dehydrogenases. In certain embodiments, arabinitol dehydrogenases disclosed herein are recombinant and/or expressed or purified from a heterologous host. Suitable heterologous hosts include for example, bacteria, yeast, and plants or plant cells. Cultures of bacteria, yeast, and plant cells in a batch reactor or a continuous flow reactor are also suitable for large-scale arabinitol dehydrogenases production.
In certain embodiments, purified arabinitol dehydrogenases disclosed herein are about 90% pure, or 95% pure or about 98% pure and generally more than about 90% pure.
Arabinitol dehydrogenases disclosed herein are capable being expressed in a variety of heterologous hosts such as bacteria, fungi, and plants. Such hosts include for example, Escherichia coli, Saccharomyces cerevisiae, and a plant cell. In certain embodiments, the heterologous hosts are engineered for increased arabinose uptake. The substrate or the source material need not be extensively or substantially purified and can include mixtures of sugars as found in plant biomass material.
The term “consisting essentially of” refers to a conserved portion of arabinitol dehydrogenases that include one or more amino acid positions disclosed herein that are important for the catalysis. For example, FIG. 6 shows a multiple sequence alignment showing conserved residues from a variety of arabinitol dehydrogenases, thus providing a structure-function relationship. Thus, the term consisting essentially of refers to that portion of the arabinitol dehydrogenases that are able to act catalytically and maintain similar efficiency.
Intermediates in the fungal pentose pathway, such as xylitol are also produced. For example, microbes are engineered to produce bulk amounts of xylitol, wherein the engineered microbes express at least one reductase and/or dehydrogenase during the synthesis of xylitol, in addition to the LADs disclosed herein. In certain embodiments, microbes are engineered to express xylose reductases (also referred to herein as XRs) and xylitol dehydrogenase (also referred to herein XDH) enzymes to produce xylitol from xylose (or xylulose) in vivo. For example, E. coli are constructed to express XDH and/or XR to produce xylitol from a substrate that includes xylose (or xylulose). Certain embodiments also provide engineered microbes capable of deriving reducing equivalents from carbon substrates (such as glucose) for the subsequent reduction of xylose or xylulose to xylitol. The contents and disclosures of co-pending PCT/US2008/069657 are herein incorporated by reference in its entirety as it relates to various xylose reductase mutants and uses thereof.
As used herein, the terms gene and polynucleotide sequence are used interchangeably. Nucleotide sequences that encode for or correspond to a particular sequence of nucleic acids (such as ribonucleic acids) or amino acids that include all or part of one or more products (such as polypeptides, proteins, or enzymes), and may or may not include regulatory sequences, such as promoter sequences, which determine, for example, the conditions under which the gene is expressed.
Many small variations in the nucleotide sequence of a gene do not significantly change the catalytic properties of the encoded arabinitol dehydrogenases disclosed herein. For example, many changes in nucleotide sequence do not change the amino acid sequence of the arabinitol dehydrogenases disclosed herein. Also an amino acid sequence can have variations which do not change the functional properties of arabinitol dehydrogenases disclosed herein, in particular they do not prevent arabinitol dehydrogenases from carrying out its catalytic function. Such variations in the nucleotide sequence of DNA molecules or in an amino acid sequence are known as “functionally equivalent variants”, because they do not significantly change the function of the gene to encode a protein with a particular function, e.g. catalysing a particular reaction or, respectively, of the protein with a particular function. Thus such functionally equivalent variants, including fragments, of the nucleotide sequence of SEQ ID NO: 2 and, respectively, of the amino acid sequence of SEQ ID NO: 1, are encompassed within the scope of the disclosure.
Genetically engineered DNA molecules, e.g., a recombinant DNA, a vector, e.g., an expression vector, that includes the nucleic acid encoding the arabinitol dehydrogenases disclosed herein or their catalytically active fragments thereof are expressed in host cells, i.e. microorganisms. Arabinitol dehydrogenases disclosed herein may be operably linked to a promoter. The vector can be e.g. a conventional vector, such as a virus, e.g. a bacteriophage, or a plasmid, preferably a plasmid. The construction of an expression vector is within the skills of an artisan.
