AU776922B2 - D-gluconolactone oxidase gene and method for producing recombinant D-gluconolactone oxidase - Google Patents

Description

WO 01/14574 PCTIUS00/22795 D-GLUCONOLACTONE OXIDASE GENE AND METHOD FOR PRODUCING RECOMBINANT D-GLUCONOLACTONE OXIDASE The present invention relates to novel nucleic acid molecules encoding a D-gluconolactone oxidase which enzyme is useful in methods of manufacture of erythorbic acid and related salts.

Erythorbic acid is a C-5 epimer of ascorbic acid and has essentially identical chemical properties including antioxidant activity. However, the vitamin C activity of erythorbic acid is very low compared to that of ascorbic acid and for practical purposes it is not considered to be a vitamin. Erythorbic acid has GRAS status and is used as an anti-oxidant in a number of food and other applications. Currently used chemical methods for the manufacture of erythorbic acid are based on esterification of 2-ketogluconic acid followed by basecatalyzed cyclization of the ester to produce sodium erythorbate.

It has been known since the 1960's that certain wild-type fungi belonging to the genus Penicillium produce small amounts of erythorbic acid when grown on glucose [Yagi J.

et al. Agric. Biol. Chem.31(3)340-345(1 967 Through an WO 01/14574 PCT/US00/22795 extensive chemical mutagenisis/selection program Penicillium notatum strains capable of converting glucose to erythorbic acid with yields up to 40% have been isolated [Shimizu K. et al. Agric. Biol. Chem 31 (3) 346-352 (1967)]. However, the fermentation time needed to complete the conversion is very long (1-2 weeks) making the industrial application of this process impractical.

Subsequent studies of the enzymology of the glucose-toerythorbic acid pathway in Penicillium have established that the pathway is comprised of two reactions [Takahashi T. Biotechnology and Bioengineering 11, 1157-1171 (1969)]. The first reaction is the well-known oxidation of glucose to gluconolactone by glucose oxidase. The second reaction in this pathway is the oxidation of Dgluconolactone by molecular oxygen with the formation of erythorbic acid and hydrogen peroxide. This reaction is catalyzed by D-gluconolactone oxidase (D-GLO), an enzyme detected in only several fungal species. Takahashi and co-workers [Takahashi T. et al. Agric. Biol. Chem. 121-129(1976)] have elucidated the basic enzymological properties of D-GLO from a strain of Penicillium cyaneofulvum (subsequently re-classified as Penicillium griseoroseum ATCC 1043).

The difficulties associated with the direct conversion of glucose to erythorbic acid at an economically acceptable rate remain unresolved. The present invention provides isolated nucleic acids encoding D-GLO which are useful in WO 01/14574 PCT/US00/22795 the biotechnological process for the production of erythorbic acid and its salts.

The present invention is directed to the isolation and identification of nucleic acid molecules which encode the enzyme D-gluconolactone oxidase (D-GLO) useful in the production of erythorbic acid and related salts.

Accordingly, in one embodiment, this invention is directed to newly isolated nucleic acid molecules defined by SEQ ID NO:1 (cDNA), SEQ ID NO:2(coding) and SEQ ID NO:3 (mature). The present invention further contemplates nucleic acid molecules which hybridize under stringent conditions to any one of SEQ. ID. NOS. 1-3.

In another embodiment of the present invention, vectors containing any one of the nucleic acid molecules identified herein, as well as host cells .transformed with such vectors are also provided. Recombinant methods using the identified nucleic acids to produce D-GLO are also contemplated by this invention.

A further embodiment of the present invention is directed to the D-GLO protein encoded by the nucleic acid molecules identified herein including the protein identified by SEQ ID NO:4 and proteins having at least sequence identity with SEQ ID NO:4.

WO 01/14574 PCT/US00/22795 In another embodiment of this invention, methods of producing erythorbic acid and related salts by the conversion of glucose and/or D-gluconolactone using the D-GLO of the present invention are also provided.

Figure 1 is an illustration of the plasmid pGTY (GLO) which codes for the MFal prepropepetide and the mature portion of the P. griseoroseum GLO.

Figure 2 graphically depicts cell density and GLO activity for untransformed S. cerevisiae and S.

cerevisiae transformed with pGTY(GLO).

Figure 3 is a schematic representation of the conversion of glucose to D-erythorbic acid using glucose oxidase and

D-GLO.

Figure 4 is a schematic representation of the conversion of glucose to D-erythorbic acid using glucose dehydrogenase and D-GLO.

Figure 5 is an illustration of the plasmid which contains the complete coding region of the D-GLO gene under the control of the P.pastoris promoter.

The present invention relates to isolated nucleic acid molecules encoding a D-GLO of fungal origin. Preferably, the fungus is of the genus Penicillium, Penicillium WO 01/14574 PCT/US00/22795 griseoroseun, Penicillium notatum, Penicillium cyaneum and Penicillium decumbens. The term "D-gluconolactone oxidase" (D-GLO) as used herein refers to and includes any natural or man-made variant of fungal D-GLO. For example, the nucleic acid molecules of the present invention which encode the fungal D-GLO can have the sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; or can have a sequence that hybridizes under stringent conditions to an isolated nucleic acid molecule having SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; or can have a sequence that encodes a protein having a sequence identity of about 70% or greater when compared to the amino acid sequence set forth in SEQ ID NO:4. Amino acid sequence identities of about 70% or greater can be defined as a percentage of positives identified by the BLASTP algorithm as implemented at the Internet site http://www.ncbi.n/m.nih.gov/egi-bin/BLAST/ in a search using the default parameters of the program (Matrix Blosum 62; Gap existence cost 11; Gap extension cost 1).

More specifically, the nucleic acid molecules of the present invention include variations of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, such as deletions, insertions, additions and mutations wherein such sequences encode a protein which retains the enzymatic activity of naturally occurring D-GLO, the ability to convert Dgluconolactone to erythorbic acid.

Vectors and transformed host cells or transgenic organisms containing such nucleic acid molecules of the WO 01/14574 PCT/US00/22795 present invention are also included within the scope of this invention. By "vectors or expression vectors" is meant any nucleic acid molecule or virus containing regulatory elements or reporter genes for the purpose of, but not limited to, expression in prokaryotic or eukaryotic cells or organisms. 'By "transformed host cell" is meant a host cell into which (or into an ancestor of which) has been introduced, by means of molecular biological techniques, a nucleic acid encoding a D-GLO, preferably from a fungal source and most preferably from the genus Penicillium. After introduction into the cell, this nucleic acid can exist extrachromosomally or become integrated into the host genome. It is routine for those skilled in the art to construct expression vectors into which a nucleic acid molecule of the present invention is placed in operable linkage to a desired promoter in order to effect the expression and secretion of the D-GLO proteins encoded by such nucleic acid molecules in a wide variety of host cells. Sambrook, et al, (Molecular Cloning, A Laboratory Manual, Sambrook, Fritsch, and Maniatis, 2 nd ed. (1989) Cold Spring Harbor Laboratory Press).

Host cells useful for the practice of the present invention can be any available host cell which is amenable to transformation.procedures. For example, bacterial cells can be used such as bacteria belonging to genera Eschericha, Erwinia, Pantoea, Bacillus, Lactobacillus or Pseudomonas. Yeasts can also be used as host cells and include, for example, yeast belonging to the genera Saccharomyces, Kluyveromyces, Pichia, WO 01/14574 PCT/US00/22795 Hansenula, Candida and Schwanniomyces. Unicellular algae may also be used such as, for example, those of the genera Synechosystis, Chlamydomonas, and Euglena. Higher plant cells can also be transformed with the isolated nucleic acids of the present invention. Methods for transforming plant cells with naked DNA or with vectors comprising promoters which function in plants operably linked to heterologous genes, are widely known in the art. Exemplary plants include soybean, maize, potato, tomato, sugarbeet and the like. Mammalian cells may also be used as host cells. Preferably, the expressed GLO should be targeted to the culture medium or one of the organells, such as vacuole, chloroplast, microsome, peroxisome and the like.

The term "nucleic acid or nucleic acid molecule" encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic chemically synthesized or modified) DNA. The nucleic acid molecules of the present invention can be double-stranded or single-stranded.

Where single stranded, the nucleic acid can be a sense strand or an antisense strand. The term "isolated nucleic acid" refers to a nucleic acid which may be flanked by non-natural sequences, such as those of a plasmid or virus. Thus, the nucleic acid can include none, some, or all of the non-coding promoter) sequences which are immediately contiguous.to the coding sequence. The term, therefore, includes, for example, a recombinant DNA which is incorporated into a vector including an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote WO 01/14574 PCT/US00/22795 other than Penicillium, or which exists as a separate molecule a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. This term also includes a recombinant DNA or RNA which is part of a hybrid gene encoding an additional polypeptide sequence. Moreover, the term is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.

These recombinant nucleic acids may further include any of the varieties of sequences which increase, regulate or modify the transcription of the D-GLO coding sequence in the various recombinant hosts; namely, constitutive or regulated promoters, transcriptional enhancers and terminators and other sequences regulating the expression of D-GLO by various known mechanisms such as transcriptional repressor binding, attenuation or antitermination.

By "hybridizes under stringent conditions" is meant, the conditions in which a nucleic acid forms a stable, sequence-specific, non-covalent bond with the nucleic acid of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 in solution or on a solid support under the low salt and high temperature conditions, regarded as stringent and set forth in Sambrook, et al, (Molecular Cloning, A Laboratory Manual, Sambrook, Fritsch, and Maniatis, 2 nd ed. (1989) Cold Spring Harbor Laboratory Press). For example, reference nucleic acids such as SEQ ID NO:1 can be immobilized on nitrocellulose filters, and WO 01/14574 PCT/US00/22795 any other nucleic acids specifically and non-covalently binding to the immobilized reference nucleic acids in the presence of 0.2 X SSC (1.75 g/l NaC1, 0.88 g/1 sodium citrate dihydrate; pH 7.0) and 0.1% sodium dodecylsulfate at 68°C are considered to be hybridized under stringent conditions.

A GLO preparation or D-GLO protein obtained from a host cell transformed with the nucleic acid molecules of this invention is also included within the scope of the present invention. Host cells transformed with the nucleic acid molecules of the present invention can be cultured and the D-GLO recovered from the cultured cells. The D-GLO may be secreted or expressed within the host cells; the whole D-GLO coding region or parts of the coding region can be expressed without any additional modifications or after adding various C- and N-terminal extensions, such as an initiator codon or an oligohistidine sequence. Fusions of D-GLO with other proteins which retain the enzymatic activity of D-GLO are within the scope of the current invention as well as mutated forms of all of the D-GLO-related proteins identified herein. These mutant forms may be obtained by random or directed mutagenesis. The preparations of such proteins having D-GLO activity may be crude or purified, used in solution or be immobilized on various carriers known in the art. Likewise, recombinant host cells expressing said proteins can be used to catalyze the conversion of D-gluconolactone into erythorbic acid during the fermentation of the host, or even in a resting state of the host. Notably, killed recombinant host cells of the WO 01/14574 PCT/US00/22795 present invention can also be used as a catalyst of Dgluconolactone oxidation in the conversion of glucose and/or D-gluconolactone to erythorbic acid. Specifically, glucose can be converted to D-gluconolactone in the presence of glucose oxidase or glucose dehydrogenase; the D-gluconolactone can then be converted to erythorbic acid by contacting the D-gluconolactone substrate with the D- GLO of the present invention for a time, and under conditions sufficient to produce erythorbic acid. Dgluconolactone, as a substrate for D-GLO of the present invention, can be prepared through the chemical conversion of gluconic acid or by the conversion of glucose by glucose oxidase or glucose dehydrogenase. Dgluconolactone is thus converted to erythorbic acid by contacting the substrate with D-GLO for a time and under conditions sufficient to produce erythorbic acid.

