WO2021183818A1 - Recombinant oxalate oxidase and uses thereof - Google Patents

Abstract

Aspects of the disclosure relate to compositions and methods for producing recombinant proteins in yeast expression systems. The disclosure is based, in part, on expression vectors encoding certain oxalate oxidase proteins and methods of purifying such proteins. In some embodiments, expression constructs described herein comprise one or more N-terminal signal sequences, such as secretion signal sequences.

Description

RECOMBINANT OXALATE OXIDASE AND USES THEREOF

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of the filing date of US provisional Application Serial Number 62/988,603, filed March 12, 2020, entitled “RECOMBINANT OXALATE OXIDASE AND USES THEREOF”, the entire contents of which are incorporated herein by reference.

BACKGROUND

Urinary oxalate, an integral part of the 24-hour urine stone profile, is used to calculate mineral supersaturations and estimate kidney stone risk. However, there is no commercially available source of recombinant oxalate oxidase - an enzyme that could be used to create a diagnostic test strip to identify the presence of kidney stones.

SUMMARY

Aspects of the disclosure relate to compositions and methods for producing recombinant proteins in yeast expression systems. The disclosure is based, in part, on expression vectors encoding certain oxalate oxidase (OxOx) proteins and methods of purifying such proteins. In some embodiments, expression vectors described herein comprise a chimeric secretion signal encoding sequence. In some embodiments, transforming yeast cells with expression constructs comprising a chimeric secretion signal linked to a recombinant OxOx protein surprisingly results in increased recovery of secreted heterologous protein (e.g., recombinant OxOx protein) from the yeast cells.

Accordingly, in some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 1,

2, and 7-9.

In some embodiments, an isolated nucleic acid encodes a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11.

In some embodiments, an expression construct further comprises an alpha factor (a-factor) signal sequence operably linked to a nucleic acid sequence (e.g., the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9). In some embodiments, an a-factor signal sequence is positioned 5’ relative to a nucleic acid sequence (e.g., the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9). In some embodiments, an a-factor signal sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments, an expression construct comprises a non-yeast signal sequence that encodes a peptide comprising the sequence set forth in any one of SEQ ID NOs: 17-19.

In some embodiments, an expression construct further comprises one or more purification tag sequences. In some embodiments, one or more purification tag sequences are positioned 3’ relative to a nucleic acid sequence (e.g., the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9). In some embodiments, one or more purification tag sequences encodes a c- myc epitope tag or a polyhistidine tag. In some embodiments, an expression construct does not comprise (e.g., lacks) a purification tag sequence.

In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct comprising a nucleic acid sequence encoding an oxalate oxidase (OxOx) protein operably linked to a non-yeast protein transduction domain sequence.

In some embodiments, an OxOx protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11. In some embodiments, a nucleic acid sequence encoding a protein comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9.

In some embodiments, a protein transduction domain sequence comprises bacterial transduction domain sequence. In some embodiments, a bacterial transduction domain sequence comprises a twin-arginine translocation (Tat) pathway signal sequence. In some embodiments, a Tat pathway signal sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 19.

In some embodiments, an expression construct further comprises a promoter operably linked to an a-factor signal sequence or a nucleic acid sequence (e.g., the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9). In some embodiments, a promoter is an AOX1 promoter.

In some embodiments, an expression construct further comprises a transcription terminator sequence. In some embodiments, a transcription terminator sequence is an AOX1 transcription terminator sequence.

In some aspects, the disclosure provides a plasmid or a host cell comprising an isolated nucleic acid as described herein. In some embodiments, a plasmid comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 14-16. In some embodiments, a host cell is a yeast cell. In some embodiments, a yeast cell is a Pichia pastoris cell (X-33 or GS115 strains) or Saccharomyces cerevisiae cell.

In some aspects, the disclosure provides a container housing a population of yeast cells, wherein at least one yeast cell comprises an isolated nucleic acid or plasmid as described herein.

In some aspects, the disclosure provides a method for producing a recombinant protein, the method comprising introducing into a cell an isolated nucleic acid as described herein under conditions under which the cell expresses a recombinant oxalate oxidase (OxOx) protein.

In some embodiments, a recombinant OxOx protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11, or is encoded by the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9.

In some embodiments, a method further comprises purifying a recombinant OxOx protein from a cell. In some embodiments, purifying comprises lysing a cell to produce cell lysate and an insoluble fraction. In some embodiments, the insoluble fraction is further treated to separate recombinant OxOx protein from yeast cellular membrane.

In some embodiments, purifying comprises contacting a cell lysate or insoluble fraction with an anion exchange media. In some embodiments, an anion exchange media comprises a Q- Sepharose anion exchange column. In some embodiments, purifying comprises providing conditions under which recombinant OxOx protein binds to a column.

In some embodiments, a method further comprises eluting bound recombinant OxOx protein from a column to produce a purified protein fraction. In some embodiments, a purified protein fraction comprises >90% recombinant OxOx protein.

In some aspects, the disclosure provides an isolated nucleic acid comprising a nucleic acid sequence encoding a protein comprising a chimeric secretion signal having the amino acid sequence set forth in SEQ ID NO: 26.

In some embodiments, a chimeric secretion signal comprises a pre region encoded by the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a chimeric secretion signal comprises a pro region encoded by the nucleic acid sequence set forth in SEQ ID NO: 23.

In some embodiments, a protein comprises an oxalate oxidase (OxOx) protein. In some embodiments, an OxOx protein is a barley OxOx protein. In some embodiments, a protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11. In some embodiments, an isolated nucleic acid further comprises a promoter operably linked to a nucleic acid sequence encoding a protein. In some embodiments, a promoter comprises an AOX1 promoter.

In some embodiments, an isolated nucleic acid further comprises a transcription terminator sequence, optionally wherein the transcription terminator sequence is an AOX1 transcription terminator sequence.

In some aspects, the disclosure provides a protein comprising a chimeric secretion signal having the amino acid sequence set forth in SEQ ID NO: 26.

In some embodiments, a protein comprises an oxalate oxidase (OxOx) protein, optionally a barley OxOx protein or a wheat OxOx protein. In some embodiments, a protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, or 11.

In some embodiments, a protein further comprises a purification tag (e.g., a poly Histidine tag, etc.).

In some aspects, the disclosure provides a cell comprising an isolated nucleic acid or protein (e.g., protein comprising a chimeric secretion signal) as described herein. In some embodiments, a cell is a yeast cell. In some embodiments, a yeast cell is a Pichia pastoris cell.

BRIEF DESCRIPTION OF DRAWINGS

FIGs. 1A-1C show OxOx plasmid construction and confirmation. FIG. 1A shows synthesis of a codon optimized barley-derived oxalate oxidase gene and cloned into the EcoRI/Xbal sites of a pPICZaA expression vector. FIG. IB shows sequencing of the OxOx gene insert. FIG. 1C shows the NCBI nucleotide BEAST® alignment tool was used to confirm the integrity of the inserted OxOx gene. SEQ ID NO: 2 is shown.

FIGs. 2A-2D show OxOx expression and purification. FIG. 2A shows accumulation of recombinant OxOx in the insoluble fraction of yeast lysate. FIG. 2B shows. P. pastoris culture media sample western blotting using an anti-OxOx antibody. FIG. 2C shows purification of recombinant OxOx from culture media that was dialyzed overnight against distilled water, pH adjusted to 9.0 using a tris buffer, applied to a Q-sepharose anion exchange column, and purified eluted with 1M NaCl. FIG. 2D shows the eluted fraction separated by SDS-PAGE and total protein was visualized with Coomassie stain to determine purity. FIG 3 shows Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis. Following trypsin digestion, LC-MS/MS was used to confirm the identity of the major band from the Coomassie gel in FIG. 2C. A peptide “AGETFVIPR” (SEQ ID NO: 20) was identified with 100% accuracy that is unique to the barley-derived oxalate oxidase (OxOx) protein and that is not present in the P. pastoris proteome.

FIGs. 4A-4B show representative data relating to reaction kinetics of oxalate oxidase (OxOx). FIG. 4A shows a Michaelis-Menten saturation curve. Kinetic analysis was performed in a 96-wel plate at 37°C. Purified OxOx (400 pg/ml) was added to buffer pH 3.1 containing MBTH (0.11 mM), DMAB (1.6 mM), and HRP (0.1 U/ml) at designated potassium oxalate concentrations. Absorbance at 590 nm was measured and plotted against substrate concentration. FIG. 4B shows a Lineweaver-Burk plot. A linear transformation for the data described in FIG. 1A was plotted. Enzyme Km was estimated to be 256 mM.