The following examples are for illustrative purposes only and are not intended to limit the scope of the disclosure.
Example 1
N. crassa LAD Gene Identification
LADs from Hypocrea jecorina (GenBank accession number AF355628.1) and Aspergillus oryzae (AB116938.2) were used as templates for a protein BLAST search. Utilizing the whole-genome sequence of N. crassa, a postulated hypothetical protein NCU00643.1 (EAA36547.1) was discovered that had the greatest sequence identity ( ̃80%). This protein was later designated as N. crassa LAD and had significant homology (72 to 80% identity) with other LADs. Among the conserved residues were those that formed the active site and the structural Zn2+-binding site and the glycine fingerprint found in polyol dehydrogenases, as well as the majority of those shown to bind substrate in the SDH homologues.
This example demonstrates that a hypoethetical sequence was specifically selected from a myriad of sequences and subsequently demonstrated to have the functional properties of an arabinitol dehydrogenase.
Example 2
N. crassa LAD Cloning and Expression
N. crassa RNA purification, reverse-transcription PCR, cloning, and N. crassa LAD expression are described in this example. Reverse transcription-PCR (RT-PCR) performed on total RNA isolated from L-arabinose-induced N. crassa 10333 showed the expected size of a gene product (FIG. 5). The reverse transcription-PCR product was subcloned into pGEX-4T-3 vector (Amersham Biosciences) using EcoRI and NotI restriction sites and was transformed into Escherichia coli BL21(DE3). This construct (pGEX-lad1) encoded N. crassa LAD as an N-terminal glutathione S-transferase (GST)-tagged fusion with a thrombin cleavage site. Cell lysates of isopropyl-β-D-thiogalactopyranoside (IPTG) induced cultures of these cells were prepared, analyzed by SDS-PAGE, and assayed for LAD activities. The construct produced soluble GST-tagged N. crassa LAD at  ̃16% of the total soluble cellular proteins (FIG. 1), which was then purified in a single step with a GST-Bind kit (Novagen) according to manufacturer's protocol. The purified protein was desalted by ultrafiltration with several washes of 50 mM morpholinepropanesulfonic acid (MOPS) buffer (pH 7.25). After digesting with biotinylated thrombin (Novagen), the enzyme was incubated with streptavidin agarose to remove the thrombin, and then passed through GST-Bind resin again to remove the cleaved GST-tag. GST-tagged LAD cleaved with thrombin was used for characterization purposes, as it had about 65%-greater specific activity than the tagged LAD enzyme. LAD stocks were stored frozen with 10% (v/v) glycerol at −80° C. Protein concentrations were determined by the Bradford method and by using an estimated extinction coefficient (San Diego Supercomputer Center Biology Workbench) of 35.3 mM-1 cm-1 at 280 nm with similar results. The purity of the protein was analyzed by an SDS-PAGE gel stained with Coomassie brilliant blue (FIG. 1). The final yield of protein was 30 mg/liter of culture ( ̃9 mg/g of E. coli) of >95% pure LAD with a molecular mass of  ̃39 kDa, consistent with the predicted value of 39.6 kDa.
Example 3
Steady-State Kinetics
Initial rates were determined using a Varian Cary 100 Bio UV-visible spectrophotometer (Varian) at 25° C. in 50 mM Tris (pH 8.0). Purified N. crassa LAD displayed activity with NAD+ as the preferred cofactor (Table 1), although there was small yet detectable activity with NADP+, which was verified by high performance liquid chromatography (HPLC, FIG. 7). This is the first reported detection of NADP+ utilization by LAD, although it is still considered a strongly NAD+-dependent enzyme. Kinetic measurements with substrate L-arabinitol and cofactor NAD+ were taken in a 5-by-5 matrix format, with substrate and cofactor concentrations varied from below their Km to 10-fold higher than their Km. The kinetic data were analyzed with a modified version of Cleland's program (Cleland, W. W. (1979) Statistical analysis of enzyme kinetic data, Methods Enzymol 63, 103-138.). Vmax and Km for both L-arabinitol and NAD+ were obtained by fitting the data to a sequential ordered mechanism with NAD+ binding first, based on the proposed mechanism for sorbitol dehydrogenase (Lindstad et al. (1992) The kinetic mechanism of sheep liver sorbitol dehydrogenase, Eur J Biochem 210, 641-647.)