The full-length D-GLO coding sequence may be used without any modifications or may be modified by methods well known in the art. For example, the N- or C-terminal amino acid sequences may be deleted or substituted with various known pre- or prepro-peptides (signal peptides) improving the secretion of D-GLO in suitable hosts.

Various other amino acid sequences regulating the sorting and targeting of the D-GLO polypeptide may be fused with full-length D-GLO or parts of D-GLO coding sequences retaining the GLO activity.

The D-GLO of the present invention can also be expressed as a fusion protein. In particular, it can be fused to protein domains which provide an auxiliary and/or WO 01/14574 PCT/US00/22795 associated enzymatic activity, such as glucose oxidase, catalase and the like. Likewise, D-GLO can be fused to domains which provide other known useful functions such as high affinity for a specific ligand (e.g.

streptavidin, maltose-binding protein, cellulose- and other polysaccharide-binding domains) or improved stability and/or solubility of the fusion protein (e.g.

superoxide dismutase or glutathione S-transferase).

1 0 Erythorbic acid can be produced using the recombinant D- GLO of the present invention as a key element in the enzymatic process, preferably in an efficient host such as a filamentous fungus a fungus belonging to the genera Aspergillus or Penicillium), or a yeast species with high secretory potential those belonging to the genera Pichia or Hansenula), to convert glucose to erythorbic acid. In addition to D-GLO, such process incorporates at least two other enzymes, preferably including glucose oxidase (or an enzyme with overlapping specificity, such as glucose dehydrogenase, hexose oxidase or pyranose oxidase) and a catalase. The sequence of chemical reactions underlying the process based on glucose oxidase and GLO is illustrated by Fig.

3. Briefly, glucose is oxidized by molecular oxygen to Dglucono-d-lactone. This reaction is catalyzed by the glucose oxidase (or, hexose oxidase, pyranose oxidase, glucose dehydrogenase and the like) and generates hydrogen peroxide as a by-product. D-GLO subsequently catalyzes the conversion of D-glucono-d-lactone to erythorbic acid. Also in this reaction, molecular oxygen is consumed and hydrogen peroxide is formed. D-glucono-d- WO 01/14574 PCT/US00/22795 lactone in water solution is known to be in. equilibrium with gluconic acid and D-glucono-y-lactone. D-glucono-ylactone is also a substrate of D-GLO. Both Dgluconolactones are oxidized by GLO to the same product, erythorbic acid. The spontaneous reaction of gluconolactone hydrolysis is relatively slow and if sufficiently high concentrations of GLO and molecular oxygen are present in the reaction mixture, the hydrolysis reaction can be minimized. Moreover, because of the reversibility of this reaction, gluconic acid eventually forms a lactone that can be oxidized to erythorbic acid. Both the reaction catalyzed by the glucose oxidase and that catalyzed by D-GLO, generate hydrogen peroxide as a by-product. Hydrogen peroxide is a highly reactive substance that can cause inactivation of the enzymes involved in the process. Therefore, the hydrogen peroxide has to be continuously removed from the reaction mixture. The preferred method for the removal of hydrogen peroxide is through the use of a catalase. The use of other known scavengers which remove various activated oxygen species, e.g. superoxide dismutase, can also be advantageous in practicing this invention.

If glucose dehydrogenase is used to catalyze the conversion of glucose to gluconolactone, the removal of hydrogen peroxide can be coupled to the regeneration of the NAD+ which is consumed in the glucose dehydrogenasecatalyzed reaction. Fig. 4 illustrates the overall reaction scheme of this particular implementation of the present invention. The preferred manner of converting glucose to erythorbic acid according to the present -12- WO 01/14574 PCT/US00/22795 invention is to conduct the whole process in a single reactor wherein the oxidation of glucose and the oxidation of gluconolactone proceed concurrently.

However, implementation, wherein the con.ersion of glucose to gluconolactore and the conversion of gluconolactone to erythorbic acid are conducted separately, is also acceptable. It is also satisfactory to use gluconolactone, gluconic acid or a mixture of the two as the starting material for production of erythorbic acid using the recombinant D-GLO of the present invention.

The D-GLO nucleic acids of the present invention can also be expressed in hosts already capable of converting glucose to gluconolactone. A number of such microbial hosts are known in the art. Typically, such microorganisms oxidize glucose to gluconolactone using glucose oxidase many fungal species belonging to the genera Aspergillus and Penicilium) or glucose dehydrogenase. Many bacterial species belonging to several genera that produce membrane-bound glucose dehydrogenases are suitable. It is advantageous to choose hosts which also express sufficiently high levels of catalase. When such recombinant hosts expressing GLO are fermented on glucose-containing cultural media, erythorbic acid can be obtained directly from glucose.

Additionally, a recombinant D-GLO-expressing host of the present invention can be co-cultured with a different host expressing glucose oxidase, glucose dehydrogenase -13- WO 01/14574 PCT/US00/22795 and/or a catalase. In this embodiment, erythorbic acid can be produced in a one-step mixed fermentation.

Two types of recombinant hosts would be particularly suitable for direct fermentation of glucose into erythorbic acid the yeast and the filamentous fungi.

In this regard, Example 9 demonstrates that the D-GLO gene can be expressed efficiently in yeast such as Pichia pastoris. Any other yeast host known to support efficient secretion of heterologous protein, for example, Hansenula polymorpha or Kluyveromyces marxianus can also be used. The expression system used in Example 9 is based on glucose-repressible promoter and therefore is not well suited for construction of recombinant hosts fermenting glucose into erythorbic acid. However, equally efficient expression systems for P. pastoris based on strong promoters that are not repressed by glucose are known for example, one based on Stratagene's expression pGAPZ vector series and should be used for construction of such hosts. Other promoters of glycolytic genes can also be used.

In addition to expressing the GLO gene, a recombinant yeast host used in this fermentation process should also express a glucose oxidase e.g. glucose oxidase from Aspergillus niger is known to be expressed efficiently in yeast (De Iaetselier A. et al. Fermentation of a yeast producing A.niqer glucose oxidate... Bio/Technology 9, 559-561 (1991).) Also, over-expression of a preferably secreted catalase gene is a very useful additional WO 01/14574 PCT/US00/22795 genetic trait of a yeast host fermenting glucose into erythorbic acid.

In contrast to the yeast hosts, many wild-type filamentous fungi do express high levels of glucose oxidase and catalase. Therefore, genetically engineered over-expression of genes coding for these two enzymes is not as essential as in the case of the yeast hosts. For the (over-) expression of D-GLO, promoters of highly expressed glycolytic genes are suitable. Other promoters that retain high activity during cultivation on glucose can also be used, for example, the fungal TEF1 promoter.

Unlike yeast, many filamentous fungi produce gluconolactonase. For example, in A.niger grown under high aeration conditions, gluconolactonase levels are extremely high (Whtteveen et al. Induction of glucose oxidate, catalase and lactonase in Asnergillus niger, Curr. Genetics 24, 408-416 (1993)).

Gluconolactonase competes with GLO for a common substrate and thus interferes with the conversion of glucose to erythorbic acid. Therefore, gluconolactonase should preferably be inactivated. The inactivation may be achieved either by genetic means through mutation of the gluconolactonase gene(s) or by selecting fermentation conditions which selectively inhibit gluconolactonase but not GLO. One preferable way to achieve inactivation is to add a selective inhibitor of gluconolactonase to the fermentation broth.

PA\OPER~jrr\AeadmwLA20D4-2DAmedmmu\25O2M smd pc.dw-27IO7V4 In another embodiment of this invention, erythorbic acid is produced from gluconolactone, gluconic acid or a mixture of these two substrates by fermenting each with a microbial host expressing sufficiently high levels of recombinant D-GLO.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The present invention is further illustrated, but not limited by, the following examples.

*o o* o -16- WO 01/14574 PCT/US00/22795 EXAMPLE 1 Glo Activity The activity of GLO was.measured using, as a substrate, an equilibrium mixture of glucono-5-lactone, glucono-ylactone (D-gluconolactone) and gluconic acid. This mixture was prepared by dissolving crystalline glucono-6lactone in water at 50% concentration and allowing it to stand at 50 0 C for several days. The reaction was performed in a 50 mM potassium biphthalate buffer, pH 5.6 containing 2mM hydroxyquinoline, 12 pM 2,6dichlorophenolindophenol and 70 mM substrate. Both 2,6dichlorophenolindophenol and the substrate were added to the reaction mixture in the form of stock solutions immediately before the measurement. Also, the aliquot of the substrate solution to be used in the experiment was quickly adjusted to pH 5.8 immediately before use.

The enzymatic reaction was followed by recording the time course of the absorption at 600 nm caused by the reduction of 2,6-dicholorophenolindophenol by erythorbic acid. A control, differing from the reaction mixture only in that the enzyme solution was boiled for 2 min before the assay, was included. The amount of the erythorbic acid produced in the reaction was calculated using a calibration curve obtained by adding known amounts of erythorbic acid to the reaction mixture. The activity was expressed as umoles of erythorbic acid produced per minute under the conditions described above and at 30 0

C.

-17- WO 01/14574 PCT/USOO/22795 EXAMPLE 2 Purification of the Homogenous GLO From P. griseoroseum The GLO from P. griseoroseum strain ATCC 10431 was purified to homogeneity using a procedure similar to the method published earlier Takahashi T, et al. Agric. Biol.

Chem. 40, 121-129 (1976).

Several 200 ml portions of YEPD medium bacto-peptone, 1% yeast extract, 2% glucose) in 2 liter Erlenmelyer flasks were inoculated with 2 ml of a suspension of spores of P. griseoraseum grown on potato-dextrose agar (Difco) plates for one week. The cultures.were grown on a rotary shaker at 30 0 C, 180 rpm for 2 days, 0.51 of this culture was used to innoculate a 10 1 induction medium glucose, 0.2% KHPO 4 0.1%(NH,) 2 S0 4 0.1% (NH 2 2 CO, 0.1% NaNO, 0.1% MgSO 4 .7H 2 0, 1% MnSO,.7H,0, 0.001% ZnSO,.7H10, CaCO,. pH 5.5) in a 15 1 fermentor. Chloramphenicol mg/l) and tetracycline (3 mg/l) were used in some fermentations to avoid the risk of bacterial contamination. The fermentation was allowed to proceed for 60 h at 30"C (aeration -51/min, stirring 300 rpm).