FIG. 5 shows a schematic representing expression vectors comprising yeast secretion signals. The top construct comprises a yeast a-factor secretion signal containing a “pre” region and a “pro” region. The bottom construct comprises a chimeric secretion signal containing a yeast OST “pre” region and a yeast proapp-8 “pro” region.

FIG. 6 shows representative Western blot data comparing secreted protein (“Supernatant”) and cell-bound protein (“Cell”) extracted from yeast cells transformed with expression constructs encoding recombinant OxOx linked to either a chimeric secretion signal (left) or a wild-type yeast a-factor secretion signal. Data indicate increased protein secretion from expression constructs comprising the chimeric secretion signal.

DETAILED DESCRIPTION

Aspects of the disclosure relate to compositions and methods for producing recombinant proteins in yeast expression systems, for example expression vectors encoding certain oxalate oxidase (OxOx) proteins and methods of purifying such proteins. The disclosure is based, in part, on the recognition that recombinant OxOx proteins comprising certain signal sequence tags surprisingly accumulate in the cellular membrane of yeast cells used for expressing such OxOx proteins. Recombinant Oxalate Oxidase

Oxalate oxidase (OxOx) is a germin protein found in plants such as barley ( Hordeum vulgare ) and wheat ( Triticum aestivum). Germins are developmentally-regulated plant enzymes that generate hydrogen peroxide (H2O2) from the oxidative catabolism of oxalate. Germins typically require the presence of manganese (Mn) for enzymatic activity. Structurally, germins comprise a b-barrel domain containing two conserved histidine-containing sequence motifs. In some embodiments, a Hordeum vulgare oxalate oxidase protein comprises the nucleic acid sequence set forth in NCBI Accession Number Y 14203.1, or the amino acid sequence set forth in NCBI Accession No. CAA74595.1. In some embodiments, a Triticum aestivum oxalate oxidase protein comprises the nucleic acid sequence set forth in NCBI Accession Number M63223.1, or the amino acid sequence set forth in NCBI Accession No. AAA34270.1. In the context of biotechnological applications, OxOx proteins are useful, in some embodiments, for detection of calcium oxalate kidney stones in a sample or subject. However, there is currently no commercially available source of oxalate oxidase that could be used to manufacture such diagnostic indications.

Thus, in some aspects, the disclosure provides isolated nucleic acids and vectors (e.g., plasmids, etc.) encoding a recombinant oxalate oxidase (OxOx) protein. As used herein “nucleic acid” refers to a DNA or RNA molecule. An “isolated nucleic acid” or “recombinant nucleic acid” refers to a nucleic acid (e.g., DNA or RNA) that has been prepared in vitro, for example by recombinant technology. Nucleic acids are polymeric macromolecules comprising a plurality of nucleotides. In some embodiments, the nucleotides are deoxyribonucleotides or ribonucleotides. In some embodiments, the nucleotides comprising the nucleic acid are selected from the group consisting of adenine, guanine, cytosine, thymine, uracil and inosine. In some embodiments, the nucleotides comprising the nucleic acid are modified nucleotides. Non-limiting examples of natural nucleic acids include genomic DNA and plasmid DNA. A “recombinant protein” refers to a protein expressed by an isolated nucleic acid or recombinant nucleic acid. In some embodiments, the nucleic acids of the instant disclosure are synthetic. As used herein, the term “synthetic nucleic acid” refers to a nucleic acid molecule that is constructed via joining nucleotides by a synthetic or non-natural method. One non-limiting example of a synthetic method is solid-phase oligonucleotide synthesis. In some embodiments, the nucleic acids of the instant disclosure are isolated. In some embodiments, recombinant oxalate oxidase (OxOx) protein is encoded by a nucleic acid having a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or more identical to the nucleic acid sequence set forth in NCBI Accession Number Y 14203.1. In some embodiments, a recombinant OxOx protein comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99% or more identical the amino acid sequence set forth in NCBI Accession No. CAA74595.1. In some embodiments, a recombinant OxOx protein is encoded by a nucleic acid that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or more identical the nucleic acid sequence set forth in NCBI Accession Number M63223.1. In some embodiments, a recombinant OxOx protein comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99% or more identical the amino acid sequence set forth in NCBI Accession No. AAA34270.1.

In some embodiments, an isolated nucleic acid encoding a recombinant OxOx protein comprises a codon-optimized (e.g., codon-optimized for expression in yeast cells, codon-optimized for expression in mammalian cells, etc.) coding sequence for the OxOx protein. In some embodiments, an isolated nucleic acid comprises a nucleic acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or more identical to the nucleic acid sequence set forth in SEQ ID NO:

1 or 4. In some embodiments, an isolated nucleic acid encodes a protein comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, 99% or more identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11.

In some embodiments, a recombinant OxOx protein comprises one or more amino acid substitutions, insertions, or deletions relative to a wild-type OxOx protein (e.g., a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11). In some embodiments, a recombinant OxOx protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 75, 80, 80, or 100 amino acid substitutions, insertions, or deletions relative to a wild-type OxOx protein (e.g., a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11). In some embodiments, a recombinant OxOx protein comprises more than 100 (e.g., 120m 150, 250, etc.) amino acid substitutions, insertions, or deletions relative to a wild-type OxOx protein (e.g., a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11).

In some embodiments, a recombinant OxOx protein retains functional activity. For example, a recombinant OxOx protein may retain the ability to catalyze the breakdown of oxalate into hydrogen peroxide (e.g., in the presence of Mn). The activity of a recombinant OxOx protein may vary. In some embodiments, a recombinant OxOx protein is between 2-fold and 100-fold (e.g., 2, 3, 4, 5, 10, 20, 50, 75, 100-fold, etc.) more active (e.g., by measurement of enzyme kinetics, substrate binding efficiency, etc.) than a wild-type OxOx protein (e.g., OxOx protein that has been isolated, extracted, and/or purified from barley or wheat). In some embodiments, a recombinant OxOx protein is more than 100-fold (e.g., 200, 500, 1000-fold, etc.) more active (e.g., by measurement of enzyme kinetics, substrate binding efficiency, etc.) than a wild-type OxOx protein (e.g., OxOx protein that has been isolated, extracted, and/or purified from barley or wheat). In some embodiments, a recombinant OxOx protein is between 2-fold and 100-fold (e.g., 2, 3, 4, 5, 10, 20, 50, 75, 100-fold, etc.) less active (e.g., by measurement of enzyme kinetics, substrate binding efficiency, etc.) than a wild-type OxOx protein (e.g., OxOx protein that has been isolated, extracted, and/or purified from barley or wheat). In some embodiments, a recombinant OxOx protein is more than 100-fold (e.g., 200, 500, 1000-fold, etc.) less active (e.g., by measurement of enzyme kinetics, substrate binding efficiency, etc.) than a wild-type OxOx protein (e.g., OxOx protein that has been isolated, extracted, and/or purified from barley or wheat).

Expression Constructs

Aspects of the disclosure relate to expression constructs encoding a recombinant OxOx protein. As used herein, an “expression construct” refers to an isolated nucleic acid that is engineered to express one or more nucleic acid sequences or genes of interest. An expression cassette generally includes one or more control elements and nucleic acid sequences or genes of interest to be expressed. As used herein, the term “engineered to express” refers to an isolated nucleic acid that directs expression of a sequence or gene of interest (e.g., a gene encoding recombinant OxOx protein) and, optionally, one or more expression control sequences. Examples of expression control sequences include but are not limited to promoter sequences, enhancer sequences, repressor sequences, poly A tail sequences, internal ribosomal entry sites, Kozak sequences, antibiotic resistance genes (e.g., ampR, kanR, a chloramphenicol resistance gene, a b- lactamase resistance gene, etc.), an origin of replication (ori), transcriptional start sites, transcriptional terminator sites, etc. One or more control elements (e.g., expression control sequences) are typically operably linked to the nucleic acid sequences or genes of interest. As used herein, a nucleic acid sequence (e.g., coding sequence) and regulatory control sequences are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. In some embodiments, a control sequence is directly operably linked (e.g., immediately upstream, such as 5’, or downstream, such as 3’) relative to a nucleic acid sequence or gene encoding a recombinant OxOx protein. In some embodiments, a control sequence is indirectly operably linked (e.g., upstream, such as 5’, or downstream, such as 3’, with intervening nucleic acid sequence, such as another control sequence) relative to a nucleic acid sequence or gene encoding a recombinant OxOx protein.