v=VmaxAB/(KiaKB+KAB+KBA+AB)  (EQ. 1)

where v is the initial velocity, Vmax is the maximum velocity, KA and KB are the Michaelis-Menten constants for NAD+ and L-arabinitol, respectively, A and B are the concentrations of NAD+ and L-arabinitol, respectively, and Kia is the dissociation constant for NAD+.
Table 2 displays the kinetic constants of several other sugar substrates accepted by N. crassa LAD. D-Arabinitol, adonitol, xylitol, D-sorbitol, and D-mannitol were all examined as alternative substrates for N. crassa LAD with NAD+ as the cofactor held at saturating concentration of 2 mM. Of the pentose sugar alcohols, only adonitol and xylitol acted as substrates, with Km values of 35 mM and 290 mM respectively. This pattern of substrate promiscuity is similar to those of LADs isolated from other sources.
Example 4
Temperature Dependence
The optimal temperature of activity was determined by assaying LAD activities at temperatures ranging from 12 to 65° C. The data show the optimum temperature to be between 45 and 55° C. (FIG. 2A). At higher temperatures, the enzyme inactivates rapidly, and at lowered temperatures, the rate increases with temperature according to the Arrhenius equation. Utilizing the Arrhenius equation to fit the data from 12 to 30° C., the energy of activation for L-arabinitol oxidation by N. crassa LAD was determined to be 49 kJ/mol. The stability for N. crassa LAD was relatively high, as it retained activity at room temperature for longer than one month and at 4° C. for several months. Thermal inactivation of N. crassa LAD was studied by incubating at 50° C. in 50 mM Tris (pH 8.0), with samples removed at various times and assayed for activity in saturating substrate conditions. FIG. 2B shows the percentage of residual activity versus incubation time, which followed a first-order exponential decay with a half-life of 45 min. Interestingly, when tested at a slightly lower temperature of 45° C., the enzyme was able to retain  ̃60% of its activity after 4 hr.
Example 5
pH Rate Profile
Activity was measured at pH values between 7.0 and 11.0 under saturating concentrations of NAD+ (2 mM) and L-arabinitol (200 mM) in a universal buffer (50 mM morpholineethanesulfonic acid (MES)/50 mM Tris/50 mM glycine). The pH range for N. crassa LAD activity was large, with >25% of the activity occurring with pH values of 7.0 to 11.0 (FIG. 2C). The pH optimum was around pH 9.5, and >60% of the activity remained in the pH span from 8.0 to 10.5.
Example 6
Determination of Mass and Quaternary Structure
The quaternary structure of N. crassa LAD was determined using an Agilent 1100 series HPLC system with a Bio-Sil SEC-250 column (300×7.8 mm) and a mobile phase of 0.1 M Na2HPO4, 0.15 M NaCl, and 0.01 M NaN3, pH 6.8. Based on the standardized retention times of a Bio-Rad molecular mass standard, the molecular mass of LAD was calculated from its retention time to be  ̃152 kDa (FIG. 4). Monomerization was induced in the presence of 15% SDS, causing LAD to elute as a single peak with a retention time corresponding to a molecular mass of  ̃39 kDa. The data indicates that the native LAD is a noncovalently linked tetramer, which is typical for fungal derived zinc-containing alcohol dehydrogenases.
Example 7
Metal Analysis
Samples of thrombin-cleaved N. crassa LAD were buffer exchanged with 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (pH 8.0) and lyophilized prior to submission to the Microanalytical Laboratory at the University of Illinois at Urbana-Champaign. N. crassa LAD was determined to contain very close to two mol of zinc/mol subunit using an inductively coupled plasma spectrophotometer (OES Optima 2000 DV, Perkin Elmer, Boston, Mass.). This is consistent with previously reported SDH and ADH enzymes containing both an active site zinc ion and a second zinc ion thought to be involved in stability. The verification of the second zinc atom also correlates well with the four conserved cysteine residues involved in structural zinc binding in homologous SDHs and XDHs.