The mycelium was collected by filtration on a sintered glass filter and washed with water and buffer A (10 mM phosphate buffer, pH 6.5, containing 0.1 mM EDTA). The cells were disintegrated using a glass bead mill in the same buffer containing 1 mM phenylmethylsulphonyl fluoride. The disintegration process was done in cycles with ice-water cooling between and during the cycles.

-18- WO 01/14574 PCTIUS00/22795 The length of a cycle was adjusted to maintain the temperature of the suspension within a range of 4 °C-22"C and the number of cycles was adjusted to achieve at least cell breakage (evaluated microscopically). The homogenate was centrifued for 30 min at 19000 x g and the supernatant was used for the purification of the enzyme.

Approximately 80 ml of DEAE-Sepharose FF (Pharmacia) per liter of the cell extract was added and the suspension was incubated overnight at 4°C with gentle stirring. The resin was removed by filtration and the filtrate was treated with a fresh portion of DEAE-Sepharose under the same conditions. This treatment removed a significant proportion of the ballast protein while only a minute amount of GLO was adsorbed on the resin. GLO was precipitated from the filtrate with ammonium sulfate 100% saturation). The ammonium sulfate precipitate was dialyzed against several changes of buffer A. The dialyzed enzyme solution was diluted with enough of buffer A to bring its conductivity to 1.25 mS and applied to a column of DEAE-Sepharose FF equilibrated with the same buffer (approx. 5ml column bed volume per ml of sample). The column was eluted with buffer A at 0.15 bed volume/h until the absorption at 280 nm dropped to the background value followed by elution with a linear gradient of NaC1 in buffer A (0-100 mM NaC1, total gradient volume 2 column bed volumes). The active peak fractions were pooled, concentrated using an Amicon ultrafiltration device and XM 50 membrane and applied on top of a 200 cm column of Sephacryl S-300 HR (Pharmacia) -19- WO 01/14574 PCT/US00/22795 equilibrated with buffer A. The column was eluted at a linear rate of 0.5 cm/min. Active fractions from the gel filtration column were collected and applied on top of a hydroxyapatite column (Bio-Gel HT, Bio-Rad) equilibrated with buffer A. The column bed volume was 0.13 of the sample volume and elution rate 0.1 bed volume per min.

GLO was eluted from the column with a linear gradient of ammonium sulfate in buffer A. The total volume of gradient was approximately 36 column bed volumes. The fractions with the highest activity of GLO were pooled and the pool was analyzed by polyacrylamide gel electrophoresis. A single strong diffuse band corresponding to apparent molecular weights of 68-80 kDa and a very weak band at approximately 34 kDa were observed. The results of a typical purification experiment are summarized in Table 1.

WO 01/14574 PCT/US00/22795 EXAMPLE 3 Amino Acid Sequence Analysis of The GLO The analysis was done as a commercial service in the Protein Analysis Laboratory of the Institute of Biotechnology, Helsinki. The purified GLO preparation of Example 2 was found to be homogenous in terms of the Nterminal sequence analysis. The following N-terminal sequence was found: Tyr Arg Trp Phe Asn Trp Gln Phe Glu Val Thr Nnn Gln Ser Asp Ala Tyr Ile Ala Pro His Asn Glu His...(SEQ ID No.:5) ("Nnn" means that no interpretable signal was observed at this position, which is most probably explained by the presence of an underivatized cysteine residue or glycosylated amino acid residue).

The protein was further alkylated with 4-vinylpyridine, digested with Lys-C-protease and several peptides isolated from the digest using reverse phase HPLC. The following peptide sequences were determined with massspectrometry: Peptide 1 Glu His Asp Arg Met Thr Val Cys Gly Pro His Phe Asp Tyr Asn Ala Lys (SEQ ID NO:6); Peptide 2 Glu Tyr lie Cys Tyr Asp Glu Val Thr Asp Ala Ala Ser Cys Ser Pro Gln Gly Val Val (SEQ ID NO:7); Peptide 3 Cys Gin Phe Val Asn Glu Phe Leu Val Glu Gln Leu Gly Ile Thr Arg (SEQ ID NO:8).

WO 01/14574 PCT/US00/22795 EXAMPLE 4 Isolation of P. griseoroseum Chromosomal DNA P. griseoroseum was grown in YEPD medium as described in Example 2. The mycelium was washed with water and buffer A (Example 2) and freeze-dried. About 50 mg of the dry mycelium was ground in a mortar under liquid nitrogen.

The finely ground mycelium was suspended in 500 ul of extraction buffer (250 mM NaC1, 25mM EDTA, 200 mM Tris HC1, pH 8.5, 0.5% SDS), 350 ul of phenol was added and the mixture was shaken to form a homogeneous suspension.

150 il of chloroform was added to the suspension followed by a one hour high speed centrifugation (13 500 rpm in a table-top mini-centrifuge). The water phase was transferred to a new test tube and 10 ul of ribonuclease A solution was added. The mixture was incubated at 37 0 C for 1 hour. After the incubation 1/10 vol. of 5 M Na-acetate buffer (pH 5.4) was added to the solution followed by 0.6 vol. of isopropanol. The DNA was recovered by centrifugation (10 min. 13500 rpm), washed 2 times with 70% ethanol, vacuum-dried and dissolved in 100 pl of water.

WO 01/14574 PCT/US00/22795 EXAMPLE Cloning of a fragment of chromosomal DNA encoding GLO Based on the partial amino acid sequences of GLO SEQ ID NO: 5 SEQ ID NO: 8, a number of different oligonucleotides were synthesized and tested as primers in a PCR using chromosomal DNA of P. griseoroseum (Example 4) as a template.

The PCR was performed in a PTC-255 DNA Engine apparatus (MJ Research Inc., MA, USA) using the following program: 2 min at 94 0 C; 10 cycles of (30 sec at 94 0 C; 45 sec at 3 min at 72 0 C) followed by 30 cycles of (30 sec at 94 0 C; 45 sec at 60 0 C; 3 min at 72 0 Each reaction was performed in 15 ul of a solution containing about 25 ng of template DNA, 0.75 unit of Taq DNA polymerase (Boehringer Mannheim), 0.75 pM of each of the oligonucleotide primers, 200 pM of each of the four deoxynucleoside triphosphates (dATP, dTTP, dCTP, dGTP), pl of the 10 X buffer concentrate (supplied by the manufacturer of the Taq polymerase). The products of the PCR were analyzed by agarose gel electrophoresis using conventional techniques. (Maniatis, T. et al. (1982) Molecular cloning. Cold Spring Harbor Laboratory].

One pair of oligonucleotide primers produced the best results: a sense oligonucleotide TAYCGITGGTTYAAYTGGCA (SEQ ID NO: 9) and an antisense oligonucleotide CCIARYTGYTCIACIARRAAYTCRTTIACRAAYTGRCA (SEQ ID NO: In these sequences represents an inosine phosphate WO 01/14574 PCT/US00/22795 residue, R a mixture of adenosine and guanosine phosphate residues, and Y a mixture of thymidine and cytosine phosphate residue. The PCR product (approximately 1.2 kb) obtained with this pair of oligonucleotides was purified by agarose gel electrophoresis and cloned into pCR2.1-TOPO vector (Invitrogen) using the TOPO TA Cloning kit supplied by the same manufacturer resulting in plasmid pCR(GLO).

-24- WO 01/14574 PCT/US00/22795 EXAMPLE 6 Construction of a P. griseoroseum cDNA library Total RNA was isolated from P. griseoroseum mycelium grown on a mineral medium under conditions inducing GLO production (Example The mycelium (stored frozen at 0 C) was ground in a mortar under liquid nitrogen. 3 g of the finely ground mycelium was suspended with vigorous shaking in 10 ml of ice-cold RNA-extraction buffer (4 M guanidine thiocyanate, 0.5% Na laurylsarcosine, 25 mM Na citrate, 100 mM P-mercaptoethanol). The mixture was centrifuged at 4 0 C and 10000 rpm (SS-34 rotor, Sorvall) for 6 min. 10 ml of the supernatant was transferred to a new tube and 4 g of CsCl was added to it. All solutions used at subsequent steps were prepared using diethylpyrocarbonate-treated water and glassware. The RNA-containing solution was layered on the top of 1.2 ml of 5.7 M CsC1, 0.1M EDTA, pH 7 and centrifuged at and 33000 rpm for about 20 h. The precipitate was quickly rinsed with a small amount of water and dissolved in 100 pl of water. mRNA was isolated from this preparation using Oligotex Midi Kit (Qiagen) according to the manufacturer's instructions. A cDNA library was prepared from the P. griseoroseum mRNA using Stratagen's cDNA Synthesis Kit and XZAP-cDNA Gigapack III Gold Cloning Kit according to the instructions supplied with these kits.

WO 01/14574 PCT/US00/22795 EXAMPLE 7 Isolation of the full-length GLO cDNA from the P.

griseoroseum cDNA library The 1.2 kb DNA fragment containing part of the chromosomal GLO gene was isolated from the plasmid pCR(GLO) (Example 5) by EcoRI restriction and preparative agarose gel electrophoresis. Standard genetic engineering techniques were used for restrictase digestion, isolation of plasmid DNA and DNA fragments etc. [Maniatis, T. et al. (1982) Molecular cloning. Cold Spring Harbor Laboratory], This fragment was radioactively labeled using the Random Primed DNA labeling kit (Boehringer Mannheim) and [aP 32 -dCTP.

The X-phage library of Example 6 was plated and screened by DNA hybridization using the labeled 1.2 kb fragment according to the manual provided by Stratagene with the XZAP-cDNA Gigapack III Gold Cloning Kit. A number of positive plaques were identified and the recombinant phages from twenty of them were purified and converted to the plasmid form according to the protocols from the same manual. These 20 plasmids were analyzed by restriction with EcoRI and XhoI and found to have inserts of different sizes. One small DNA fragment was present in all plasmids suggesting that all of the cDNA clones are derived from the same gene. The largest plasmid (named pGLO 1.8) contained an insert of about 1.8 kb size. The whole insert was sequenced using the commercial service WO 01/14574 PCT/USOO/22795 of Eurogentec Bel. S.A. (Belgium). Sequence analysis revealed that the insert of the plasmid pGLO 1.8 contained the complete coding region of the GLO cDNA (1443 bp, including the stop codon), 70 bp of the untranslated sequence and 261 bp of the 3'-untranslated sequence (SEQ ID NO: The sequence encoded a protein of 480 amino acid residues (SEQ ID NO: 4).

The deduced amino acid sequence of P. griseoroseum GLO was compared to the known protein amino acid sequences using the BLAST service provided by GenBank over the Internet htlp://www.nclhi.lmn.nih.gov/c i-hin/B1LAST/nplhnewhlast?.lfoi=0). A number of homologous protein sequences were identified. If only the proteins with established function are considered, the highest homology was observed with the other lactone oxidases, such as Darabinonolactone oxidase from Candida albicans and Lgulonolactone oxidase from rat.

