MODIFICATION OF PROTEIN GLYCOSYLATION IN MICROORGANISMS

Abstract

The present disclosure contemplates methods for modifying post-translational modification of proteins recombinantly expressed a microbial host to improve one or more properties of the recombinant protein.

Claims

1.-41. (canceled)

42. A method of producing a consumable composition comprising: a. recombinantly expressing a nutritional protein in a host cell, wherein the nutritional protein is secreted from of the host cell; b. recombinantly expressing an α-1,2-mannosidase in the host cell; wherein the α-1,2-mannosidase reduces the glycosylation of greater than 50% of the nutritional protein secreted from the host cell and, wherein the nutritional protein with reduced glycosylation is mixed with at least one more component to form the consumable composition.

43. The method of claim 42, wherein the α-1,2-mannosidase has a sequence of SEQ ID No: 7, a functional equivalent thereof or a sequence 85% or more identical to SEQ ID No: 7.

44. The method of claim 42, wherein the α-1,2-mannosidase has a sequence of SEQ ID No: 150, a functional equivalent thereof or a sequence 85% or more identical to SEQ ID No: 150.

45. The method of claim 42, wherein the nutritional content of the consumable composition is equal to or greater than the nutritional content of a control composition wherein the control composition is produced using the same protein isolated from a native source or the recombinant nutritional protein un-modified by the α-1,2-mannosidase.

46. The method of claim 45, wherein the nutritional content is a protein content of the composition.

47. The method of claim 46, wherein the protein content of the consumable composition is at least 5%, at least 10% or at least 20% higher than the control composition.

48. The method of claim 42, wherein at least 75% of the nutritional protein secreted from the host cell has reduced glycosylation as compared to a control protein wherein the control protein is isolated from a native source or is the recombinant nutritional protein un-modified by the α-1,2-mannosidase.

49. The method of claim 48, wherein at least 80% of the nutritional protein secreted from the host cell has reduced glycosylation as compared to the control protein.

50. The method of claim 49, wherein at least 90% of the nutritional protein secreted from the host cell has reduced glycosylation as compared to the control protein.

51. The method of claim 42, wherein a thermal stability of the nutritional protein is increased as compared to a control composition wherein the control composition is produced using the same protein isolated from a native source or the recombinant nutritional protein un-modified by the α-1,2-mannosidase.

52. The method of claim 42, wherein the host cell is Pichia pastoris.

53. The method of claim 42, wherein the nitrogen to carbon ratio of the nutritional protein is equal to or greater than the ratio of the nutritional protein isolated from its native source.

54. The method of claim 42, wherein the nutritional protein is an animal or avian protein.

55. A consumable composition produced using the method of claim 42

56. The consumable composition of claim 55, wherein the composition is a beverage.

57. The consumable composition of claim 55, wherein the composition is a foodstuff.

58. A host cell used for the expression of a recombinant nutritional protein comprising: c. a first promoter driving expression of a nutritional protein; d. a second promoter driving expression of an α-1,2-mannosidase with sequence of SEQ ID Nos: 7 or 150, a functional equivalent thereof or a sequence 85% or more identical to SEQ ID Nos: 7 or 150; wherein the mannosylation of the nutritional protein is reduced as a result of the expression of the α-1,2-mannosidase.

59. The host cell of claim 58, wherein the host cell is Pichia pastoris.

60. The host cell of claim 58, wherein the nutritional protein and the α-1,2-mannosidase are expressed using one or more expression cassettes.

61. The host cell of claim 58, wherein the nutritional protein and the α-1,2-mannosidase are expressed on separate expression constructs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0045] FIGS. 1A-1D illustrate Man.sub.xGlcNAc.sub.2 substructures.

[0046] FIG. 2 illustrates an exemplary vector comprising a promoter operably linked to a transgene.

[0047] FIGS. 3A-B illustrate mass spectra results for samples showing the relative amounts of each glycoform present in samples.

[0048] FIGS. 4A-B illustrate SDS-Page band patterning of Strain 2 (a TrMDS2 expressing strain) compared to its parent strain Strain 1 in SF17 (a) and SF22 (b). The 2 strains produce a similar amount of OVD. Strain 1 produces the characteristic OVD pattern seen in K. phaffii thus far with 7 main bands labeled in (a). With the exception of bands 6 and 7, all the main bands appear to have shifted.