Example 8
Cofactor Specificity
The cofactor specificity of N. crassa LAD was examined by HPLC. The separation of NAD+, NADP+, NADH, and NADPH was carried out using standard methods. No discernible cross-contamination of oxidized cofactors was observed. 20 μL reaction mixtures consisting of equal parts of 1 mM NAD(P)+ and 25 mM L-arabinitol in 50 mM Tris (pH 8.0) were set up, and following addition of approximately 1 μg of enzyme, the reaction was allowed to proceed for 20 min at 37° C. When NAD+ was used as the cofactor, the products were analyzed by HPLC, and a single peak (UV 340 nm) was observed that had the same retention time as authentic NADH. The same process was carried out for NADP+ as the cofactor, and a small yet detectable peak was observed with a retention time corresponding to an authentic sample of NADPH. This indicated the strong preference for NAD+ as the cofactor of N. crassa LAD.
Example 9
Homology Modeling
Using the coordinates for NAD+-dependent human SDH (PDB accession code 1PL8) (Pauly et al. (2003) X-ray crystallographic and kinetic studies of human sorbitol dehydrogenase, Structure 11, 1071-1085) and NADP+-dependent SDH from silverleaf whitefly, Bemisia argentifolii (PDB accession code 1E3J) (Banfield et al. (2001) Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 A resolution, J Mol Biol 306, 239-250), a homology model was created with Insight II software. The model was docked with NAD+ and the catalytic zinc ion, and subjected to energy minimization by using the Molecular Operating Environment (MOE) program. The model was verified for consistency with known protein folds and allowed ψ and φ angles. The resulting model was very similar to the human SDH crystal structure in overall fold and binding of coenzyme, as illustrated in FIG. 3. The only major deviation between the backbone of these two structures is the N-terminal region of amino acids 1 through 8 (FIG. 3). However, this may be due to the different conformations of the N-terminus between being in solution and forming dimerization contacts found to be present in SDH. The conserved catalytic zinc binding residues C53, H78, E79, and E163 from SDH (FIG. 3A) have similar orientations and locations in the N. crassa LAD model (FIG. 3B). When comparing proposed substrate binding residues from SDH to N. crassa LAD, the majority—S55, F127, T130, E163, R308, Y309—are strictly conserved and configured in similar orientations. However, one substrate binding residue, F59, was different from the homologous tyrosine residue in SDH, making the N. crassa LAD binding pocket slightly more hydrophobic (FIG. 6).
Example 10
F59 Mutant Kinetic Analysis
Activity assays were run for three mutants of N. crassa LAD (F59A, F59S, and F59Y) to study the effect of mutation of this active site, putative substrate binding residue homologous to tyrosine in other SDHs. All assays were carried out similar to substrate specificity profile for the wild type enzyme (described in Steady-state kinetics example), with the cofactor NAD+ held at saturating concentration of 2 mM for all assays. The mutants were still found to have activity with L-arabinitol, xylitol, and adonitol, and their kinetic parameters are displayed in Table 4. D-Sorbitol was also tested but showed no significant activity over the wild type N. crassa LAD.