The N-terminal amino acid sequence determined using the purified GLO from P. griseoroseum (Example 3, SEQ ID NO: is identical to the deduced sequence of GLO (SEQ ID NO: 4) starting at amino acid residue 21. This observation and the predominance of hydrophobic amino acid residues in the area 1-20 of the translated GLO coding sequence strongly suggest that P. griseoroseum GLO contains an N-terminal signal peptide. The presence of a signal peptide in GLO is unexpected, since other lactone oxidases do not have signal peptides and are not known to be secreted. Furthermore, the deduced sequence of P. griseoroseum GLO contains 8 tentative -linked WO 01/14574 PCT/US00/22795 glycosylation sites. The isolated GLO appears as a diffuse band on the polyacrylamide gel suggesting that is indeed a glycoprotein. However, we have isolated GLO from the cell extracts of P. griseoroseum. It may be speculated that in its native host GLO is either directed from the Golgi apparatus to one of the intracellular organelles or is secreted but remains associated with the cells.

-28- WO 01/14574 PCT/US00/22795 EXAMPLE 8 Expression of the P. griseoroseum GLO gene in a heterologous host Since the deduced sequence of GLO displayed many features of a secreted protein, a yeast secretory expression seemed to be the most suitable for testing the functionality of the cloned GLO cDNA. The expression vector used for the expression of GLO is based on the well known yeast-E.coli shuttle vector pJDB207 [Beggs.

in Wiliamson Genetic Engineering 2, Academic Press (1981)]. Construction of the expression vector was accomplished in two stages. Firstly, pAC109 was constructed by simultaneously ligating three DNA fragments: a 0.45 kb BamHI-Eco47III fragment from the promoter area of the Saccharomyces cerevisiae PH05 gene; a 0.38 kb HaeIII-HindIII fragment from the S.

cerevisiae MFal gene containing 116 bp of the 3'noncoding area of the MFal gene and part of coding area corresponding to the sequence of the prepropeptide of the yeast a-factor precursor protein (Mfal-prepropeptide); an approximately 6.5 kb fragment of pJDB207 obtained by restriction with BamHI and HindIII. Secondly, a synthetic polylinker was inserted into the HindIII site of pAC109.

The polylinker was composed of two oligonucleotides: the top strand nucleotide AGCTCTCGAGATCTCCCGGGA (SEQ ID NO: 11) and the bottom strand nucleotide AGCTTCCCGGGAGATCTCGAG (SEQIDNO:12). The plasmid that has -29- WO 01/14574 PCT/US00/22795 the polylinker inserted in such an orientation that the HindIII site is located proximally to the Mfal preproarea was selected and named pGTY.

The DNA sequence of the cloned GLO gene was modified into a form suitable for constructing a fusion with the ME alprepropeptide by conducting a PCR with the plasmid pGLOl.8 as template and the two oligonucleotide primers, the "sense" primer: GAAGAAGCTTACCGGTGGTTCAATTGGCAGTTTTTGGT (SEQ ID NO: 13) and the "anti-sense" primer: CACGACGTTGTAAAACGACGGCCAG (SEQ ID NO: 14), annealing in the vector downstream of the GLO gene. At this step, a modification was introduced into the GLO gene. All of the sequence and the sequence corresponding to the amino acid residues 1-20 of the deduced GLO coding sequence was deleted and a HindIII site was introduced in a position allowing for the in-frame fusion of the Mfolprepropeptide and mature GLO. The product of this PCR reaction was-digested with HindIII and XhoI and ligated with pGTY digested with the same pair of restrictases.

The resulting plasmid pGTY(GLO) (Fig. 1) codes for a fusion protein composed of the MFal prepropeptide and the presumptive mature part of the P. griseoroseum GLO (SEQ ID NO: S.cerevisiae strain GRF18 (ATCC 64667, genotype: MATa, leu2-3, leu2-112, his3-11, his3-15 was transformed to leucine prototrophy using the "lithium" transformation method [Sherman F. et al. Laboratory Course Manual for WO 01/14574 PCT/US00/22795 Methods in Yeast Genetics pp-121-122, Cold Spring Harbor Laboratory (1986)].

One of the transformed clones as well as the recipient strain used as a control were grown until early stationary phase (rotary shaker, 180 rpm, 30 0 C) in 0.3 1 SC-his medium (0.67% Yeast Nitrogen Base w/o amino acids, Difco, 2% Glucose, 100 mg/1 histidine; for the nontransformed control strain leucine was also added at 100 mg/l). The yeast cells from these cultures were used to inoculate two identical 15 1 fermentors each containing 1 of a low-phosphate (PEP) medium. To prepare the PEP medium an 8% solution of bacto-peptone (Difco) was treated with CaCI 2 (added to 0.4M concentration) at pH 11 and 100 0 C for 5 min. The peptone solution was cooled to room temperature, adjusted to pH 5.5, filtered through paper and 0.4 pm pore-size membrane and used as the stock solution of phosphate-depleted peptone. PEP medium contained 2% phosphate-depleted peptone and 5% glucose.

The. fermentation conditions were: stirring 300 rpm, aeration 5 1/min, pH 5.5 maintained by addition of 4M NaOH. Samples of the cultures were taken at suitable intervals. Cell density was followed by measured the absorption at 600 nm. The GLO activity was measured after removing the cells by centrifugation, concentrating the samples of the medium about 500-fold using Centriplus membranes (Amicon) and removing the low molecular weight components of the fermentation medium by gel filtration using disposable EconoPack 10 DG mini-columns (BioRad).

The peak levels of GLO (about 2.4 mU/ml) were measured after about 60-70 hours of fermentation. No GLO activity WO 01/14574 PCT/US00/22795 was found in the control fermentation of the untransformed recipient strain.

The results of this experiment show conclusively that the cDNA clone of the GLO gene in the plasmid pGLO 1.8 is indeed functional. S.cerevisiae is known to be a relatively inefficient host for secretion of heterologous proteins. Therefore, much higher expression levels of recombinant GLO may be expected when the GLO gene is introduced into other fungal species filamentous fungi or other yeast with higher secretory potential.

WO 01/14574 PCT/US00/22795 Example 9 Expression of the GLO gene in methylotrophic yeast The coding region of the GLO gene was amplified by PCR using the oligonucleotide primers: CAAAGCTTCTAGAGCCTCAGACCACTCATATCACATC (SEQ. ID No: 14) and CCAACAATTGATGCTGAGCCCTAAGCCGGCTTTCCTGC (SEQ. ID No: 16). The resulting DNA fragment was digested with restriction endonucleases Xbal and MfeI and ligated with the plasmid pPIC3.5K (Multi-Copy Pichia Expression Kit, Invitrogen Corp.) digested with AvrII and EcoRI. The resulting plasmid PPIC3.5K (GLO 51-3) contains the complete coding region of the GLO gene under control of the P.pastoris AOXI promoter (Figure P.pastoris strain GS115 was transmfored with 51-3) using the method recommended by Invitrogen.

Several independently obtained transformed clones were cultivated in a rotary shaker (30°C, 200rpm) in BMGY (yeast extract 1% peptone 2% potassium phosphate buffer, pH 6.0-100mM, 1% glycerol, 1.34% Yeast Nitrogen Base (Difco), 0.4 mg/1 biotin). After reaching early stationary phase, the cells were collected by low speed (4000 rpm) centrifugation, re-suspended and cultivated overnight in an equal volume of BMMY is identical to BMGY except that the glycerol is replaced with 0.5% methanol.

The highest GLO expression levels measured in culture supernatant in these experiments were about 0.4-0.5 U/ml.

wo 01/14574 WO~l/ 4574PCT/US00122795 This value is approximately 200 times higher than the GLO expression levels in S.cerevisiae.

-34- WO 01/14574 PCT/US00/22795 EXAMPLE Purification of recombinant GLO produced P.pastoris For the preparative isolation of recombinant GLO, a recombinant P.pastoris strain GS115::pPIC3.5(GLO51-3) was cultivated in a 101 fermentor using a fed-batch mode essentially as described in Sreckrishna, et al.

Biochemistry, 1989, 28, 4117-4125).

After the cultivation, the cells were separated by centrifugation, the pH of the clarified culture medium was adjusted to 6.5 and 500 ml of DEAE Sepharose FF was added. The suspension was stirred overnight at 4 oC after which the DEAE Sepharose was collected by sedimentation, packed into a column and eluted with 0-0.2 M gradient of NaC1 in 10Mm sodium phosphate buffer (pH 6.5) containing ImM EDTA.

The fractions containing the highest activity of GLO were pooled and subjected to hydroxyapatite chromatography under conditions described in Example 2. The fractions containing the highest GLO activity were analyzed by acrylamide gel electrophoresis and found to contain an almost homogeneous GLO preparation (approximately 80-90% purity as judged by the intensity of Coomassie Brilliant Blue G250 staining).

The specific activity of this preparation was about 24 U/mg protein, i.e. approximately 4-times higher than the WO 0 1/14574 PCTUSOO/22795 specific activity of homogeneous GLO purified from P. cyaneo -fulvus.

WO 01/14574 PCT/USOO/22795 EXAMPLE 11 Immobilization of the recombinant GLO 1 ml of N-hydroxysuccinimide-activated Sepharose 4 FF (Pharmacia) was incubated with 0.5 ml of 0.35 mg/ml solution of GLO purified according to Example 10 and adjusted to pH 8.0. The coupling reaction and subsequent treatment were carried out according to the instructions of the manufacturer of the resin.

The activity of immobilized GLO was measured by a slight modification of our standard assay (Example 1 ml of the GLO-Sepahrose was gently shaken with 10 ml of the substrate solution (Example 1) for 30 min, the resin was separated by sedimentation and the amount of erythorbic acid formed was measured in a reaction with 2,6dichlorophenolindophenol.

It was found that GLO, the activity of immobilized GLO (about 2.5 U/ml resin) and the GLO activity remaining unbound approximately (5.5 U) match the amount of enzyme used in the reaction. Thus, GLO retains most of its enzymatic activity after immobilization on Nhydroxysuccinimde-activated Sepharose. Therefore, immobilized GLO can be used in the production of erythorbic acid from gluonolactone.

WO 01/14574 PCT/US00/22795 EXAMPLE 12 Enzymatic conversion of glucose into erythorbic acid 120 jil of a reaction mixture containing 600U/ml glucose oxidate, 1.2 U/ml of catalase and 1% glucose in 100 mM potassium phosphate buffer, pH 6.0 was incubated for 1 hour at 35"C. At this point, 120 pl of potassium phthalate buffer pH 5.6 and 100 l of purified recombinant GLO solution containing about 0.8 activity units (Example 10) was added and the reaction was allowed to proceed for an additional 1 hour. The reaction was terminated by freezing and erythorbic acid was analyzed by HPLC (using the conditions described by L. W. Doner, K. Hicks, Anal. Biochem. 115, 225-230 (1981)) of by measuring the reduction of 2,6-dichlorophenolindophenol.