In some embodiments, one or more isolated nucleic acid is operably linked to a promoter sequence. A promoter can be a constitutive promoter or an inducible promoter. In some embodiments, a promoter is a constitutive promoter. Examples of constitutive promoters include but are not limited to constitutive E. coli s⁷⁰ promoters, constitutive E. coli o^s promoters, constitutive E. coli s³² promoters, constitutive E. coli s⁵⁴ promoters, constitutive B. subtilis s^A promoters, constitutive B. subtilis s^B promoters, constitutive bacteriophage T7 promoters, constitutive bacteriophage SP6 promoters, constitutive yeast promoters, etc. In some embodiments, the promoter is a yeast promoter. Examples of yeast promoters include but are not limited to AOX1, SAS, FLD1, ICL1, PH089, THI11, ADH1, ENOl, GUT1, GAP, TEF1, PGK1, GCW14, Gl, G6, etc.

In some embodiments, a promoter is an inducible promoter (e.g., induced in the presence of a small molecule, such as IPTG or tetracycline). Examples of inducible promoters include but are not limited to a promoter comprising a tetracycline responsive element (TRE), a pLac promoter, a pBad promoter, alcohol-regulated promoters (e.g., AlcA promoter), steroid-regulated promoters (e.g., LexA promoter), temperature-inducible promoters (e.g., Hsp70- or Hsp90-derived promoters, light-inducible promoters (e.g., YFI), etc. In some embodiments, an inducible promoter is a methanol-regulated promoter, for example AOX1 promoter as described by Yang et al. Scientific Reports (2018) 8:1401. In some embodiments, an AOX1 promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, an isolated nucleic acid comprises a transcription initiation sequence operably linked to nucleic acid sequences or genes of interest (e.g., a gene encoding a recombinant OxOx protein). In some embodiments, an isolated nucleic acid comprises a transcription terminator sequence. Examples of transcription terminator sequences are described by Vogl et al. (2014) ACS Synth Biol 3(3): 1880191. In some embodiments, an isolated nucleic acid comprises a transcriptional terminator sequence. Examples of transcriptional terminator sequences include but are not limited to AOX1, CYC1, TEF1, PGK1, FUM1, SED1, GND1, GYP7, CYC7, YGR127W, PSY4, GSY2, MRP4, VPS 13, TPS1, ECM10, Y0L036W, GRE3, HUG1, TIPI, AIP1, YJR085C, HSP26, PDC6, ADH1, UBX6, SPOl, GAT2, LSC2, PRM5, IDP1, SPG5, HIS5, CPS1, PRM9, etc. In some embodiments, the transcriptional terminator sequence is an AOX1 transcriptional terminator sequence, for example as described by Vogl et al. (2018) Applied and Environmental Microbiology 84 (6) e02712-17. An isolated nucleic acid described by the disclosure may further comprise a polyadenylation (poly A) sequence.

Aspects of the disclosure relate inclusion of one or more signal sequences (e.g., signal peptide sequences) in an expression construct encoding a recombinant OxOx protein. In some embodiments, a signal sequence is a secretion signal sequence. Secretion signals are typically short peptides that are present at the N-terminus of a protein and assist in directing the protein into the secretory pathway of a cell. In some embodiments, a signal sequence facilitates translocation of the protein across cellular membrane. A secretion signal may be a yeast secretion signal. Examples of yeast secretion signals include but are not limited to PHOl, a-MF, USC2, PHA-E, KILM1, pGKL, CLY-L8, SCW, DSE, EXG, Pirl, HBFI, etc. In some embodiments, a secretion signal is a yeast alpha-factor (a-factor) signal sequence. Yeast alpha-factor (a-factor), as described, for example by Lin-Cereghino et al. (2013) Gene 519(2):311-317. In some embodiments, a yeast a-factor signal sequence is encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO: 12.

Aspects of the disclosure relate to expression constructs comprising one or more (e.g., 1, 2, 3, 4, 5, or more) chimeric signal sequences. A “chimeric signal sequence” generally refers to a nucleic acid sequence encoding a signal peptide that comprises a first region from one gene and a second region from a different gene. For example, in the context of yeast secretion signals, a chimeric signal sequence may comprise a first nucleic acid sequence encoding a “pre” region of a first gene and a second nucleic acid sequence encoding a “pro” region of a second gene. In some embodiments, an expression construct comprises an Oligosaccharyl transferase subunit 1 (OST1) “pre” region, such as a S. cerevisiae “pre” region, or a variant thereof, for example as described by Barrero et al. Microb. Cell Fact. (2018) 17:161. A variant of a “pre” region may have 1, 2, 3, 4, 5, 6, or more amino acid substitutions relative to a wild-type “pre” region. In some embodiments, an OST1 “pre” region is encoded by the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, an OST1 “pre” region comprises the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, an expression construct comprises a yeast a-factor “pro” region or a variant thereof, for example as described by Rakestraw et al. Biotechnol Bioeng. 2009 August 15; 103(6): 1192-1201. doi:10.1002/bit.22338. In some embodiments, a “pro” region variant comprises a proapp-8 “pro” region. In some embodiments, a “pro” region is encoded by the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a “pro” region comprises the amino acid sequence set forth in SEQ ID NO: 24.

In some embodiments, an expression construct comprises a chimeric signal sequence comprising an OST1 “pre” region and a proapp-8 “pro” region. In some embodiments, an expression construct comprises a chimeric signal sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, an expression construct comprises a chimeric signal sequence having the amino acid sequence set forth in SEQ ID NO: 26.

In some embodiments, a secretion signal sequence is a non-yeast secretion signal (e.g., a secretion signal that is not naturally present in yeast or is heterologous to yeast). The disclosure is based, in part on the recognition that inclusion of one or more signal sequences that are not native to yeast (e.g., that are not naturally present in yeast, or are heterologous to yeast) in expression constructs described herein improves expression of recombinant OxOx protein in yeast cells relative to expression constructs that do not include the one or more signal sequences. In some embodiments, an expression construct described herein comprises a bacterial signal peptide sequence (e.g., a secretion signal sequence naturally present in bacteria) operably linked to a nucleic acid encoding a recombinant OxOx protein. In some embodiments, the non-yeast signal sequence is a twin-arginine translocation pathway (Tat pathway) signal sequence. In some embodiments, a Tat pathway signal sequence is a Pseudomonas aeruginosa, Legionella pneumophila, Yersinia pseudotuberculosis, or E. coll 0157:H7 Tat pathway signal sequence. In some embodiments, a Tat pathway signal sequence is encoded by the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a protein comprising a Tat pathway signal sequence is transported (or capable of being transported) across a cellular membrane in a folded state.

A non-yeast signal sequence may also be derived from the same organism as the recombinant gene of interest. For example, in some embodiments, an expression construct encoding recombinant OxOx protein may comprise a plant signal sequence. In some embodiments, the plant signal sequence is a barley or wheat plant signal sequence. In some embodiments, the plant signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 17 or 18 (or a nucleic acid encoding such a peptide). Additional examples of non-yeast signal sequences include but are not limited to twin arginine translocation (TAT) sequences, protein transduction domain (PTD) sequences, etc.

An expression construct may further comprise one or more nucleic acid sequences encoding a peptide purification tag. Examples of peptide purification tags include but are not limited to FLAG tag, c-myc epitope tag, polyhistidine (poly-His) tag, etc. The nucleic acid sequence encoding the purification tag may be positioned upstream (e.g. 5’ or N-terminal) or downstream (e.g., 3’ or C-terminal) with respect to the nucleic acid sequence encoding the recombinant OxOx protein. In some embodiments, an expression construct encoding a recombinant OxOx protein lacks a protein purification tag.

In some embodiments, an isolated nucleic acid engineered to express a protein is a component of a vector. Examples of vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. In some aspects, one or more isolated nucleic acids engineered to express a protein (e.g., recombinant OxOx protein, etc.) are located (e.g., situated) on a plasmid, for example a bacterial plasmid or yeast plasmid. In some embodiments, the vector is a high-copy plasmid. In some embodiments, the vector is a low-copy plasmid. In some embodiments, a yeast cell comprises one or more plasmids comprising the one or more isolated nucleic acids. For example, a plasmid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 isolated nucleic acids. In some embodiments, a plasmid comprises 1, 2, or 3 isolated nucleic acids. Methods

In some embodiments, the disclosure relates to methods of producing recombinant OxOx protein in a host cell as described by the disclosure. In some embodiments, the host cell is a bacterial cell, mammalian cell, insect cell, or yeast cell. In some embodiments, the host cell is a yeast cell. Yeast cell-based recombinant protein expression systems are known in the art and include, for example Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, and Schizosaccharomyces pombe expression systems, for example as described by Baghban et al. (2019) Mol Biotechnol 61(5):365- 384.