TABLE 1







Kinetic parameters for N. crassa LADa.











N. crassa
Km for


Km for


LAD with
NAD(P)

kcat/Km 
L-arabinitol


indicated
(mean ± SD)
kcat (mean ± SD)
for NAD(P)
(mean ± SD)


coenzyme
(μM)
(min−1)
(μM−1 min−1)
(mM)





NAD
174 ± 24
1,206 ± 54
6.9
16 ± 3


NADP
—
—
6.5 × 10−7
—





aAll assays were performed at 25° C. in 50 mM Tris, pH 8.0













TABLE 2







Kinetic parameters for N. crassa LAD with other substratesa.











N. crassa






LAD with


kcat/Km


indicated
kcat (mean ± SD)
Km (mean ± SD)
(mM−1 


substrate
(min−1)
(mM)
min−1)
% Activity














L-Arabinitol
1,210 ± 30
18 ± 2
67
100


Xylitol
  970 ± 40
290 ± 27
3.3
   4.9


Adonitol
1,080 ± 30
35 ± 3
31
 46


D-Arabinitol
—
—
NDb
 0


D-Sorbitol
 —c
 —c
 —c
  0c


D-Mannitol
—
—
ND
 0





aAll assays were performed at 25° C. in 50 mM Tris, pH 8.0, at saturated NAD+ concentration


bND, not detected


ctrace activity at 2M D-sorbitol concentration, possibly due to substrate contamination













TABLE 3







Kinetic parameters of LAD from various source organisms.
















kcat/




Specific


Km,L-arabinitol



Organism
activity
kcat
Km,L-arabinitol
(mM−1 
Km,NAD+


(reference)
(U/mg)
(min−1)
(mM)
min−1)
(μM)















N. crassa
31
1,206
16
75
174


H. jecorina
1.6
N/Aa
40
N/A
180


H. jecorina
0.013
51
4.5
11
N/A


A. niger
96
N/A
89
N/A
 50


A. oryzae
0.04
N/A
N/A
N/A
N/A





aN/A, not determined













TABLE 4







Kinetic parameters of F59 mutantsa.















kcat/Km




kcat (mean ± SD)
Km (mean ± SD)
(mM−1 


Substrate
Enzyme
(min−1)
(mM)
min−1)














L-Arabinitol
WT
1,210 ± 30
 8 ± 2
67



F59Y
  840 ± 30
42 ± 5
20



F59S
  60 ± 3
62 ± 9
0.97



F59A
—b
>400
0.12


Xylitol
WT
  970 ± 40
290 ± 27
3.3



F59Y
—b
>880
1.2



F59S
—b
>1,400
0.04



F59A
—b
>1,850
0.01


Adonitol
WT
1,080 ± 30
35 ± 3
31



F59Y
1,420 ± 50
193 ± 11
7.4



F59S
 120 ± 5
430 ± 48
0.28



F59A
—b
>1,110
0.03





aAll assays were performed at 25° C. in 50 mM Tris, pH 8.0.


bSaturation of substrate was not reached.