Aproximately 1.5 mg/ml of erythorbic acid was found in the reaction mixture corresponding to about 40% yield.

In another experiment, 1 ml of a reaction mixture containing 0.1 mg/ml glucose, 0.06 U/ml glo, 24 U/ML catalase, 800 U/ml glucose oxidate in 0.1M potassium phosphate buffer was incubated at room temperature in an.

open test tube. About 0.3 mg/ml of erythorbic acid was formed corresponding to about 30% yield.

Notably, no attempt to optimize the conversion yield was made in these experiments. For example, the yield of erythorbic acid could be further improved by introduction of automatic pH control and other protocols well known to those skilled in the art.

EDITORIAL NOTE APPLICATION NUMBER 70627/00 The following Sequence Listing pages 1 to 19 are part of the description. The claims pages follow on pages "39" to "42".

11/08/04 WO 01/14574 WO 0114574PCT/USOO/22795 <110> <120> <130> <140> <141> <160> <170> <210> <211> <212> <213> <220> <223> SEQUENCE LISTING Danisco Cultor America, Inc.

D-GLUCONOLACTONE OXIDASE GENE AND METHOD FOR PRODUCING RECOMBINANT D-GLUCONOLACTONE OXIDASE Andrei Masnikov, et al.

18 Patentln Ver. 2.1 1 1774

DNA

Unknown Organism Description of Unknown Organism: Penicillium D-gluconolactcone cDNA <400> 1 gactattcgca ctatatcaaa cggc tcggcc tattgctcct ctcccatatc caat-gctctc tatcgataag ccaagagc Ec ctttgtgggt aacccagatz gaagcacaaL cacggagttg gaa tgccacg cgaccgcatg ggaatttatc atgctgagcc taccgctggt cataatgagc aaagttgttg actgaaac aagaac ttga a agg cca ac g gctgcctcca atcgggctg~c gcggaagaat actatcaagg tccgactat actgtctggg ttgtgagacg ctaagccggc tcaactggca atgccgccgc ggaatggtca ccacctacar ccgtcacctt act tgtcctt caggtaccca gtgtattgga tgaaggcttC tccagcctac caaaga tgta gtcctcactu atcttgttca Lttcctgctg gtttgaggtc cgagttcctc tggatttggt cgtttctctc tLggtgC'Cggc cagcaatc tc cggttctgga c tcacaggga ccgtatcagc ccaac tcc tg caatgagct t cgaCtggaat tagtttgagc ttgctgctgc acttgccagt aaggaacagt aacctcacta accaacctga tgggatgtcg ggtgttgagc tccgacc tcg ggcctgcgtg cttggtgccc aagaagacca gcccagctgt gcaaagtc tc attcctattc acgcagtgtL ctgatgccta accctaagag cctgtgtcga agaagc Ecca atgaccttat gtgttcagaa ggaatatcgc tcatcaacga tcgguttaat ccaaggtctt acaaggagca agagct~ggqa 120 180 240 300 360 420 480 540 600 660 720 780 840 W001/14574 WO~l/ 4574PCT/USOO/22795 ccttgagcct c tgc accc tc cgacgaagtc cgccgagatc tattttccaa gcagcaccgt tggcccagac caacttcacc tgaaacc tac Cctqgagaaa gtgccagt tc tatctctgta agaggtgatg cc taagaatt ctcttggtgg tcatctacca act tac ttcc aattactgcg actgatgcgg gagcacttcc cagggccaga tcgc tcaagg cttagcggtg actctctggc aacgctcgct ttccccaagc gttaacgaat taagatgtga tggtcgtcaa tttgagtgga gtttcctagt ccagttaaaa tctcttactg ccaacggctg cgtcctgctc ttcctataga cgtctcgcat gtgatgatac tttttggagt agaaccagga cgcactggaa tgcccgagtt tcctggttga tatgagtggt tatgtcagtt atgggtcatt tatgtacata aaaaaaaaaa ggagccaacc cggtgactgc tcctcaaggt atatttcgcg gaaggcaccc atacttgtcc catcgagatt attggccc at caagatgagc cttggccatc gcagcttgga ctgaggcact ggcaaacacc gaatgagctt tatagtttct aaaa aactacaccg aagaaggagt gtctgttCCC gaagccgcca tacaacaagc ccagttaaca gactggatcc gaattcctcc gctcctaatg cagaagcgtc attacgcgc t cttttctttt ttttccaccg cgtgtcggac gagatagctt gtgttcgcaa acatttgcta ggggcttcta ccaactacac agatggtcat cctacaacct aagaatacaa ctcagtttgg ccacttatac aggaccccaa gtg'caaacta cttttttgtC caacttttgt ttggtggcac catgaccaat 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1774 <210> 2 <211> 1443 <212> DNA <213> Unknown Organism <220> <221> CDS <222> (1)..(1440) <220> <223> Description of Unknown organism: Penicillium D-gluconolactone cDNA <400> 2 atg ctg agc cct aag ccg gct ttc ctg ctg ttg ctg ctg cac gca gtg Met Leu Ser Pro Lys Pro Ala Phe Leu Leu Leu Leu Leu His Ala Val 1 5 10 ttc ggc tcg gcc tac cgc tgg ttc aac tgg cag ttt gag gtc act tgc Phe Gly Ser Ala Tyr Arg Trp Phe Asn Trp Gin Phe Glu Val Thr Cys WO 01/14574 WO 0114574PCTIUSOO/22795 cag Gin ttc Phe tct gat Ser Asp gcc tat att gct Ala Tyr Ile Ala cct cat aat gag cat gcc gcc gcc gag Pro His Asn Glu His Ala Ala Ala Glu 40 aag agc tcc cat atc aaa gtt gtt ggg Lys Ser Ser His Ile Lys Val Val Gly C tc Leu aat ggt Asn Gly aag gaa cag tac cct Lys Glu Gin Tyr Pro cat gga ttt ggt aac His Gly Phe Gly Asn 70 ctc act acc tgt Leu Thr Thr Cys 75 act Thr cat His gac aat gct ctc Asp Asn Ala Leu ctg aag aag ctc Leu Lys Lys Leu gag aag ccc acc Glu Lys Pro Thr atc gat aag aag Ile Asp Lys Lys tac atc gtt tct ctc Tyr Ile Val Ser Leu 90 aac ttg acc gtc acc Asn Leu Thr Val Thr 105 caa gag ctc aag gcc Gin Glu Leu Lys Ala ac c Thr aac Asn 100 gtc gat gac ctt Val Asp Asp Leu ttt ggt gcc ggc tgg gat Phe Gly Ala Gly Trp Asp 110 aac gac ttg tcc ttc agc Asn Asp Leu Ser Phe Ser 144 192 240 288 336 384 432 480 528 576 624 atc Ile aat ctc Asn Leu 130 ggt gtt Gly Val gag cgt gtt Glu Arg Val 120 cag aac ttt gtg ggt Gin Asn Phe Val Giy 140 125 gct gcc tcc aca Ala Ala Ser Thr ggt acc Gly Thr 145 atc ggg Ile Gly cac ggt tct His Gly Ser ctg cgt gta Leu Arg Val gga Gly 150 135 tcC Ser gac ctc ggg Asp Leu Gly gag aag cac aat Giu Lys His Asn 180 gcc ctc ggt tta.

Ala Leu Giy Leu 195 165 gcg Ala atc ttg gac tca cag gga Leu Asp Ser Gin Gly 170 gaa gaa ttg aag gct Glu Glu Leu Lys Ala 185 acg gag ttg act atc 'Ph' Clu Leu Thr Ile aat aLc gca acc Asn Ile Ala Thr 155 ggc ctg cgt gtc Gly Leu Arg Val ttc cgt atc agt Phe Arg Ile Ser 190 aag gtc cag cct Lys Val Gin Pro 205 c ag Gin atc aac Ile Asn 175 ctt ggt Leu Gly acc caa Thr Gin att Ile 160 200 WO 01/14574 WO 0114574PCTIUSOO/22795 ctc ctg Leu Leu 210 aag atg Lys Met aag aag acc acc aag Lys Lys Thr Thr Lys 215 Lac aat gag ctt gcc Tyr Asn Glu Leu Ala 230 tgg ggt cct cac ttc Trp Gly Pro His Phe gtc ttg aat gcc acg Val Leu Asn Ala Thr 220 cag ctg Lac aag gag Gin Leu Tyr Lys Giu tcc gac tat tca Ser Asp Tyr Ser cac gac cgc atg His Asp Arg Met 240 tct cag agc tgg Ser Gin Ser Trp, 255 225 act Thr gac Asp ac c Thr gtc Val gac tgg aat Asp Tr-p Asn 245 ctt gag cct act Leu Giu Pro Thr Lac ttc ctc Ltt Tyr Phe Leu Ser 265 tgc acc ctc aat Cys Thr Leu Asn 250 ggt gtt Gly Val 275 260 cgc aac Arg Asn Lac tgg gag cca. acc aac tac Tyr Trp, Glu Pro Thr Asn Tyr 270 Lac tgc gcc aac ggc tgc ggt Tyr Cys Ala Asn Gly Cys Gly 285 gac gaa gtc act gat gcg gcg Asp Glu Val Thr Asp Ala Ala gac tgc Asp Cys 290 Lcc tgc Ser Cys 305 aag Lys aag gag tac att Lys Giu Tyr Ile 280 tgc tac Cvs Tyr 672 720 768 816 864 912 960 1008 1056 1104 1152 Let cct caa ggt Ser Pro Gin Gly 310 295 gtc Val tgt Lee cgg ggc Cys Ser Arg Gly 315 tat ttc gcg gaa Tyr Phe Ala Glu ttc Lac gcc gag atc Phe Tyr Ala Glu Ile 320 gcc gcc acc aac tac Ala Ala Thr Asn Tyr 335 gag cac ttc ctt cct ata Giu His Phe Leu Pro Ile 325 act att ttc caa cag ggc Thr Ile Phe Gin Gin Gly 340 aag cag atg gtc atg cag Lys Gin Met Val Met Gin 355 ttg tcc cca gtt aac acc Leu Ser Pro Val Asn Thr 370 gaa Glu cag acg Gin Thr cac cgt His Arg 360 tac aac Tyr Asn 375 330 cgc Arg tcg ctc Ser Leu ctt ggc Leu Giy atg aag gca ccc tac aac Met Lys Ala Pro Tyr Asfl 350 aag ggt gat gat aca Lac Lys Gly Asp Asp Thr Tyr 365 cca gac ctt agc ggt gtt Pro Asp Leu Ser Gly Val 380 ttt Phe 385 act Thr ggt Gly aat As n gc C Ala ctg Leu 465 taa W0 01/14574 gga gtc E Gly ValI ctc tgg c Leu Trp C gaa acct Glu Thx gcc actt Ala Thr 435 atc cag Ie Gin 450 gtt gag Val Giu itc lie :ag ;in :ac [yr :at [yr iag cag "ln gag Giu aac Asn 405 aac Asn acc Thr cgt Arg ctt Leu att gac Ile. Asp 390 cag gaa Gin Giu gct cgc Ala Arg ctg gag Leu Giu cag gac Gin Asp 455 gga att Giy Ile 470 tgg Trp ttg Leu tcg Ser aaa Lys 440