A yeast cell-based expression system can be a methylotrophic yeast expression system. In some embodiments, the methylotrophic yeast expression system is a Pichia pastoris (also referred to as Komagataella pastoris ) expression system. Pichia expression systems are known in the art and are described, for example, by Ahmad et al. (2014) Appl Microbiol Biotechnol 98(12):5301- 5317. In some embodiments, a Pichia yeast cell is selected from the following strains: CBS7435, CBS704, X-33, GS115, KM71, KM71H, BG09, GS190, GS200, JC220, JC220, JC254, JC227, JC300, JC301, JC202, JC303, JC304, JC305, JC306, JC307, JC308, YJN165, CBS7435 his4a, SMD1 163, BG21, etc. In some embodiments, a yeast cell is a Saccharomyces cerevisiae cell.

Typically, methods described by the disclosure comprise the steps: transforming a yeast cell with an isolated nucleic acid engineered to express a recombinant OxOx protein; and culturing (e.g., growing) the yeast cell. Methods of introducing vectors into microorganisms, such as yeast, are well known in the art and described, for example, in Current Protocols in Molecular Biology, Ausubel et al. (Eds), John Wiley and Sons, New York, 2007. In some embodiments of methods described by the disclosure, a yeast cell is transformed with one or more isolated nucleic acids comprising the sequence set forth in any one of SEQ ID NOs: 1, 2, 7-9, and 14-16.

The skilled artisan recognizes that the conditions (e.g., temperature, CO2 concentration, humidity, presence or absence of inducers, such as methanol, etc.) under which a recombinant OxOx protein is expressed by the yeast cell as described herein is maintained may affect the production and/or stability and/or function of the recombinant OxOx protein by the yeast cell(s). In some embodiments, a yeast cell transformed with an isolated nucleic acid as described herein is incubated at temperatures between 10 to 30 °C ( e.g ., 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C).

The length of time a yeast cell culture is grown after transformation with an isolated nucleic acid as described herein can vary. In some embodiments, a yeast cell is cultured for between about 10 hours and 120 (e.g., 10, 12, 24, 48, 72, 84, 96, etc.) hours after being transformed with an isolated nucleic acid as described herein. In some embodiments, the yeast cell is cultured in the presence of methanol for a portion (or all) of the culture period. In some embodiments, a yeast cell is cultured without methanol in complete medium to expand the yeast culture. In some embodiments, the yeast culture is expanded 10-fold every 24 hours. The length of this non methanol culture depends on vessel volume. After attaining the desired growth, the culture volume is reduced. In some embodiments, the culture volume is reduced between the ranges of about 2:1 to about 10:1. For example, a 500ml culture is reduced to 250ml, or as low as 50ml.

In some embodiments, the yeast culture is induced (e.g., induced by addition of methanol).

In some embodiments, induction media is a minimal media with 0.5% methanol. In some embodiments, induction media is added to the culture on a daily basis. In some embodiments, inducer (e.g., methanol) is continuously infused into a yeast culture. In some embodiments, methanol induction ranges from about 1 to 10 days.

The disclosure is based, in part, on the recognition that recombinant OxOx protein expressed in yeast cells surprisingly accumulates in the cell membrane of the yeast cells. Thus, methods described by the disclosure may further comprise a step of extracting, isolating, and/or purifying recombinant OxOx protein from the yeast cell by which it was produced. Recombinant OxOx protein can be separated or purified from yeast cells by any suitable methodology, for example physical separation or chemical separation. Examples of suitable separation methodologies include but are not limited to chromatography (affinity chromatography, high pressure liquid chromatography (HPLC), anion exchange chromatography, etc.), mass spectrometry (MS), electrophoresis (e.g., 2D or 3D gel electrophoresis, capillary electrophoresis, etc.), etc.

Cells (e.g., yeast cells) are typically lysed prior to isolation or purification of recombinant proteins (e.g., recombinant OxOx protein). In some embodiments, methods described by the disclosure further comprise the step of lysing yeast cells that have been transformed with an isolated nucleic acid encoding a recombinant OxOx protein. In some embodiments, the cells are lysed using a lysis buffer selected from CelLytic Y, YeastBuster Protein Extraction Reagent, Y-PER Yeast Protein Extraction Reagent, etc. In some embodiments, the cell lysate is separated from an insoluble fraction, which typically comprises cellular membrane components. In some embodiments, an insoluble fraction comprises recombinant OxOx proteins. In some embodiments, an insoluble fraction is treated with a detergent (e.g., sodium dodecaylsulfate, SDS) prior to a purification step.

In some embodiments, a recombinant OxOx protein (e.g., a cell lysate or insoluble fraction comprising a recombinant OxOx protein) is treated by anion exchange chromatography. Anion exchange chromatography is a process that separates molecules (e.g., proteins) from a sample based on their electro-chemical charges by contacting the sample to a positively-charged solid substrate (e.g., a resin or beads). Examples of anion exchange chromatography solid substrates include Diethylaminoethyl (DEAE), which is typically characterized as a “weak” anion exchanger, and quaternary ammonium (e.g., Q Sepharose), which is typically characterized as a “strong” anion exchanger. The disclosure is based, in part, on the recognition that recombinant OxOx proteins are characterized by several hydrophobic regions and that performing chromatography using “strong” anion exchangers provides improved purity of recombinant OxOx protein samples relative to “weak” anion exchangers. In some embodiments, a cell lysate or insoluble fraction comprising recombinant OxOx protein is first subjected to anion exchange chromatography using a “weak” anion exchanger, and subsequently subjected to anion exchange chromatography using a “strong” anion exchanger. In some embodiments, a recombinant OxOx protein is bound to an anion exchange solid substrate using a buffer having a pH ranged from 8.0-10.0 (e.g., 8, 8.5, 9, 9.5, or 10). In some embodiments, the buffer is pH 9.0. In some embodiments, recombinant OxOx protein is eluted from a substrate using a high salt” buffer, for example 1M NaCl. In some embodiments, a buffer comprises Tris buffer, phosphate buffer, sodium chloride buffer, glycine buffer, etc.

EXAMPLE

This example describes an expression system for recombinant oxalate oxidase (OxOx) protein using a yeast expression system.

A codon optimized synthetic OxOx gene derived from Hordeum vulgare (barley) was generated and cloned into the EcoRI/Xbal sites of the pPICZaA expression vector downstream of the N-terminal alpha mating factor secretion signal peptide sequence and used for expression in Pichia pastoris X-33 strain (FIG. 1A). This expression system is capable of generating disulfide bonds and addition of glycans required for the functional expression of the OxOx enzyme. FIG. IB shows confirmatory sequencing of the OxOx gene insert. FIG. 1C shows data from a NCBI nucleotide BLAST® alignment used to confirm the integrity of the inserted OxOx gene by comparing the recombinant OxOx sequence to Wild-type Hordeum vulgare oxalate oxidase nucleic acid sequence.

P. pastoris was transformed with the expression plasmid and induced to express active enzyme. Purification was carried out as follows: following protein expression, yeast cultures are centrifuged. Cell-free culture media is collected and dialyzed overnight against distilled water. Because the isoelectric point of the enzyme is approximately 5.5, an alkaline Tris buffer was used to induce a strong negative charge, allowing the recombinant OxOx enzyme to bind to a strong anion exchange column. The bound recombinant OxOx protein is eluted using 1M NaCl.

FIG. 2A shows presence of recombinant OxOx in the insoluble fraction of yeast lysate, indicating accumulation of recombinant protein in yeast cellular membranes. FIG. 2B shows. P. pastoris culture media sample western blotting using an anti-OxOx antibody. Data indicate the production of recombinant OxOx protein in the yeast system. Culture media was subsequently dialyzed overnight against distilled water. One advantage of using this yeast secretion system is that it functions as a first step in the process of protein purification. Since the isoelectric point (pi) of OxOx enzyme is approximately 5.5, anion exchange chromatography was used for purification (Q-sepharose: equilibration/binding with tris buffer pH 9.0 and elution with 1M NaCl). FIG. 2C shows purification of recombinant OxOx using the Q-sepharose anion exchange column. FIG. 2D shows the eluted fraction separated by SDS-PAGE. Total protein was visualized with Coomassie stain to determine purity. A composition having a purity of over 90% recombinant OxOx was produced. FIG. 3 shows Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis.