Materials and Methods
Strains, Plasmids and Reagents
Materials. The Neurospora crassa genomic sequence and LAD protein sequences were accessed from the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov). N. crassa 10333 was obtained from the American Type Culture Collection (ATCC). Escherichia coli BL21(DE3), GST-Bind kit, biotinylated thrombin, and streptavidin agarose were purchased from Novagen (Madison, Wis.). E. coli μM1788 was kindly provided by William Metcalf at the University of Illinois (Urbana, Ill.). GST gene fusion expression vector pGEX-4T-3 was purchased from Amersham Biosciences (Piscataway, N.J.). SuperScriptTM One-Step RT-PCR with Platinum® Taq kit was obtained from Invitrogen (Carlsbad, Calif.). Shrimp alkaline phosphatase, and PCR grade dNTPs were obtained from Roche Applied Sciences (Indianapolis, Ind.). Phusion High-Fidelity DNA Polymerase and DNA-modifying enzymes DNase I, EcoRI, NotI, and T4 DNA ligase and their appropriate buffers were purchased from New England Biolabs (NEB) (Beverly, Mass.). L-arabinitol, D-arabinitol, adonitol, xylitol, D-sorbitol, D-mannitol, ampicillin, isopropyl β-D-thiogalactopyranoside (IPTG), NADH, NADP+, and NADPH were purchased from Sigma (St. Louis, Mo.). NAD+ was a gift from Julich Fine Chemicals. Other required salts and reagents were purchased from Fisher (Pittsburgh, Pa.) or Sigma-Aldrich. The QIAprep spin plasmid mini-prep kit, QIAquick gel purification kit, RNeasy midiprep kit, and QIAquick PCR purification kit were purchased from Qiagen (Valencia, Calif.). Various oligonucleotide primers were obtained from Integrated DNA Technologies (Coralville, Iowa). SDS-PAGE gel materials, electrophoresis equipment, protein size markers, size exclusion standards (catalog number 151-1901) and Bio-Sil SEC-250, 300×7.8 mm column were purchased from Bio-Rad (Hercules, Calif.).
RT-PCR and cloning. N. crassa 10333 was grown on rich potato media at 30° C. for 24 h, and induced with 150 mM L-arabinose for 2 h. Since the predicted gene contained one intron, RT-PCR was utilized to isolate the processed gene. Total RNA was purified from collected cells (RNeasy purification kit, Qiagen) and treated with DNase I to remove residual genomic DNA. RT-PCR was performed using SuperScriptTM One-Step RT-PCR with Platinum® Taq (Invitrogen) following the manufacturer's guidelines and suggestions for controls. Sequencing results determined at the Biotechnology Center of the University of Illinois showed the product had four silent mutations compared with the predicted sequence from the NCBI database. A control reaction consisted of the same protocol except the SuperScriptTM enzyme mix was heated to 95° C. for ten minutes to thermally inactivate the reverse transcriptase enzyme and the reverse transcription thermocycler step was omitted. The primers used for the RT-PCR were: Forward 5′-GTA GCT ACG TCA GAA TTC CAT GGC TTC TAG CGC TTC C-3′ (SEQ ID NO: 3) and Reverse 5′-GCT GAT TCT GCG GCC GCT TAC TCC AGA CTC TGG ATC-3′ (SEQ ID NO: 4). The forward primer contained an EcoRI restriction site (shown in bold), while the reverse primer contained a NotI restriction site (shown in bold) and stop codon (underlined). The resulting RT-PCR product was isolated by a QIAquick agarose gel purification kit and amplified by an additional 20 cycles of PCR. The product was digested with EcoRI and NotI restriction enzymes and purified again by agarose gel electrophoresis. It was then ligated into pGEX-4T-3 which had been previously prepared by EcoRI and NotI digestion, dephosphorylation by shrimp alkaline phosphatase, and gel purification. The ligation mixture was precipitated with n-butanol, and resuspended in water.