CCC

Pro acg Thr atc Ile gcc Ala cac His 425 ttc Phe aag Lys cgC Arg c aa Gin cat His 410 tgg Tr-p

CCC

Pro tgc Cys tgt Cys gaa Glu 395 gaa Giu aac Asn aag Lys cag Gin gc a Ala 475 PCT/US00122795 tac aac aac ttc acc 1200 Tyr Asn Asn Phe Thr 400 ttc ctC cct cag ttt 1248 Phe Leu Pro Gin Phe 415 aag atg agc gCt cct 1296 Lys Met Ser Ala Pro 430 ctg ccc gag ttc ttg 1344 Leu Pro Giu Phe Leu 445 ttc gtt aac gaa ttc 1392 Phe Val Asn Giu Phe 460 aac tat atc tct gta 1440 Asn Tyr Ile Ser Val 480 1443 <210> 3 <211> 480 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: Peniciiiiuu D-gluconoiactone protein <400> 3 Met Leu Ser Pro Lys Pro Aia Phe Leu Leu Leu Leu Leu His Ala Val 1 5 10 Phe Gly Ser Ala Tyr Arg Trp Phe Asn Trp Gin Phe Glu Val Thr Cys 25 Gin Ser Asp Ala Tyr Ie Ala Pro His Asn Giu His Ala Ala Ala Giu 40 Phe Leu Lys Giu Gin Tyr Pro Lys Ser Ser His Ile Lys Val Val Gly 55 WO 01/14574 WO 0114574PCT/USOO/22795 Asn Gly His Gly Phe Thr Glu His Ile Val Asp Asn Leu 130 Gly Thr 145 Ile Gly Glu Lys Ala Leu Leu Leu 210 Lys Met 225 Thr Val Asp Leu Thr Gly Asp Cys 290 Ser Cys 305 Glu His Thr Ile Lys Asp Asp 115 Gly His Leu His Gly 195 Lys Tyr Trp Glu Val 275 Lys Ser Phe Phe Pro Lys 100 Leu Val Gly Arg Asn 180 Leu Lys Asn Gly Pro 260 Arg Lys Pro Leu Gin 340 Thr Lys Ile Glu Ser Val1 165 Ala Ilie Thr Glu Pro 245 Thr Asn Giu Gin Pro 325 Gin Gly 70 Tyr Asn Gin Arg Gly 150 Leu Glu Thr Thr Leu 230 His Tyr Cys Tyr Gly 310 Ile Gly Asn Ile Leu Giu Val1 135 Ser Asp Giu Giu Lys 215 Ala Phe Phe Thr Ile 295 Val1 Glu Gin Leu Val Thr Leu 120 Gin Asp Ser Leu Leu 200 Val Gin

ASP

Leu Leu 280 Cys Cys Tyr Thr Thr S er Val1 105 Lys Asn Leu Gin Lys 185 Thr Leu Leu Trp Ser 265 Asn Tyr Ser Phe Ser 345 Thr Leu 90 Thr Ala Phe Giy Gly 170 Ala Ile Asn Tyr Asn 250 Tyr Tyr Asp Arg Ala 330 Arg Cys 75 Thr Phe Asn Val Asn 155 Giy Phe Lys Ala Lys 235 Ala Trp Cys Giu Gly 315 Glu Met Val Asn Gly Asp Gly 140 Ile Leu Arg Val Thr 220 Giu Lys Giu Ala Val 300 Phe Ala Lys Asp Leu Ala Leu 125 Al a Ala Arg Ile Gin 205 Ser His Ser Pro Asn 285 Thr Tyr Aila Ala Asn Lys Gly 110 Ser Ala Thr Val1 Ser 190 Pro Asp Asp Gin Thr 270 Giy Asp Ala Thr Pro 350 Ala Lys Trp Phe Ser Gin Ile 175 Leu Thr Tyr Arg Ser 255 Asn Cys Ala Glu Asn 335 Tyr Leu Leu Asp Ser Thr Ile 160 Asn Gly Gin Ser Met 240 Trp Tyr Gly Ala Ile 320 Tyr Asn Lys Gin Met 355 Val Met Gin His Arg 360 Ser Leu Lys Gly ASP Thr Tyr Leu Phe 385 Thr Gly Asn Ala Leu 465 WO 01/14574 Ser Pro Val 370 Gly Val Ilie Leii Trp Gin Glu Thr Tyr 420 Ala Thr Tyr 435 Ile Gin Lys 450 Val Giu Gin Asn Giu Asn 405 Asn Thr Arg Leu Thr Ile 390 Gin Ala Leu Gin Gly 470 Tyr 375 Asp Giu Arg Giu Asp 455