Following trypsin digestion, LC-MS/MS was used to confirm the identity of the major band from the Coomassie gel in FIG. 2C. A peptide that is unique to the barley-derived oxalate oxidase (OxOx) protein and that is not present in the P. pastoris proteome, “AGETFVIPR” (SEQ ID NO: 20), was identified with 100% accuracy. Kinetic assays were performed at optimal enzyme conditions as noted in FIGs. 4A-4B. Purified OxOx enzyme displayed standard Michaelis-Menten kinetics at substrate concentrations up to 400mM (after which substrate inhibition occurs) and had an estimated Km value of 256mM based on linear regression analysis using a Lineweaver-Burk plot.

One challenge of recombinant OxOx expression in yeast is that the expressed heterologous protein is often not secreted properly. For example, recombinant OxOx expressed in yeast cells often is trapped in subcellular compartments (e.g., endoplasmic reticulum, Golgi apparatus, etc.) instead of being secreted from the cells. Thus, recombinant OxOx expression vectors comprising alternative yeast secretion signals were tested. FIG. 5 shows a schematic representing expression vectors comprising yeast secretion signals. The top construct comprises a wild-type yeast a-factor secretion signal containing a “pre” region and a “pro” region upstream of a codon-optimized OxOx nucleic acid sequence. The a-factor secretion signal shown in the top vector comprises the sequence set forth in SEQ ID NO: 12. The bottom construct comprises a chimeric secretion signal containing a yeast OST “pre” region (SEQ ID NO: 21) and a yeast proapp-8 “pro” region (SEQ ID NO: 23) upstream of a codon-optimized OxOx nucleic acid sequence. The entire secretion signal sequence of the bottom construct is represented by SEQ ID NO: 25. Yeast cells were transformed with the OxOx expression constructs and protein secretion was measured by Western blot. Surprisingly, the expression construct comprising the chimeric secretion signal resulted in increased secretion of recombinant OxOx protein relative to the wild-type a-factor secretion signal (FIG. 6).

SEQUENCES

>Codon-optimized Hordeum vulgare OxOx nucleic acid sequence 1 no leader (SEQ ID NO:l)

TCTGACCCAGATCCATTGCAGGATTTCTGTGTTGCTGACTTGGACGGTAAGGCCGTTTC

TGTTAACGGTCACACTTGTAAGCCAATGTCTGAAGCTGGTGACGACTTCCTGTTCTCCT

CAAAGTTGACTAAGGCTGGTAACACCTCCACTCCAAACGGTTCTGCTGTTACCGAATTG

GACGTTGCTGAATGGCCTGGAACTAACACTTTGGGTGTCTCCATGAACAGAGTTGACTT

TGCTCCAGGTGGTACAAACCCACCACATATTCATCCAAGAGCTACCGAGATCGGTATG

GTCATGAAGGGTGAGTTGTTGGTCGGTATCTTGGGTTCTTTGGACTCCGGTAACAAGCT

GTACTCCAGAGTTGTTAGAGCCGGTGAGACTTTCGTTATCCCAAGAGGTTTGATGCACT TCCAGTTCAACGTTGGTAAGACCGAGGCCTACATGGTTGTGTCCTTCAACTCTCAAAAC

CCCGGTATCGTTTTCGTCCCATTGACTTTGTTCGGTTCTGACCCACCAATTCCAACTCCA

GTTTTGACCAAGGCTTTGAGAGTCGAGGCTGGTGTTGTTGAATTGCTGAAGTCTAAGTT

CGCCGGTGGTTCC

>Codon-optimized Hordeum vulgare OxOx nucleic acid sequence 2 with leader (SEQ ID NO: 2)

AGTGACCCTGACCCACTTCAGGACTTCTGTGTGGCTGACCTGGACGGAAAAGCTGTGTC

TGTAAACGGCCATACGTGCAAACCTATGTCAGAGGCTGGTGATGACTTCCTGTTCTCTT

CT AAACT AACT AAGGC AGGTAAC ACC AGT ACCCCC AATGGAAGTGCCGT AAC AGAATT

GGATGTAGCTGAATGGCCTGGAACAAACACGTTGGGCGTTAGTATGAATAGGGTAGAC

TTCGCACCTGGTGGAACCAATCCCCCTCATATACACCCACGTGCAACGGAAATTGGCAT

GGTGATGAAGGGCGAGCTGCTTGTAGGCATACTTGGAAGTCTGGACTCTGGCAATAAA

CTATACTCCAGAGTCGTCAGAGCAGGAGAAACTTTCGTCATTCCCAGAGGACTTATGC

ACTTCCAGTTCAACGTAGGCAAGACGGAAGCATATATGGTCGTCAGTTTCAACAGTCA

GAACCCTGGCATTGTATTCGTTCCATTAACTCTGTTTGGCAGTGATCCCCCAATCCCAA

CCCCTGTTTTAACAAAAGCCTTGAGAGTAGAAGCAGGCGTCGTGGAATTATTGAAGAG

TAAATTCGCAGGTGGCTCA

>Wild-type Hordeum vulgare oxalate oxidase nucleic acid sequence no leader (SEQ ID NOG)

TCCGACCCAGACCCACTCCAGGACTTCTGCGTCGCGGACCTCGATGGCAAGGCGGTCT

CGGTGAACGGGCATACGTGTAAGCCCATGTCGGAGGCCGGCGACGACTTCCTCTTCTC

GTCCAAGCTGACCAAGGCCGGCAACACGTCCACCCCGAACGGCTCGGCCGTGACGGAG

CTCGACGTGGCCGAGTGGCCCGGTACGAACACGCTGGGCGTGTCCATGAACCGTGTGG

ACTTCGCGCCGGGGGGCACCAACCCGCCGCACATCCACCCGCGTGCAACCGAGATCGG

CATGGTGATGAAAGGTGAGCTCCTCGTTGGAATCCTCGGCAGCCTTGACTCCGGAAAC

AAGCTCTACTCCAGGGTGGTGCGTGCCGGAGAGACTTTCGTCATCCCGCGCGGCCTCAT

GCACTTCCAGTTCAACGTTGGTAAGACGGAAGCCTACATGGTTGTGTCCTTCAACAGCC

AGAACCCTGGCATCGTCTTCGTGCCGCTCACACTCTTCGGCTCCGACCCTCCCATCCCC

ACGCCCGTGCTCACCAAGGCTCTCCGGGTGGAGGCCGGAGTCGTGGAACTTCTCAAGT

CCAAGTTCGCCGGTGGGTCT >Wild-type Hordeum vulgare oxalate oxidase nucleic acid sequence with leader (SEQ ID NO: 4)

ATGGGTTACTCTAAAAACCTAGGGGCTGGCCTGTTCACCATGCTGCTCCTTGCTCCGGC

CATCATGGCTACCGACCCTGACCCTCTACAGGACTTCTGCGTCGCGGACCTCGATGGCA

AGGCGGTCTCGGTGAACGGGCATACGTGTAAGCCCATGTCGGAGGCCGGCGACGACTT

CCTCTTCTCGTCCAAGCTGACCAAGGCCGGCAACACGTCCACCCCGAACGGCTCGGCC

GTGACGGAGCTCGACGTGGCCGAGTGGCCCGGTACGAACACGCTGGGCGTGTCCATGA

ACCGTGTGGACTTCGCGCCGGGCGGCACCAACCCGCCGCACATCCACCCGCGTGCAAC

CGAGATCGGCATGGTGATGAAAGGTGAGCTCCTCGTTGGAATCCTCGGCAGCTTTGAC

TCCGGAAACAAGCTCTACTCCAGGGTGGTGCGTGCCGGAGAGACTTTCGTCATCCCGC

GCGGCCTCATGCACTTCCAGTTCAACGTTGGTAAGACGGAAGCCTACATGGTTGTGTCC

TTCAACAGCCAGAACCCTGGCATCGTCTTCGTGCCGCTCACACTCTTCGGTTCCAACCC

GCCCATCCCCACACCGGTGCTCACCAAGGCTCTTCGGGTGGAGGCCGGGGTCGTGGAA

CTTCTCAAGTCCAAGTTCGCCGGTGGGTCT

>H. vulgare Oxalate Oxidase amino acid sequence no leader (SEQ ID NO: 5) SDPDPLQDFCVADLDGKAVSVNGHTCKPMSEAGDDFLFSSKLTKAGNTSTPNGSAVTELD V AEWPGTNTFG V S MNRVDF APGGTNPPHIHPR ATEIGM VMKGEFFV GIFGS EDS GNKF Y S RVVRAGETFVIPRGLMHFQFNV GKTE A YM VVSFN S QNPGIVFVPLTLFGSDPPIPTPVLTKA LRVEAGVVELLKS KFAGGS