The new construct was used to transform E. coli WM1788 by electroporation. Positive clones were selected on Luria-Bertani (LB) solid media with ampicillin at 37° C. overnight. All colonies were then removed from the plates and grown to saturation in 5 mL liquid LB from which the plasmids were purified using a QIAprep spin plasmid miniprep kit, which were used to transform E. coli BL21 (DE3) by heat shock. Positive clones were selected on LB solid media with ampicillin, picked individually, and assayed for LAD activity by the cell lysate assay described below. Plasmids were sequenced using the BigDye® Terminator sequencing method and an ABI PRISM 3700 sequencer (Applied Biosystems, Foster City, Calif.).
Lysate assay. E. coli BL21 (DE3) harboring pGEX-4T-3 derived vector were grown to maximum OD600 at 37° C. with shaking at 250 rpm. 50 μl was used to inoculate a new culture, which was grown at 37° C. with shaking at 250 rpm until an OD600 of  ̃0.6 was reached. The cultures were then induced with 0.3 mM IPTG and shaken at 250 rpm at 25° C. for 4 h. Cell density was then normalized to a constant OD600 and 1 mL of cells was harvested by centrifugation and lysed by resuspension in 1 mL of 1 mg/mL lysozyme/50 mM Tris (pH 8.0). The cells were frozen at −80° C. and thawed at room temperature. The resulting lysate was vortexed thoroughly and centrifuged to remove cell debris. Ten μL of the lysate was used in an assay with 200 mM L-arabinitol and 2 mM NAD+ as the substrates as described below in the Kinetics section. To determine soluble and insoluble expression, lysozyme was utilized as the lysis reagent for the induced and normalized cells following the manufacturer's recommendations and samples were subsequently analyzed by SDS-PAGE.
GST-tag removal. The GST-tag was removed by incubation with biotinylated thrombin overnight at 4° C., incubation with streptavidin agarose for 30 min at 4° C. to remove thrombin, and passing mixture through GST-Bind resin to remove GST-tag, leaving five residues (GlySerProAsnSer) (SEQ ID NO: 9) attached to the N-terminus of the N. crassa LAD sequence.
To determine the effect of removal of the GST-tag, the purified LAD was incubated with and without thrombin at 4° C. overnight. Complete cleavage of the 25.7 kDa tag was verified by SDS-PAGE. The specific activities of the cleaved and noncleaved samples were compared. It was determined that removal of the GST-tag enhanced activity by about 65%. Because of this significant difference in activity, the cleaved enzyme was used in all subsequent assays.
Kinetics. The data were used to calculate the kinetic constants for various substrates by fitting the Michaelis-Menten equation using Origin 5.0. N. crassa LAD displayed typical Michaelis-Menten type kinetics with respect to all active substrates tested except D-sorbitol. The data represent averages of assays performed in duplicate or triplicate on two separate occasions.
HPLC analysis. The enzyme (1 μg) was incubated in a mixture of buffer and 25 mM L-arabinitol with 1 mM NAD(P)+ at 37° C. for 20 min. The sample was eluted on a Zorbax 3.0×150 mm C-18 (3.5 μm) column with a UV detector (Agilent 1100 series). The eluent consisted of two components: 0.1 M KH2PO4 containing 5 mM tetrabutylammonium hydrogen sulfate (pH 5.5) (buffer A) and 100% methanol (buffer B). The most suitable gradient was an initial isocratic step for 6 min at 93% buffer A, a gradient for 5 min from 7 to 30% buffer B, and a final isocratic step for 5 min at 30% buffer B.
Homology modeling. Insight II was used to prepare the model (Insight II, version 2000; Accelrys Inc., San Diego, Calif.) and MOE (Chemical Computing Group Inc., Montreal, Canada) was used for optimization. To verify the model, the overall fold was checked using Profiles3-D (Insight II), and the allowed states for φ and ψ angles and bond distances were checked using ProStat (Insight II), both with default settings. The Profiles3-D (Insight II, default parameters) check resulted in a self-compatibility score of 139.94, which compares well to the scores of 150.53 and 145.49 for the coordinates from 1PL8 and 1E3J, respectively. The ProStat check of φ and ψ angles were determined to be 81.2% within their core expected values, comparing well to the 83.3% and 82.4% for the same analysis of PDB structures 1PL8 and 1E3J, respectively.