TIP

Asn Trp Leu Ser Lys 440 Pro Thr Leu Ile Ala His 425 Phe Lys Arg Pro Giu 395 Glu Asn Lys Gin Ala 475 Leu Asn Leu Met Pro 445 Val Tyr Ser Asn Pro Ser 430 Giu Asn Ile PCT/USOO/22795 Giy Val Phe Thr 400 Gin Phe 415 Ala Pro Phe Leu Giu Phe Ser Vai 480 <210> 4 <211> 1386 <212> DNA <213> Unknown Organism <220> <221> CDS <222> (1)..(1383) <220> <223> Description of Unknown Organism: Peniciiiiun D-giuconoiactone mature cDNA <400> 4 atg tac cgc tgg ttc aac tgg cag ttt gag gtc act tgc met Tyr Arg Trp Phe Asn Trp Gin Phe Giu Val Thr Cys 1 5 10 gcc tat att gct cct cat aat gag cat gcc gcc gcc gag Ala Tyr Ile Ala Pro H-is Asn Giu His Ala Ala Ala Glu 25 gaa cag tac cct aag agc tcc cat atc aaa gtt gtt ggg Giu Gin Tyr Pro Lys Ser Ser His Ile Lys Val Vai Gly 40 gga ttt ggt aac ctc act acc tgt gtc gac aat gct ctc Gly Phe Gly Asn Leu Thr Thr Cys Val Asp Asn Ala Leu 55 cag Gin ttc Phe aa t Asn act Thr tct Ser c tc Leu ggt Gly gag Glu ga t Asp aag Lys cat His aag Lys 48 96 144 192 WO 01/14574 WO 0114574PCT/EJS0O22795 ccc Pro aag Lys cc Leu acc tac atc gtt tct Thr Tyr Ile Val Ser 70 aag aac ttg acc gtc Lys Asn Leu Thr Val atc caa gag ctc aag Ile Gin Giu Leu Lys ctc acc aac ctg aag Leu Thr Asn Leu Lys 75 acc ttt ggt gcc ggc Thr Phe Gly Ala Gly 90 aag ctc cat atc gat Lys Leu His Ile Asp tgg gat gtc gat gac Trp Asp Val Asp Asp gcc aac gac Aia Asn Asp 100 gtt gag cgt gt Val Giu Arg Val 115 10~5 cag aac ttt gtg ggt Gin Asn Phe Val Gly 120 ttg tcc Etc agc aat ctc ggt Leu Ser Phe Ser Asn Leu Gly 110 gct gcc tcc aca ggt acc cac Ala Ala Ser Thr Gly Thr His 125 gca acc cag att atc ggg ctg Ala Thr Gin Ile Ile Gly Leu ggt cct Gly Ser cgt Arg 145 aa t Asn i3 0 gta Val gcg Ala gga tcc gac ctc ggg Gly Ser Asp Leu Gly 135 ttg gac Eca cag gga Leu Asp Ser Gin Gly 150 gaa gaa ttg aag gct Giu Giu Leu Lys Ala aa t Asn 140 ggc ctg cgt gtc atc aac gag Gly Leu Arg Val Ile Asn Glu 155 ttc cgt atc agE ctt ggt gc Phe Arg Ile Ser Leu Gly Ala atc Ile 240 288 336 384 432 480 528 576 624 672 720 aag cac Lys His 160 ctc ggt Leu Gly 175 165 170 tta atc acg gag Leu Ile Thr Giu 180 aag acc acc aag Lys Thr Thr Lys 195 aat gag ctt gcc Asn Giu Leu Ala ttg act atc aag gtc Leu Thr Ile Lys Vai 185 gtc ttg aat gcc acg Val Leu Asn Ala Thr 200 cag ccg tac aag gag Gin Leu Tyr Lys Giu cag cct acc caa ctc ctg aag Gin Pro Thr Gin Leu Leu Lys 190 tcc gac tat tca aag atg tac Ser Asp Tyr Ser Lys Met Tyr 210O ggt cct Gly Pro 225 215 cac Etc gac Egg aat His Phe Asp Trp Asn 230 gca aag- Ala Lys cac gac cgc His Asp Arg 220 tct cag agc 205 atg act gtc Egg Met Thr Val Trp tgg gac ctt gag Ser Gin Ser Trp Asp Leu 235 Glu 240 WO 01/14574 WO 01/ 4574PCTIUSOO/22795 cct act tac ttc ctc Pro Thr Tyr Phie Leu 245 cgc aac tgc acc ctc Arg Asn Cys Thr Leu tct tac tgg gag cca Ser Tyr Trp Giu Pro 250 aat tac tgc gcc aac Asn Tyr Cys Ala Asn acc aac tac acc ggt gtt Thr Asn Tyr Thr Gly Vai 255 ggc tgc ggt gac tgc aag Gly Cys Gly Asp Cys Lys 270 aag gag tac Lys Giu Tyr 275 cct caa ggt Pro Gin Gly 260 att Ilie gtc Val tgc tac gac gaa Cys Tyr Asp Giu 280 tgt tcc cgg ggc Cys Ser Arg Giy 265 gtc Val t tc Phe act gat gcg gcg tcc tgc tct Thr Asp Aia Aia Ser Cys Ser 285 tac gcc gag atc gag cac ttc Tyr Ala Glu Ile Giu His Phe 290 ctt cct Leu Pro 295 300 305 c aa Gln ata gaa tat ttc Ile Giu Tyr Phe 310 ggc cag acg tct Gly Gin Thr Ser cag Gin gcg gaa gcc gcc Ala Glu Ala Ala cgc atg aag gca Arg Met Lys Ala 330 ctc aag ggt gat Leu Lys Gly Asp acc Thr 315 aac Asn tac act att Tyr Thr Ile 325 t tc Phe 320 gtc atg cag cac Val Met Gin His 340 gtt aac acc tac Val Asn Thr Tyr 355 cgt Arg tcg Ser aac ctt ggc cca Asn Leu Gly Pro 360 345 gac ctt Asp Leu tac aac Tyr Asn ccc tac aac aag cag atg Pro Tyr Asfl Lys Gin met 335 gat aca tac ttg tcc cca Asp Thr Tyr Leu Ser Pro 350 agc ggt gtt ttt gga gtc Ser Giy Val Phe Gly Val 365 aac ttc acc act ctc tgg Asn Phe Thr Thr Leu TrP 768 816 864 91i2 960 1008 1056 1104 1152 1200 1248 atc gag Ile Giu 370 cag aac Gin Asn att gac tgg atc caa Ile Asp Trp Ile Gin 375 cag gaa ttg gcc cat Gin Glu Leu Ala His gaa Glu gaa 380 ttc ctc cct cag ttt Phe Leu Pro Gin Phe ggt gaa Giy Glu acc Thr 400 385 tac 390 cac 395 aac gct cgc tcg tgg aac aag'atg agc gct cct aat gcc act Tyr Asn Ala Arg Ser His Trp, 405 TyrAsnAlaArgSerHisTrpAsn Lys Met Ser Ala Pro Asn Ala Thr 405410 415 WO001/14574 PCT/USO0122795 tat acc ctg gag aaa ttc ccc aag ctg ccc gag ttc ttg gcc atc cag 1296 Tyr Thr Leu Glu Lys Phe Pro Lys Leu Pro Giu Phe Leu Ala Ile Gin 420 425 430 aag cgt cag gac ccc aag tgc cag ttc gtt aac gaa ttc ctg gtt gag 1344 Lys Arg Gin Asp Pro Lys Cys Gin Phe Val Asn Giu Phe Leu Val Glu 435 440 445 cag ctt gga att acg cgc tgt gca aac tat atc tct gta taa 1386 Gin Leu Giy Ile Thr Arg Cys Ala Asn Tyr Ile Ser Val 450 455 460 <210> <2ii> 461 <212> PRT <213> Unknown organism <220> <223> Description of Unknown Organism: Penicillium D-giuconoiactone mature protein <400> Met Tyr Arg Trp Phe Asn Trp Gin Phe Giu Val Thr Cys Gin Ser Asp 1 5 10 Ala Tyr Ile Ala Pro His Asn Giu His Ala Ala Ala Giu Phe Leu Lys 25 Giu Gin Tyr Pro Lys Ser Ser His Ile Lys Val Val Gly Asn Gly His 40 Giy Phe Gly Asn Leu Thr Thr Cys Val Asp Asn Ala Leu Thr Glu Lys 55 Pro Thr Tyr Ile Val Ser Leu Thr Asn Leu Lys Lys Leu His Ile Asp 70 75 Lys Lys Asn Leu Thr Val Thr Phe Giy Ala Gly Ti-p Asp Val Asp Asp 90 Leu Ile Gin Glu Leu Lys Ala Asn Asp Leu Ser Phe Ser Asn Leu Gly 100 105 110 Val Giu Arg Val Gin Asn Phe Val Giy Ala Ala Ser Thr Gly Thr His 115 120 125 Gly Ser Gly Ser Asp Leu Gly Asn Tie*Ala Thr Gin Ile Ile Gly Leu 130 135 140 Arg Val Leu Asp Ser Gin Giy Gly Leu Arg Val Ile Asn Glu Lys His 145 150 155 160 WO 01/14574 PCT/USOO/22795 Asn Ala Giu Glu Leu Lys Asn Gly 225 Pro Arg Lys Pro Leu 305 Gin Val Val Ile Gin 385 Tyr Tyr Lys Ile Thr Thr Thr 195 Glu Leu 210 Pro His Thr Tyr Asn Cys Glu Tyr 275 Gin Gly 290 Pro Ile Gin Gly Met Gin Asn Thr 355 Glu Ile 370 Asn Gin Asn Ala Thr Leu Arg Gin 435 Glu 180 Lys Ala Phe Phe Thr 260 Ile Val Glu Gin His 340 Tyr Asp Glu Arg Glu 420 Asp Leu 165 Leu Val Gin Asp Leu 245 Leu Cys Cys Tyr Thr 325 Arg Asn Trp Leu Ser 405 Lys Pro Thr Leu Leu Trp 230 Ser Asn Tyr Ser Phe 310 Ser Ser Leu Ile Ala 390 His Phe Lys Ile Asn Tyr 215 Asn Tyr Tyr Asp Arg 295 Ala Arg Leu Gly Gin 375 His Trp Pro Cys Lys Ala Phe Arg Lys Ala 200 Lys Ala Trp Cys Glu 280 Gly Glu Met Lys Pro 360 Glu Glu Asn Lys Gin 440 Val 185 Thr lu Lys Glu Ala 265 Val Phe Ala Lys Gly 345 Asp Tyr Phe Lys Leu 425 Phe Ile 170 Gin Ser His Ser Pro 250 Asn Thr Tyr Ala Ala 330 Asp Leu Asn Leu Met 410 Pro Val Pro Asp Asp Gin 235 Thr Gly Asp Ala Thr 315 Pro Asp Ser Asn Pro 395 Ser Glu Asn Thr Tyr Arg 220 Ser Asf Cys Ala Glu 300 Asn Tyr Thr Gly Phe 380 Gin Ala Phe Glu Ser Leu Gly Ala Gin Ser 205 Met Trp Tyr Gly Ala 285 Ile Tyr Asn Tyr Val 365 Thr Phe Pro Leu Phe 445 Leu Lys rhr Asp rhr Asp 270 Sex Glu Thr Lys Leu 350 Phe Thr Gly Asn Ala 430 Leu Leu 175 Leu Met Val Leu Gly 255 Cys Cys His Ile Gin 335 Ser Gly Leu Glu Ala 415 Ile Val Gly Lys Tyr rrp Glu 240 Val Lys Sex Phe Phe 320 Met Pro Val Trp Thr 400 Thr Gin Glu Gin Leu 450 Gly Ile Thr Arg Cys 455 Ala Asn Tyr Ile Ser Vai 460 4 WO 01/14574 PTU0129 PCTIUSOO/22795 <210> 6 <211> 480 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: Penicillium D-giuconolactone protein <400> 6 Met Leu Ser-Pro Lys Pro Ala Phe 1 Phe Gin Phe Asn Thr His Val1 Asn Gly 145 Ile Giu Ala Leu Lys 225 Gly Ser Leu Gly Glu Ile Asp Leu 130 Thr Gly Lys Leu Leu 210 Ser

ASP

Lys His Lys Asp Asp 115 Gly His Leu His Gly 195 Lys Ala Ala Glu Gly Pro Lys 100 Leu Val1 Gly Arg Asn 180 Leu Lys 5 Tlyr Tyr Gin Phe Thr Lys Ile Glu Ser Val 165 Ala Ile Thr Arg Ile Tyr Gly 70 Tyr Asn Gin Arg Gly 150 Leu Glu Thr Thr Trp Ala Pro 55 Asn Ile Leu Glu Val1 135 Ser Asp Glu Glu Lys 215 Phe Pro 40 Lys Leu Val1 Thr Leu 120 Gin Asp Ser Leu Leu 200 Val1 Leu Asn 25 His Ser Thr Ser Val 105 Lys Asn Leu Gin Lys 185 Thr Leu Leu 10 Trp Asn Ser Thr Leu 90 Thr Ala Phe Gly Giy 170 Ala Ile Asn Leu Gin Glu His Cys 75 Thr Phe Asn Val Asn 155 Gly Phe Lys Ala Leu Phe His Ile Val1 Asn Gly Asp Gly 140 Ile Leu Arg Val1 Thr 220 Leu Glu Al a Lys Asp Leu Ala Leu 125 Ala Ala Arg Ile Gin 205 Ser His Val Ala Val Asn Lys Gly 110 Ser Ala Thr Val Ser 190 Pro

ASP

Ala rhr Ala Val1 Al a Lys Trp Phe Ser Gin Ile 175 Leu Thr Tyr Val1 Cys Glu Gly Leu Leu Asp Ser Thr I le 160 Asn Gly Gin Ser Met Tyr Asn Giu Leu Ala Gin Leu Tyr 230 Lys 235 Glu His Asp Arg Met 240 WO 01/14574 WO 0114574PCT1US00/22795 Thr Val Trp Gly Pro His Phe Asp Trp 245 Asp Thr Asp Ser 305 Giu Thr Lys Leu Phe 385 Thr Gly Asn Ala Leu 465 Leu Gly Cys 290 Cys His Ile Gin Ser 370 Giy Leu Giu Ala Ile 450 Giu Val 275 Lys Ser Phe Phe Met 355 Pro Val1 Trp Thr Thr 435 Gin Pro 260 Arg Lys Pro Leu Gin 340 Val1 Val1 Ile Gin Tyr 420 Tyr Lys Thr Asn Giu Gin Pro 325 Gin Met Asn Glu Asn 405 Asn Thr Arg Tyr Cys Tyr Gly 310 Ile Gly Gin Thr Ile 390 Gin Ala Leu Gin Gly 470 Phe Thr Ile 295 Va. 1 Giu Gin His Tyr 375 Asp Giu Arg Giu Asp 455 Leu Leu 280 Cys Cys Tyr Thr Arg 360 Asn Trp Leu Ser Lys 440 Pro Ser 265 Asn Tyr Ser Phe Ser 345 Ser Leu Ile Ala His 425 Phe Lys Asn 250 Tyr Tyr Asp Arg Ala 330 Arg Leu Giy Gin His 410 Trp Pro Cys Ala Lys Ser Gin Trp Giu Cys Ala Giu Vai 300 Giy Phe 315 Giu Ala Met Lys Lys Gly Pro Asp 380 Giu Tyr 395 Giu Phe Asn Lys Lys Leu Gin Phe 460 Pro Thr 270 Asn Gly 285 Thr Asp Tyr Ala Ala Thr Ala Pro 350 Asp Asp 365 Leu Ser Asn Asn Leu Pro Met Ser 430 Pro Giu 445 Val Asn Ser Trp, 255 Asn Tyr Cys Gly Ala Ala Giu Ile 320 Asn Tyr 335 Tyr Asn Thr Tyr Giy Val Phe Thr 400 Gin Phe 415 Ala Pro Phe Leu Giu Phe Val Giu Gin Leu Ile Thr Arg Cys Ala Asn Tyr Ile Ser 475 Val 480 <210> 7 <211> 24 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: Peniciiiiun -13- WO01/14574 PCT/US00/22795 D-gluconolactone peptide <220> <221> UNSURE <222> (12) <223> Xaa at position 12 is cysteine or any glycosylated amino acid residue <400> 7 Tyr Arg Trp Phe Asn Trp Gin Phe Glu Val Thr Xaa Gin Ser Asp Ala 1 5 10 Tyr Ile Ala Pro His Asn Glu His <210> 8 <211> 17 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: Penicillium D-gluconolactone peptide <400> 8 Glu His Asp Arg Met Thr Val Cys Gly Pro His Phe Asp Tyr Asn Ala 1 5 10 Lys <210> 9 <211> <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: Penicillium D-gluconolactone peptide <400> 9 Glu Tyr Ile Cys Tyr Asp Glu Val Thr Asp Ala Ala Ser Cys Ser Pro 1 5 10 Gin Gly Val Val <210> <211> 16 <212> PRT <213> Unknown Organism <220> WO 01/14574 PCT/US00/22795 <223> Description of Unknown Organism: Penicillium D-gluconolactone peptide <400> Cys Gin Phe Val Asn Glu Phe Leu Val Glu Gin Leu Gly Ile Thr Arg 1 5 10 <210> <211> <212> <213> <220> <223> 11