>H. vulgare Oxalate Oxidase amino acid sequence with leader (SEQ ID NO: 6) MGYSKNLGAGLFTMLLLAPAIMATDPDPLQDFCVADLDGKAVSVNGHTCKPMSEAGDDF LFS S KLTKAGNT S TPN GS A VTELD V AE WPGTNTLG V S MNRVDF APGGTNPPHIHPRATEIG M VMKGELLV GILGSFDS GNKLYSRVVRAGETFVIPRGLMHFQFNV GKTE A YM VV S FN S QN PGIVFVPLTLFGSNPPIPTPVLTKALRVEAGVVELLKS KFAGGS

>Wild-type Triticum aestivum oxalate oxidase nucleic acid sequence no leader (SEQ ID NO: 7)

GTCTTGGCCACCGACCCAGACCCTCTCCAGGACTTCTGTGTCGCCGACCTCGACGGCAA

GGCGGTCTCGGTGAACGGGCACACGTGCAAGCCCATGTCGGAGGCCGGCGACGACTTC CTCTTCTCGTCCAAGTTGGCCAAGGCCGGCAACACGTCCACCCCGAACGGCTCCGCCGT

GACGGAGCTCGACGTGGCCGAGTGGCCCGGTACCAACACGCTGGGTGTGTCCATGAAC

CGCGTGGACTTTGCTCCCGGAGGCACCAACCCACCACACATCCACCCGCGTGCCACCG

AGATCGGCATCGTGATGAAAGGTGAGCTTCTCGTGGGAATCCTTGGCAGCCTCGACTC

CGGGAACAAGCTCTACTCGAGGGTGGTGCGCGCCGGAGAGACGTTCCTCATCCCACGG

GGCCTCATGCACTTCCAGTTCAACGTCGGTAAGACCGAGGCCTCCATGGTCGTCTCCTT

CAACAGCCAGAACCCCGGCATTGTCTTCGTGCCCCTCACGCTCTTCGGCTCCAACCCGC

CCATCCCAACGCCGGTGCTCACCAAGGCACTCCGGGTGGAGGCCAGGGTCGTGGAACT

TCTCAAGTCCAAGTTTGCCGCTGGGTTT

>Wild-type Triticum aestivum oxalate oxidase nucleic acid sequence with leader (SEQ ID NO: 8)

ATGGGGTACTCCAAAACCCTAGTAGCTGGCCTGTTCGCAATGCTGTTACTAGCTCCGGC

CGTCTTGGCCACCGACCCAGACCCTCTCCAGGACTTCTGTGTCGCCGACCTCGACGGCA

AGGCGGTCTCGGTGAACGGGCACACGTGCAAGCCCATGTCGGAGGCCGGCGACGACTT

CCTCTTCTCGTCCAAGTTGGCCAAGGCCGGCAACACGTCCACCCCGAACGGCTCCGCCG

TGACGGAGCTCGACGTGGCCGAGTGGCCCGGTACCAACACGCTGGGTGTGTCCATGAA

CCGCGTGGACTTTGCTCCCGGAGGCACCAACCCACCACACATCCACCCGCGTGCCACC

GAGATCGGCATCGTGATGAAAGGTGAGCTTCTCGTGGGAATCCTTGGCAGCCTCGACT

CCGGGAACAAGCTCTACTCGAGGGTGGTGCGCGCCGGAGAGACGTTCCTCATCCCACG

GGGCCTCATGCACTTCCAGTTCAACGTCGGTAAGACCGAGGCCTCCATGGTCGTCTCCT

TCAACAGCCAGAACCCCGGCATTGTCTTCGTGCCCCTCACGCTCTTCGGCTCCAACCCG

CCCATCCCAACGCCGGTGCTCACCAAGGCACTCCGGGTGGAGGCCAGGGTCGTGGAAC

TTCTCAAGTCCAAGTTTGCCGCTGGGTTT

>Wild-type Triticum aestivum oxalate oxidase codon optimized nucleic acid sequence (SEQ ID NO: 9)

ACCGACCCTGATCCACTTCAAGACTTCTGTGTGGCAGACTTGGATGGTAAGGCCGTGTC AGT A A AT GGT C AC ACTT GCA A ACC A AT G AGT G A AGCT GG AG ATG ATTT CTT ATTT AGT A GTAAGTTGGCTAAAGCTGGCAACACCTCTACACCTAACGGTTCCGCAGTCACCGAGCT AGACGTAGCAGAATGGCCAGGAACAAATACGTTAGGCGTCTCAATGAACAGGGTGGA TTTCGCCCCTGGAGGTACGAACCCCCCACATATCCACCCTCGTGCTACCGAAATTGGAA

TCGTGATGAAGGGCGAACTACTGGTCGGAATCCTAGGCAGTTTGGATTCCGGTAACAA

ACTTTATTCTCGTGTCGTTAGAGCAGGTGAAACCTTCTTAATTCCAAGGGGTTTAATGC

ACTTTCAATTCAATGTCGGTAAGACGGAAGCCTCAATGGTGGTCTCTTTCAACTCTCAG

AATCCAGGTATCGTTTTTGTTCCTCTGACATTATTCGGTTCCAACCCTCCTATACCCACG

CCTGTGCTTACAAAAGCCTTGAGGGTAGAAGCCCGTGTAGTGGAGCTATTAAAAAGTA

AGTTTGCCGCAGGTTTC

> T. aestivum Oxalate Oxidase amino acid sequence no leader (SEQ ID NO: 10)

TDPDPLQDFC V ADLDGKA V S VN GHTCKPMS E AGDDFLF S S KLAK AGNT S TPN GS A VTELD V AEWPGTNTFG V S MNRVDF APGGTNPPHIHPR ATEIGI VMKGEFF V GIFGS EDS GNKF Y S R VVRAGETFLIPRGLMHFQFNVGKTEASMVVSFNSQNPGIVFVPLTLFGSNPPIPTPVLTKAL RYE ARVVELLKS KFAAGF

> T. aestivum Oxalate Oxidase amino acid sequence with leader (SEQ ID NO: 11) MGYSKTLVAGLFAMLLLAPAVLATDPDPLQDFCVADLDGKAVSVNGHTCKPMSEAGDDF LFS S KLAKAGNT S TPNGS A VTELD V AEWPGTNTLG V S MNRVDF APGGTNPPHIHPRATEIGI VMKGELL V GILGS LDS GNKL Y S RV VR AGETFLIPRGLMHF QFN VGKTE AS M VV S FN S QNP GIVFVPLTLFGSNPPIPTPVLTKALRVE ARVVELLKS KFAAGF

>S. cerevisiae wild-type a-factor signal nucleic acid sequence; pre region is underlined and pro region is bolded (SEQ ID NO: 12)

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCA

TCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAAC

AGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAA

AGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCT

>AOXl promoter nucleic acid sequence (SEQ ID NO: 13) GATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACA

GGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGAC

CGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAA

AACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGC

TACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTT

GTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGC

TTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTT

AAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAG

TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGC

ATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCT

TAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGG

GAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCT

GGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCT

ACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATC

ATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTA

ACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACG

> H. vulgare OxOx Expression Construct 1 nucleic acid sequence (SEQ ID NO: 14)

AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCAC

AGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGA

CCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAA

AAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGG

CT ACT AAC ACC ATGACTTT ATT AGCCTGTCT ATCCTGGCCCCCCTGGCGAGGTTC ATGTT

TGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGG

CTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTT

TAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAA

GTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGG

CATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATG

CTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATG

GGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGAT TCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACC

CCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTT

ATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATT

TTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACGA

TGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC

CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGG

TTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAA

ATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGG

GTATCTCTCGAGAAAAGAGAGGCTGAAGCTGAATTCAGTGACCCTGACCCACTTCAGG

ACTTCTGTGTGGCTGACCTGGACGGAAAAGCTGTGTCTGTAAACGGCCATACGTGCAA

ACCTATGTCAGAGGCTGGTGATGACTTCCTGTTCTCTTCTAAACTAACTAAGGCAGGTA

ACACCAGTACCCCCAATGGAAGTGCCGTAACAGAATTGGATGTAGCTGAATGGCCTGG

AACAAACACGTTGGGCGTTAGTATGAATAGGGTAGACTTCGCACCTGGTGGAACCAAT

CCCCCTCATATACACCCACGTGCAACGGAAATTGGCATGGTGATGAAGGGCGAGCTGC

TTGTAGGCATACTTGGAAGTCTGGACTCTGGCAATAAACTATACTCCAGAGTCGTCAGA

GCAGGAGAAACTTTCGTCATTCCCAGAGGACTTATGCACTTCCAGTTCAACGTAGGCA

AGACGGAAGCATATATGGTCGTCAGTTTCAACAGTCAGAACCCTGGCATTGTATTCGTT

CCATTAACTCTGTTTGGCAGTGATCCCCCAATCCCAACCCCTGTTTTAACAAAAGCCTT

G AG AGT AG A AGC AGGCGTCGT GG A ATT ATT G A AG AGT A A ATT C GC AGGT GGCT CAT G A

TCTAGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCAT

CATCATCATTGAGTTTGTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTT

ACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCT

GAGAGATGC AGGCTTC ATTTTTGAT ACTTTTTT ATTTGT AACCT AT AT AGT AT AGGATTT

TTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTG

ATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTA

TTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCGGATCCC

CCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGA

CTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTC

TTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAG

ACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTT TTCTTGAAATTTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATATTTAAGTT

AATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTT

TTTACTTCTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGGCGGTGTTGAC

AATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAA

CCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGC

GGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTC

GCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGG

TGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGA

GTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAG

ATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCG

TGCACTTCGTGGCCGAGGAGCAGGACTGACACGTCCGACGGCGGCCCACGGGTCCCAG

GCCTCGGAGATCCGTCCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCT

TACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACC

TG A AGT CT AGGT CCCT ATTT ATTTTTTT AT AGTT AT GTT AGT ATT A AG A AC GTT ATTT AT

ATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAA

AACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACC

AACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTG

GCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCA

GAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC

CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCT

TCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT

CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCT

TATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCA

GCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCT

TGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTG

CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA

CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGG

ATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACT

CACGTTAAGGGATTTTGGTCATGAGATC > H. vulgare OxOx Expression Construct 2 nucleic acid sequence (SEQ ID NO: 15)

AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCAC

AGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGA

CCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAA

AAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGG

CT ACT AAC ACC ATGACTTT ATT AGCCTGTCT ATCCTGGCCCCCCTGGCGAGGTTC ATGTT

TGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGG

CTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTT

TAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAA

GTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGG

CATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATG

CTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATG

GGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGAT

TCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACC

CCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTT

ATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATT

TTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACGA

TGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC

CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGG

TTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAA

ATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGG

GTATCTCTCGAGAAAAGAGAGGCTGAAGCTGAATTCTCTGACCCAGATCCATTGCAGG

ATTTCTGTGTTGCTGACTTGGACGGTAAGGCCGTTTCTGTTAACGGTCACACTTGTAAG

CCAATGTCTGAAGCTGGTGACGACTTCCTGTTCTCCTCAAAGTTGACTAAGGCTGGTAA

CACCTCCACTCCAAACGGTTCTGCTGTTACCGAATTGGACGTTGCTGAATGGCCTGGAA

CTAACACTTTGGGTGTCTCCATGAACAGAGTTGACTTTGCTCCAGGTGGTACAAACCCA

CCACATATTCATCCAAGAGCTACCGAGATCGGTATGGTCATGAAGGGTGAGTTGTTGG

TCGGTATCTTGGGTTCTTTGGACTCCGGTAACAAGCTGTACTCCAGAGTTGTTAGAGCC

GGTGAGACTTTCGTTATCCCAAGAGGTTTGATGCACTTCCAGTTCAACGTTGGTAAGAC

CGAGGCCTACATGGTTGTGTCCTTCAACTCTCAAAACCCCGGTATCGTTTTCGTCCCATT GACTTTGTTCGGTTCTGACCCACCAATTCCAACTCCAGTTTTGACCAAGGCTTTGAGAG

TCGAGGCTGGTGTTGTTGAATTGCTGAAGTCTAAGTTCGCCGGTGGTTCCTAATCTAGA

ACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCAT

CATTGAGTTTGTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGA

AGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGA

TGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGT

CATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATA

TCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCAC

TCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCGGATCCCCCACACAC

CATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGC

ATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTCTTTCTTCC

TCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCT

CGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGA

A ATTTTTTTTTTT AGTTTTTTT CTCTTT C AGT G ACCT CC ATT GAT ATTT A AGTT A AT A A AC

GGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTT

CTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGGCGGTGTTGACAATTAA

TCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGG

CCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGA

GTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTG

TGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGA

CAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCG

GAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCG

AGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTT

CGTGGCCGAGGAGCAGGACTGACACGTCCGACGGCGGCCCACGGGTCCCAGGCCTCGG

AGATCCGTCCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTC

ACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTC

T AGGT CCCT ATTT ATTTTTTT AT AGTT AT GTT AGT ATT A AG A AC GTT ATTT AT ATTT C A A

_{ATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTG}

CTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGT

GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG

CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGC

GCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAA

GCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC

TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG

TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC

ACT GGT A AC AGG ATT AGC AG AGC G AGGT AT GT AGGC GGT GCT AC AG AGTTCTTG A AGT

GGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAA

GCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCT

GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTC

AAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGT

TAAGGGATTTTGGTCATGAGATC

> H. vulgare OxOx Expression Construct 3 nucleic acid sequence (SEQ ID NO: 16)

AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCAC

AGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGA

CCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAA

AAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGG

CT ACT AAC ACC ATGACTTT ATT AGCCTGTCT ATCCTGGCCCCCCTGGCGAGGTTC ATGTT

TGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGG

CTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTT

TAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAA

GTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGG

CATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATG

CTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATG

GGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGAT

TCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACC

CCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTT

ATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATT

TTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACGA TGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC

CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGG

TTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAA

ATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGG

GTATCTCTCGAGAAAAGAGAGGCTGAAGCTGAATTCATGGGTTACTCTAAAAACCTAG

GGGCTGGCCTGTTCACCATGCTGCTCCTTGCTCCGGCCATCATGGCTACCGACCCTGAC

CCTCTACAGGACTTCTGCGTCGCGGACCTCGATGGCAAGGCGGTCTCGGTGAACGGGC

ATACGTGTAAGCCCATGTCGGAGGCCGGCGACGACTTCCTCTTCTCGTCCAAGCTGACC

AAGGCCGGCAACACGTCCACCCCGAACGGCTCGGCCGTGACGGAGCTCGACGTGGCCG

AGTGGCCCGGTACGAACACGCTGGGCGTGTCCATGAACCGTGTGGACTTCGCGCCGGG

CGGCACCAACCCGCCGCACATCCACCCGCGTGCAACCGAGATCGGCATGGTGATGAAA

GGTGAGCTCCTCGTTGGAATCCTCGGCAGCTTTGACTCCGGAAACAAGCTCTACTCCAG

GGTGGTGCGTGCCGGAGAGACTTTCGTCATCCCGCGCGGCCTCATGCACTTCCAGTTCA

ACGTTGGTAAGACGGAAGCCTACATGGTTGTGTCCTTCAACAGCCAGAACCCTGGCAT

CGTCTTCGTGCCGCTCACACTCTTCGGTTCCAACCCGCCCATCCCCACACCGGTGCTCA

CCAAGGCTCTTCGGGTGGAGGCCGGGGTCGTGGAACTTCTCAAGTCCAAGTTCGCCGG

TGGGTCTTAATCTAGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGA

CCATCATCATCATCATCATTGAGTTTGTAGCCTTAGACATGACTGTTCCTCAGTTCAAGT

TGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGC

CATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGT

ATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCT

CGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTT

TCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTG

CGGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATT

TTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAAT

TTTCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAA

AAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCAC

GTTTCTTTTTCTTGAAATTTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATAT

TTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTAC

AACTTTTTTTACTTCTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGGCGG TGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGG

AACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCG

CCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGA

CGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGAC

CAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGT

ACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCAT

GACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGG

CAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTCCGACGGCGGCCCACG

GGTCCCAGGCCTCGGAGATCCGTCCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTAT

GTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTA

GACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGT

TATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTA

TACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTG

GAGACCAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCG

TTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC

AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA

AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT

CTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGT

GTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT

GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA

CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG

AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGC

GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAAC

AAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA

AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACG

AAAACTCACGTTAAGGGATTTTGGTCATGAGATC

> H. vulgare OxOx signal peptide sequence (SEQ ID NO: 17) MGY S KNLGAGLFTMLLLAPAIMA > T. aestivum OxOx signal peptide sequence (SEQ ID NO: 18) MGY S KTLVAGLFAMLLLAPA VLA

>Bacterial Tat pathway signal peptide sequence (SEQ ID NO: 19) SRRQFLK

>OxOx peptide sequence (SEQ ID NO: 20) AGETFVIPR

>S. cerevisiae OST1 pre-region encoding nucleic acid sequence (SEQ ID NO: 21)