  CV
  =
  
    
      
        σ
        x
      
      
        <
        x
        >
      
    
    ×
    100
    ⁢
    
      
    
    ⁢
    %
  












N. crassa LAD amino acid sequence:



(SEQ ID NO: 1)










1
MASSASKTNI GVFTNPQHDL WISEASPSLE SVQKGEELKE GEVTVAVRST GICGSDVHFW



 


61
KHGCIGPMIV ECDHVLGHES AGEVIAVHPS VKSIKVGDRV AIEPQVICNA CEPCLTGRYN


 


121
GCERVDFLST PPVPGLLRRY VNHPAVWCHK IGNMSYENGA MLEPLSVALA GLQRAGVRLG


 


181
DPVLICGAGP IGLITMLCAK AAGACPLVIT DIDEGRLKFA KEICPEVVTH KVERLSAEES


 


241
AKKIVESFGG IEPAVALECT GVESSIAAAI WAVKFGGKVF VIGVGKNEIQ IPFMRASVRE


 


301
VDLQFQYRYC NTWPRAIRLV ENGLVDLTRL VTHRFPLEDA LKAFETASDP KTGAIKVQIQ


 


361
SLE


 








N. crassa LAD nucleic acid sequence:



(SEQ ID NO: 2)










1
atggcttcta gcgcttccaa gaccaacatt ggcgttttca ccaaccctca gcatgatctg



 


61
tggatcagcg aggcctctcc ctctctcgag agcgtccaaa agggcgaaga gctgaaggaa


 


121
ggcgaggtca ctgttgccgt ccgaagcaca ggcatttgcg gatccgacgt ccacttctgg


 


181
aagcatggtt gcatcggccc catgatcgtc gaatgcgatc atgtcctcgg ccacgagtcg


 


241
gcaggcgagg tcattgctgt ccatcccagc gtcaagagca tcaaggtcgg cgacagggtt


 


301
gccattgagc cccaagtcat ctgcaatgcc tgcgagccct gcctgactgg ccgttacaac


 


361
ggatgcgagc gcgttgactt cctctctacg ccccctgtgc ccggccttct ccgccgctac


 


421
gttaaccacc ctgccgtgtg gtgccacaaa atcggtaaca tgtcctatga gaacggtgcc


 


481
atgctcgagc ccctttccgt ggcgctggcc ggtcttcaga gagccggtgt tcgtctgggc


 


541
gaccctgtcc tcatctgtgg tgccggcccc attggtctga tcaccatgct ctgcgccaag


 


601
gccgctggtg cctgccctct tgtcattacc gacattgacg aaggccgctt gaagttcgcc


 


661
aaggagatct gccccgaggt cgtcacccac aaggtcgagc gcctgtcggc cgaggagtcg


 


721
gccaagaaga tcgtcgagag ctttggtgga atcgagcccg cggtggctct cgagtgtact


 


781
ggtgtcgaga gcagtatcgc ggctgctatc tgggccgtca agttcggcgg caaggtgttc


 


841
gtcatcggcg tgggcaagaa cgagatccag attcctttca tgcgcgccag tgtgcgcgag


 


901
gtcgacctgc agttccagta ccgttactgc aacacttggc ccagggccat tcgcctggtc


 


961
gagaatggcc tcgttgacct caccaggctg gtgacgcacc gtttcccgtt ggaggatgcg


 


1021
ctcaaggcgt tcgagacggc gtcagacccc aagacgggtg ccatcaaggt gcagatccag


 


1081
agtctggagt aa

## Claims

1. A method of oxidizing L-arabinitol to L-xylulose, the method comprising:
(a) obtaining a purified arabinitol dehydrogenase wherein the arabinitol dehydrogenase comprises a tetramer of a polypeptide, where the polypeptide comprises an amino acid sequence that is at least 95% similar to SEQ ID NO: 1; and
(b) contacting a substrate containing arabinose to oxidize arabinitol with the arabinitol dehydrogenase.

2. The method of claim 1, wherein the L-xylulose is further converted to xylitol.

3. The method of claim 2, wherein the production of xylitol utilizes a phosphite dehydrogenase-based NADP regeneration system.

4. The method of claim 2, wherein the arabinitol dehydrogenase is expressed in a bacterium.