DNA

Unknown Organism Description of Unknown Organism: D-gluconolactone oligonucleotide <220> <221> unsure <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> (3) Y at position unsure (12) Y at position unsure Y at position unsure (6) N at position 3 is a mixture of T and C 12 is a mixture of T and C 15 is a mixture of T and C 6 is an inosine phosphate residue <400> 11 taycgntggt tyaaytggca <210> <211> <212> <213> <220> <223> <220> <221> <222> <223> 12 38

DNA

Unknown Organism Description of Unknown Organism: D-gluconolactone oligonucleotide unsure (6) Y at position 6 is a mixture of T and C WO 01/14574 PCT/USOO/22795 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> unsure (9) Y at position unsure (21) Y at position unsure (33) Y at position unsure (3) N at position unsure (12) N at position unsure N at position unsure (27) N at position 9 is a mixture of T and C 21 is a mixture of T and C 33 is a mixture of T and C 3 is an inosine phosphate residue 12 is an inosine phosphate residue 15 is an inosine phosphate residue 27 is an inosine phosphate residue <400> 12 ccnarytgyt cnacnarraa ytcrttnacr aaytgrca <210> 13 <211> 21 <212> DNA <213> Unknown Organism <220> <223> Description of Unknown Organism: D-gluconolactone oligonucleotide <400> 13 agctctcgag atctcccggg a <210> 14 <211> 21 WO 0 1/1 4574 PCTIJSOOI22795 £<212> DNA <213> unknown organism <220> <223> Description of Unknown Organism: D-gluconolactone oligonucleotide <400> 14 agcttcccgg gagatctcga g 21 <210> <211> 38 <212> DNA <213> unknown organism <220> <223> Description of Unknown organism: D-gluconolactone oligonucleotide sense primer <400> gaagaagctt accggtggtt caattggcag tttttggt 38 <210> 16 <211> 37 <212> DNA <213> Unknown Organism <220> <223> Description of Unknown Organism: D-gluconolactone oligonucleotide anti-sense primer <400> 16 caaagcttct agagcctcag accactcata tcacatc 37 <210> 17 <211> 549 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: D-gluconolactone fusion protein <400> 17 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gin 25 ~WO 1/14574 PTUO~l9 PCTfUSOO/22795 Ie Pro Ala Glu Ala Val Ile Asp Val Phe Ile Ser Leu Phe Leu His Ala Ilie Lys 130 Val Asp 145 Asn Leu Gly Ala Asp Leu Gly Ala 210 Ie Ala 225 Leu Arg Arg Ilie Val Gin Thr Ser 290 Glu His 305 Ala Asn Asp Val Ala 115 Val1 Asn Lys Gly Ser 195 Ala Thr Val Ser Pro 275 Asp Asp Val1 Thr Lays Thr 100 Ala Val1 Ala Lys Trp 180 Phe Ser Gin Ile Leu 260 Thr Tyr Arg Leu Pro Thr Ilie 70 Arg Glu Cys Gin Giu Phe Gly Asn Leu Thr 150 Leu His 165 Asp Val Ser Asn Thr Gly Ile Ile 230 Asn Glu 245 Gly Ala Gin Leu Ser Lays Met Thr 310 Phe 55 Ala Ala Ser Leu Gly 135 Giu Ile Asp Leu Thr 215 Gly Lays Leu Leu Met 295 Val Gly 40 Ser Ser Giu Asp Lys i2 0 His Lys Asp Asp Gly 200 His Leu His Gly Lys 280 Tyr Trp Tyr Asn Ile Ala Ala 105 Glu Gly Pro Lays Leu 185 Vai Gly Arg Asn Leu 265 Lays Asn Gly S er Ser Ala Tyr 90 Tyr Gin Phe Thr Lays 170 Ile Giu Ser Val Ala 250 Ile Thr Glu Pro Asp Thr Al a 75 Arg Ile Tyr Gly Tyr 155 Asn Gin Arg Giy Leu 235 Giu Thr Thr Leu His 315 Leu Asn Lys Trp Ala Pro Asn 140 Ile Leu Giu Val Ser 220 Asp Giu Giu Lys Ala 300 Phe Giu Asn Glu Phe Pro Lys 125 Leu Val Thr Leu Gin 205 Asp Ser Leu Leu Val1 285 Gin Asp Gly Gly Giu Asn His 110 Ser Thr Ser Val Lys 190 Asn Leu Gin Lys Thr 270 Laeu Leu Trp Asp Laeu Gly Trp Asn Ser Thr Leu Thr 175 Ala Phe Gly Gly Ala 255 Ile Asn Tyr Asn Phe Leu Val Gin Giu His Cys Thr 160 Phe Asn Val1 Asn Gly 240 Phe Lys Ala Lys Ala 320 Lys Ser Gin Ser Trp 325 Asp Leu Giu Pro Thr Tyr Phe Leu Ser 330 Tyr Trp 335 -18- WO 01/14574 WO 01/4574PUS00122795 Glu Pro Thr Asn Tyr Thr Gly Val 340 Asn Cys Thr Leu Asn Tyr Cys 350 Ala Asn Gly Cys Gly Asp Cys Lys Lys Glu Tyr Ile Cys Tyr 355 360 365 Val Thr Asp Ala Ala Ser Cys Ser Pro Gin Gly Val Cys Ser 370 375 380 Phe Tyr Ala Giu Ile Giu His Phe Leu Pro Ile Glu Tyr Phe 385 390 395 Ala Ala Thr Asn Tyr Thr Ilie Phe Gin Gin Gly Gin Thr Ser 405 410 Lys Ala Pro Tyr Asn Lys Gin Met Val Met Gin His Arg Ser 420 425 430 Gly Aso Asp Thr Tyr Leu Ser Pro Val Asn Thr Tyr Asn Leu 435 440 445 Asp Leu Ser Gly Val Phe Gly Val Ile Glu Ilie Asp Trp, Ile 450 455 460 Tyr Asn Asn Phe Thr Thr Leu Trp Gin Asn Gin Giu Leu Ala 465 470 475 Phe Leu Pro Gin Phe Gly Giu Thr Tyr Asn Ala Arg Ser His 485 490 Lys Met Ser Ala Pro Asn Ala Thr Tyr Thr Leu Giu Lys Phe 500 505 510 Leu Pro Giu Phe Leu Ala Ile Gin Lys Arg Gin Asp Pro Lys 515 520 525 Phe Val Asn Giu Phe Leu Val Giu Gin Leu Gly Ile Thr Arg 530 535 540 Asn Tyr Ile Ser Val 545 <210> 18 <211> 38 <212> DNA <213> Unknown organism <220> <223> Description of Unknown Organism: D-gluconolactofle oligonucleotide <400> 18 ccaacaattg atgctgagCC ctaagccggc tttcctgc

ASP

Arg Ala Arg 415 Leu Gly Gin His Trp 495 Pro Cys Glu Gly Giu 400 Met Lys Pro Giu Glu 480 Asn Lys Gin Cys Ala -19-

Claims (26)

1. An isolated nucleic acid molecule encoding a D-gluconolactone oxidase.

2. The nucleic acid molecule of Claim 1 isolated from a fungus.

3. The nucleic acid molecule of Claim 2 isolated from the genus Pencicillium.

4. The nucleic acid molecule of Claim 3 isolated from the species Penicillium griseoroseum, Pencillium notatum, Penicillium cyaneum or Penicillium decumbens. The nucleic acid molecule of Claim 4 isolated from the species Penicillium griseoroseum.

6. An isolated nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

7. An isolated nucleic acid molecule encoding a protein having a sequence identity of at least about 70% when compared to the amino acid sequence of SEQ ID NO:4.

8. An isolated nucleic acid molecule which hybridizes under stringent conditions to any one of the nucleic acids of Claim 6. WO 01/14574 PCT/US00/22795

9. An expression vector comprising the nucleic acid molecule of any one of Claims 1-8. A transformed host cell comprising the expression vector of Claim 9.

11. The isolated nucleic acid molecule of Claim 6 further comprising at least one addition, deletion, insertion or mutation, wherein said nucleic acid molecule encodes enzymatically active D-gluconolactone oxidase.

12. The D-gluconolactone oxidase protein encoded by the nucleic acid molecule of any one of Claims 1-8 and 11.

13. A method for producing a D-gluconolactone oxidase comprising culturing the cell of Claim 10 and recovering the D-gluconolactone oxidase from said culture.

14. A D-gluconolactone oxidase having SEQ ID NO:4. A method for the conversion of glucose to erythorbic acid comprising contacting glucose with glucose oxidase to form D-gluconolactone and contacting said D-gluconolactone with the D-gluconolactone oxidate encoded by the nucleic acid of Claims 1-8 and 11 for a time and under conditions sufficient to produce erythorbic acid. WO 01114574 PCT/US00/22795

16. A method for the conversion of D- gluconolactone to erythorbic acid comprising contacting said D-gluconolactone with the D-gluconolactone oxidase of Claims 1-8 and 11 for a time and under conditions sufficient to produce erythorbic acid.

17. The method of Claim 15 which further comprises recovering the erythorbic acid.

18. The method of Claim 16 which further comprises recovering the erythorbic acid.

19. An erythorbic acid produced by the process of Claim An erythorbic acid produced by the process of Claim 16.

21. The method of Claim 15 wherein said glucose is contacted with said glucose oxidase in the presence of a catalase.

22. The transformed host cell of Claim which further comprises a gene expressing glucose oxidase.

23. The transformed host cell of Claim which further comprises a gene expressing a catalase.

24. The transformed host cell of Claim wherein the host cell is a yeast. P XOPER W wdmnmask2004.20 A do-uA2O2988 md spe d=.27M04 The transformed host cell of Claim 10 wherein the host cell is a filamentous fungi.

26. An isolated nucleic acid molecule according to any one of Claims 1 to 8 or 11 substantially as hereinbefore described with reference to the examples and/or figures.

27. An expression vector according to Claim 9 substantially as hereinbefore described with reference to the examples and/or figures.

28. A transformed host cell according to any one of Claims 10 or 22 to substantially as hereinbefore described with reference to the examples and/or figures.

29. A D-gluconolactone oxidase according to Claim 12 or 14 substantially as hereinbefore described with reference to the examples and/or figures.

30. A method according to any one of Claims 13 or 15 to 18 or 21 substantially as hereinbefore described with reference to the examples and/or figures.

31. An erythorbic acid according to Claim 19 or 20 substantially as hereinbefore described with reference to the examples and/or figures. Dated this 27 h day of July, 2004. Danisco USA Inc. SBy their Patent Attorneys: DAVIES COLLISON CAVE -42-