ATGAGGCAGGTTTGGTTCTCTTGGATTGTGGGATTGTTCCTATGTTTTTTCAACGTGTCT

TCT

>S. cerevisiae OST1 pre-region signal peptide sequence (SEQ ID NO: 22) MRQVWFS WIV GLFLCFFNV S S

>S. cerevisiae mutant a-factor pro-region (“proapp-8”) encoding nucleic acid sequence (SEQ ID NO: 23)

GCTGCTCCAGCTAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTG

TCATCGATTACTCAGATTTAGAAGGGGATTTCGATGATGCTGATTTGCCATTGTCCAAC

AGCACAAATAACGGGTTATCGTCTACAAATACTACTATTGCCAGCATTGCTGCTAAAG

AAGAAGGGGTATCTCTCGAGTCTAGAGAGGCTGAAGCT

> S. cerevisiae mutant a-factor pro-region signal peptide sequence (SEQ ID NO: 24)

A AP ANTTTEDET AQIP AE A VID Y S DLEGDFDD ADLPLS N S TNN GLS S TNTTIAS IA AKEEG V S LESREAEA

>Chimeric signal peptide encoding nucleic acid sequence (SEQ ID NO: 25)

ATGAGGCAGGTTTGGTTCTCTTGGATTGTGGGATTGTTCCTATGTTTTTTCAACGTGTCT

TCTGCTGCTCCAGCTAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAG CTGTCATCGATTACTCAGATTTAGAAGGGGATTTCGATGATGCTGATTTGCCATTGTCC

AACAGCACAAATAACGGGTTATCGTCTACAAATACTACTATTGCCAGCATTGCTGCTAA

AGAAGAAGGGGTATCTCTCGAGTCTAGAGAGGCTGAAGCT >Chimeric signal peptide sequence (SEQ ID NO: 26)

MRQ VWFS WIV GLFLCFFN V S S A AP ANTTTEDET AQIP AE A VID Y S DLEGDFDD ADLPLS N S TNN GLS S TNTTIAS IA AKEEG V S LES RE AE A

>S. cerevisiae wild-type a-factor secretion signal peptide (SEQ ID NO: 27) MRFPS IFT A VLFA AS S AL A AP VNTTTEDET AQIP AE A VIG Y S DLEGDFD V A VLPFS N S TNN G LLFINTTIASIAAKEEGVSLEKREAEA

Claims

CLAIMS What is claimed is:

1. An isolated nucleic acid comprising an expression construct comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9.

2. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid encodes a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11.

3. The isolated nucleic acid of claim 1 or 2, wherein the expression construct further comprises an alpha factor (a-factor) signal sequence operably linked to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9.

4. The isolated nucleic acid of claim 3, wherein the a-factor signal sequence is positioned 5’ relative to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9.

5. The isolated nucleic acid of claim 3 or 4, wherein the a-factor signal sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 12.

6. The isolated nucleic acid of any one of claims 1 to 5 further comprising a non-yeast signal sequence, optionally wherein the non-yeast signal sequence encodes a peptide comprising the sequence set forth in any one of SEQ ID NOs: 17-19.

7. The isolated nucleic acid of any one of claims 1 to 6, wherein the expression construct further comprises one or more purification tag sequences positioned 3’ relative to the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9.

8. The isolated nucleic acid of any one of claims 1 to 7, wherein the expression construct further comprises a promoter operably linked to the a-factor signal sequence or the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9, optionally wherein the promoter is an AOX1 promoter.

9. The isolated nucleic acid of any one of claims 1 to 8, wherein the expression construct further comprises a transcription terminator sequence, optionally wherein the transcription terminator sequence is an AOX1 transcription terminator sequence.

10. A plasmid comprising the isolated nucleic acid of any one of claims 1 to 9.

11. The plasmid of claim 10, wherein the plasmid comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 14-16.

12. A host cell comprising the isolated nucleic acid of any one of claims 1 to 9, or the plasmid of claim 10 or 11.

13. The host cell of claim 12 , wherein the host cell is a yeast cell.

14. The host cell of claim 13, wherein the yeast cell is a Pichia pastoris cell.

15. A container housing a population of yeast cells, wherein at least one yeast cell comprises the isolated nucleic acid of any one of claims 1 to 9 or the plasmid of claim 10 or 11.

16. An isolated nucleic acid comprising an expression construct comprising a nucleic acid sequence encoding an oxalate oxidase (OxOx) protein operably linked to a non-yeast protein transduction domain sequence.

17. The isolated nucleic acid of claim 16, wherein the OxOx protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11.

18. The isolated nucleic acid of claim 16 or 17, wherein the nucleic acid sequence encoding a protein comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 7-9.

19. The isolated nucleic acid of any one of claims 16 to 18, wherein the protein transduction domain sequence comprises bacterial transduction domain sequence, optionally a twin- arginine translocation (Tat) pathway signal sequence.

20. The isolated nucleic acid of claim 19, wherein the Tat pathway signal sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 19.

21. The isolated nucleic acid of any one of claims 16 to 20, wherein the expression construct further comprises a promoter operably linked to the nucleic acid sequence encoding a protein, optionally an AOX1 promoter.

22. A plasmid comprising the isolated nucleic acid of any one of claims 16 to 21.

23. The plasmid of claim 22 comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 14-16.

24. A host cell comprising the isolated nucleic acid of any one of claims 16 to 21, or the plasmid of claim 22 or 23.

25. The host cell of claim 24 , wherein the host cell is a yeast cell.

26. The host cell of claim 25, wherein the yeast cell is a Pichia pastoris cell.

27. A container housing a population of yeast cells, wherein at least one yeast cell comprises the isolated nucleic acid of any one of claims 16 to 21 or the plasmid of claim 22 or 23.

28. A method for producing a recombinant protein, the method comprising introducing into a cell the isolated nucleic acid of any one of claims 1 to 9 or 16 to 21 under conditions under which the cell expresses a recombinant oxalate oxidase (OxOx) protein.

29. The method of claim 28, wherein the cell is a yeast cell, optionally wherein the yeast cell is a Pichia pastoris cell.

30. The method of claim 28 or 29, wherein the recombinant OxOx protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11, or is encoded by the nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 2 and 7-9.

31. The method of any one of claims 28 to 30 further comprising purifying the recombinant OxOx protein from the cell.

32. The method of claim 31, wherein the purifying comprises lysing the cell to produce cell lysate and an insoluble fraction.

33. The method of claim 32, wherein the purifying comprises contacting the cell lysate or insoluble fraction with an anion exchange media, optionally wherein the anion exchange media comprises a Q-sepharose anion exchange column under conditions under which the recombinant OxOx protein binds to the column.

34. The method of claim 33, wherein the method further comprises eluting the bound recombinant OxOx protein from the column to produce a purified protein fraction.

35. The method of claim 34, wherein the purified protein fraction comprises >90% recombinant OxOx protein.

36. An isolated nucleic acid comprising a nucleic acid sequence encoding a protein comprising a chimeric secretion signal having the amino acid sequence set forth in SEQ ID NO: 26.

37. The isolated nucleic acid of claim 36, wherein the chimeric secretion signal comprises a pre region encoded by the nucleic acid sequence set forth in SEQ ID NO: 21.

38. The isolated nucleic acid of claim 36 or 37, wherein the chimeric secretion signal comprises a pro region encoded by the nucleic acid sequence set forth in SEQ ID NO: 23.

39. The isolated nucleic acid of any one of claims 36 to 38, wherein the protein comprises an oxalate oxidase (OxOx) protein.

40. The isolated nucleic acid of claim 39, wherein the OxOx protein is a barley OxOx protein.

41. The isolated nucleic acid of any one of claims 36 to 40, wherein the protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, and 11.

42. The isolated nucleic acid of any one of claims 36 to 41 further comprising a promoter operably linked to the nucleic acid sequence encoding the protein.

43. The isolated nucleic acid of claim 42, wherein the promoter comprises an AOX1 promoter.

44. The isolated nucleic acid of any one of claims 36 to 43 further comprising a transcription terminator sequence, optionally wherein the transcription terminator sequence is an AOX1 transcription terminator sequence.

45. A protein comprising a chimeric secretion signal having the amino acid sequence set forth in SEQ ID NO: 26.

46. The protein of claim 45, wherein the protein comprises an oxalate oxidase protein.

47. The protein of claim 45 or 46, wherein the protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 6, 10, or 11.

48. The protein of any one of claims 45 to 47, wherein the protein further comprises a purification tag.

49. A cell comprising the isolated nucleic acid of any one of claims 36 to 44 or the protein of any one of claims 45 to 48.

50. The cell of claim 49, wherein the cell is a yeast cell, optionally a Pichia pastoris cell.