[0049] FIG. 5 illustrates Common N-glycosylation patterns of K. phaffii. A square indicates N-acetylglucosamine (GlcNAc) while circles indicate mannose (Man).

[0050] FIG. 6 illustrates a comparison of deglycosylation function of TrMDS2 and GgMAN1A1.

[0051] FIG. 7 illustrates a result of coexpression of TrMDS2 and GgMAN1A1.

[0052] FIG. 8 illustrates SDS-PAGE results of culture supernatants of individual transformants expressing HsORM1.

[0053] FIGS. 9A-C illustrate SDS-PAGE results of TrMDS2-induced deglycosylation of HsORM1 and the vector schematic used for transformation.

[0054] FIG. 10 illustrates SDS-PAGE results of the deglycosylation of Ovalbumin (OVA).

[0055] FIG. 11 illustrates SDS-PAGE results of native OVA and denatured OVA.

[0056] FIG. 12 illustrates SDS-PAGE results of the deglycosylation of OVA with TrMDS2.

[0057] FIG. 13 illustrates results of lack of deglycosylation activity of MDS1 on GgOVD.

[0058] FIG. 14 illustrates results of the deglycosylation activity of TrMDS2 on GgOVD.

DETAILED DESCRIPTION OF THE INVENTION

[0059] The methods, nucleic acids, expression constructs, microorganisms, compositions and methods provided herein provide tools, methods and compositions for expressing recombinant animal protein in a host and modifying the glycosylation of the expressed protein. One such host contemplated herein is Pichia sp. (now reclassified as Komagataella sp.) The present disclosure contemplates modifying a Pichia species glycosylation machinery, such as in a Pichia pastoris in any one or more of the methods described herein.

[0060] The present disclosure contemplates modifying glycosylation of the recombinant protein to alter or enhance one or more functional characteristics of the protein and/or its production.

[0061] By such modifications, a recombinant protein can be made that has a higher nutrition value as compared to the recombinant protein produced in the host microorganism absent modification to the glycosylation machinery. The recombinant animal protein may have a higher nitrogen to carbon ratio as compared to the recombinant protein produced in the host microorganism absent modification to the glycosylation machinery, and/or as compared to the same protein produced from its native source or another heterologous host. By such modifications, in concert with recombinantly expressing one or more proteins, a recombinant protein can be made that has improved expression, secretion, purification as compared to the recombinant protein produced in the host absent modification to the glycosylation machinery. By such modifications, in concert with recombinantly expressing one or more proteins, a recombinant protein can be made that has improved enzymatic functionality or activity as compared to the recombinant protein produced in the host microorganism absent modification to the glycosylation machinery.

[0062] One approach to effect glycosylation in a yeast host exploits the required alpha-1,6-Mannosyltransferase activity of OCH1 protein in the Golgi on the core Man.sub.8GlcNAc.sub.2 substrate (FIG. 1C) as a necessary step for further extending mannosylation of the glycan structure in what is deemed “outer chain elongation”. In knockouts or mutants with disrupted OCH1 function, mannosylation cannot proceed past this base substrate in the Golgi, and hypermannosylation is eliminated.

[0063] In some embodiments, the yeast host may be modified to knockout OCH1 function. In some embodiments, the yeast host may be modified to have a partial disruption or knockdown of OCH1 function.

[0064] Alternatively, or additionally, one can also knock in an ER resident, heterologous mannosidase such as Trichoderma reesei alpha-1,2 mannosidase, or other similarly functional enzymes, to cleave glycans to Man.sub.5GlcNAc.sub.2 core structures before a nascent polypeptide's translocation to the Golgi, thereby effectively eliminating the Man.sub.8GlcNAc.sub.2 substrate required for efficient alpha-1,6-Mannosyltransferase activity of OCH1. It has been suggested that OCH1's alpha-1,6-Mannosyltransferase activity is specific for the Man.sub.8GlcNAc.sub.2 glycan structure and not the Man.sub.5GlcNAc.sub.2 structure. It is therefore possible that OCH1 activity can be effectively eliminated if the majority of peptide bound ER-processed glycan structures translocated to the Golgi are cleaved to Man.sub.5GlcNAc.sub.2 structures by the activity of an ER resident, heterologous alpha-1,2-mannosidase. Following this rationale, disclosed here in a simplified method of making a microorganism with altered glycosylation relative to wild type, wherein the microorganism only comprises one or more heterologous alpha-1,2 mannosidases and in some embodiments, also retains a fully functional wild type OCH1.

[0065] In various embodiments the homogeneity of glycosylation (i.e. the proportion of proteins that carry only Man.sub.5GlcNAc.sub.2 structures on their peptide backbone) can be tuned by controlling the expression of the heterologous mannosidases. In some embodiments, the host microorganism expresses one or more heterologous alpha-1,2 mannosidases. The heterologous alpha-1,2 mannosidases may be of fungal origin, avian origin and/or mammalian origin. The heterologous alpha-1,2 mannosidase is from Trichoderma reesei, such as the MDS2 enzyme with a SEQ ID NO: 7. In some embodiments, the heterologous alpha-1,2 mannosidase is from a chicken such as from Gallus gallus, such as the SEQ Id NO: 150. In other embodiments certain alpha-1,2 Mannosidases chosen from but not limited to those proteins corresponding to SEQ ID Nos 1 to 10 and SEQ ID Nos. 145-150, an amino acid sequence encoded by SEQ ID Nos. 151-152.

[0066] In some embodiments, the proteins may have a sequence that has 80%, 85%, or more sequence identity with any of SEQ ID Nos 1 to 10 or SEQ ID Nos. 145-151. In some cases, the sequence identity may be greater than 90%, 95%, 98%. In some embodiments, the proteins may be encoded by a nucleic acid sequence having a sequence that has 80%, 85% or more sequence identity with any of SEQ ID Nos. 152-153. In some cases, the nucleotide sequence identity may be greater than 90%, 95%, 98%. The heterologous mannosidases may be one with more than 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% sequence identity with SEQ ID NO: 7. The heterologous mannosidases may be one with more than 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% sequence identity with SEQ ID NO: 150.

[0067] The mannosidases used may be a functional equivalent or functional fragment of an enzyme with any of SEQ ID Nos. 1 to 10 or SEQ ID Nos. 145-151. As used herein “functional fragment” means a polypeptide fragment of an enzyme which substantially retains the enzymatic activity of the full-length protein. A mannosidase may be a substantially equivalent functional fragment of SEQ ID No: 7. A mannosidase may be a substantially equivalent functional fragment of SEQ ID No: 150. By “substantially” is meant at least about 40%, or preferably, at least 50% or more of the enzymatic activity of the full-length α-1,2-mannosidase is retained.

[0068] Certain alpha-1,2 mannosidases can have more efficient activity on a target protein than others. In some embodiments, two or more heterologous alpha-1,2 mannosidases are recombinantly expressed. The two or more alpha-1,2 mannosidases may be from the same, similar or different origins.

[0069] The combination of two or more interventions described herein can further be used to reduce hypermannosylation of recombinant proteins. For example, one can express recombinant alpha-1,2 mannosidase in a host along with a recombinant protein in a strain that contains a mutation, deletion or otherwise reduced or eliminated expression of OCH1.

[0070] In other embodiments the resultant microorganism expressing one or more heterologous alpha-1,2 mannosidases is so designed in order to effect a desired homogeneity and or reduction in the degree of glycosylation of one or more target proteins (chosen from but not limited to those proteins or peptide subsequences corresponding to SEQ ID Nos 11 to 26) also expressed as heterologous proteins in the same microorganism.

[0071] In some embodiments herein, recombinant alpha-1,2 mannosidase is expressed in a host along with expressing one or more recombinant proteins. In some embodiments herein, expression of a recombinant alpha-1,2 mannosidase along with expressing one or more recombinant proteins results in a recombinant protein with an improved nutritional value or nutritional content. In some embodiments herein, expression of a recombinant alpha-1,2 mannosidase along with expressing one or more recombinant proteins provides a recombinant protein having a nitrogen to carbon ratio equal to or greater than the protein when isolated from its naturally-occurring source and/or from a different heterologous host. The recombinant protein may be secreted out of the host cell.

[0072] The recombinant protein may be a nutritional protein. The nutritional protein may be a protein that contains a desirable amount of essential amino acids. The nutritive protein may comprise at least 30% essential amino acids by weight. The nutritive protein may comprise at least 40% essential amino acids by weight. The nutritive protein may comprise at least 50% essential amino acids by weight. The nutritive protein may comprises or consists of a protein or fragment of a protein that naturally occurs in an edible form. The nutritional protein may be an animal protein. The nutritional protein may be an avian protein. The nutritional protein may be an egg-white protein.

[0073] In some embodiments herein, recombinant alpha-1,2 mannosidase is expressed in a host along with expressing one or more egg white proteins. In some embodiments, the proteins or peptides may have a sequence that has 80% or more sequence identity with any of SEQ ID Nos 11 to 26. In some cases, the sequence identity may be greater than 90%, 92%, 95%, 98%.

[0074] In some embodiments herein, expression of a recombinant alpha-1,2 mannosidase along with expressing one or more egg white proteins provides an egg white protein with an improved nutritional value. In some embodiments herein, expression of a recombinant alpha-1,2 mannosidase along with expressing one or more egg white proteins provides an egg white protein having a nitrogen to carbon ratio equal to or greater than the egg white protein when isolated from naturally-occurring chicken egg.

[0075] A nutritional protein may be produced recombinantly in a host cell which expresses a heterologous mannosidase enzyme in addition to the nutritional protein. Alternatively, a recombinant nutritional protein may be treated with a mannosidase described herein. The resulting recombinant protein may be a reduced glycosylated protein or deglycosylated protein.

[0076] Reduced glycosylation or deglycosylation may refer to a reduced size of the carbohydrate moiety on the recombinant glycoprotein, particularly with fewer mannose residues, when the recombinant glycoprotein is expressed in a microorganism which has been modified as described herein as compared to a wild type, unmodified strain of the microorganism. “De-glycosylated” proteins can have a level of N-linked glycosylation that is reduced by at least about 10 percent (e.g., 10 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, or 100 percent) as compared to the level of N-linked glycosylation of the same proteins that are not produced in the presence of or otherwise exposed to a mannosidase.

[0077] The enzymes used to reduce the glycosylation of one or greater proteins may include mannosidases, greater preferably an alpha-1,2 mannosidase. The enzyme may reduce the glycosylation of the recombinant proteins secreted from the host cell. For instance, a fraction of the recombinant protein may be deglycosylated by the enzyme. The enzyme may reduce the glycosylation of greater than 1% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 5% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 10% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 20% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 30% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 40% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 50% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 60% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 75% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 80% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 90% of the nutritional protein secreted from the host cell. The enzyme may reduce the glycosylation of greater than 95% of the nutritional protein secreted from the host cell.

[0078] The degree of glycosylation or the number of glycan units on a single protein may be modified in the host cell. The degree of glycosylation of the recombinant protein may be less than 90% of the degree of glycosylation of a control protein. The degree of glycosylation of the recombinant protein may be less than 80% of the degree of glycosylation of a control protein. The degree of glycosylation of the recombinant protein may be less than 75% of the degree of glycosylation of a control protein. The degree of glycosylation of the recombinant protein may be less than 50% of the degree of glycosylation of a control protein. The degree of glycosylation of the recombinant protein may be less than 30% of the degree of glycosylation of a control protein. The degree of glycosylation of the recombinant protein may be less than 20% of the degree of glycosylation of a control protein. The degree of glycosylation of the recombinant protein may be less than 15% of the degree of glycosylation of a control protein. The degree of glycosylation of the recombinant protein may be less than 10% of the degree of glycosylation of a control protein. The degree of glycosylation of the recombinant protein may be less than 5% of the degree of glycosylation of a control protein. The degree of glycosylation of the recombinant protein may be less than 1% of the degree of glycosylation of a control protein.

Compositions Comprising Recombinant Proteins

[0079] A consumable composition may comprise one or more recombinant proteins. As used herein, the term “consumable composition” refers to a composition, which comprises an isolated recombinant protein and may be consumed by an animal, including but not limited to humans and other mammals. Consumable food compositions include food products, beverage products, dietary supplements, food additives, and nutraceuticals as non-limiting examples. The consumable composition may comprise one or more components in addition to the recombinant protein. The one or more components may include ingredients, solvents used in the formation of foodstuff, beverages, etc. For instance, the recombinant protein may be in the form of a powder which can be mixed with solvents to produce a beverage or mixed with other ingredients to form a food product.

[0080] The nutritional content of the deglycosylated recombinant protein may be higher than the nutritional content of an identical quantity of a control protein. The control protein may be the same protein produced recombinantly but not treated with a mannosidase. The control protein may be the same protein produced recombinantly in a host cell which does not express a heterologous mannosidase. The control protein may be the same protein isolated from a naturally occurring source. For instance, the control protein may be an isolated an egg white protein such as OVD, OVA, or other protein that can be isolated from native egg white.

[0081] The nutritional content of a composition comprising the recombinant nutritional protein can be more than the nutritional content of the composition comprising a control protein. The nutritional content may be the protein content of the protein. The protein content of the composition may be about 1% to 80% more than the protein content of a composition comprising a control protein. The protein content of the composition may be about 1% to 5% more than the protein content of a composition comprising a control protein. The protein content of the composition may be about 1% to 10% more than the protein content of a composition comprising a control protein. The protein content of the composition may be about 1% to 20% more than the protein content of a composition comprising a control protein. The protein content of the composition may be about 1% to 50% more than the protein content of a composition comprising a control protein. The protein content of the composition may be about 1% to 80% more than the protein content of a composition comprising a control protein. The protein content of the composition may be about 5% to 10%, 5-15%, 5-20%, 5-30%, 5-50%, 5-80% more than the protein content of a composition comprising a control protein. The protein content of the composition may be about 10% to 80%, 10-20%, 10-30%, 10-50%, 10-70%, 10-80% more than the protein content of a composition comprising a control protein. The protein content of the composition may be about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% more than the protein content of a composition comprising a control protein.

[0082] Protein content of a composition may be measured using conventional methods. For instance, protein content may be measured using nitrogen quantitation by combustion and then using a conversion factor to estimate quantity of protein in a sample followed by calculating the percentage (w/w) of the dry matter.

[0083] The nitrogen to carbon ratio of a deglycosylated protein be higher than the nitrogen to carbon ratio of a control protein. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.1. The nitrogen to carbon ratio of a deglycosylated protein be higher than the nitrogen to carbon ratio of a control protein. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.25. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.3. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.35. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.4. The nitrogen to carbon ratio of a recombinant protein may be greater than or equal to about 0.5.

[0084] Solubility of a deglycosylated protein may be greater than the solubility of a control protein. Solubility of a composition comprising a deglycosylated protein may be higher than the solubility of a composition comprising the control protein. Thermal stability of the deglycosylated protein may be greater than the thermal stability of a control protein.

[0085] The degree of glycosylation of the recombinant protein may be dependent on the consumable composition being produced. For instance, a consumable composition may comprise a lower degree of glycosylation to increase the protein content of the composition. Alternatively, the degree of glycosylation may be higher to increase the solubility of the protein in the composition.

A Microorganism Carrying a Heterologously Expressed Alpha-1,2 Mannosidase

[0086] The following outlines the construction of a microorganism expressing a heterologous alpha-1,2 mannosidase.

[0087] Herein an “alpha-1,2 mannosidase” refers to any protein that recognized as catalyzing the cleavage of an alpha-1,2 glycosidic bond between mannose groups in a glycan structure that contains Man.sub.xGlcNAc.sub.2 (where x>=6) as a substructure (with reference to bonds illustrated in FIG. 1). Examples of alpha-1,2 mannosidase to those proteins encoded by any of the polynucleotide sequences or subsequences therein represented in the list comprised of SEQ ID Nos 1 to 10 and SEQ ID Nos. 145-151 or encoded by SEQ ID Nos. 152-153.

[0088] In eukaryotic organisms, precursor oligosaccharides structures (Glc.sub.3Man.sub.9GlcNAc.sub.2) synthesized in the Endoplasmic Reticulum (ER) can be added to asparagine residues of a polypeptide (at consensus Asn-X-Ser or Asn-X-Thr or Asn-X-Cys sites where X is any amino acid except a Proline) in the first step of what is known as N-glycosylation. In the lumen of the ER, the precursor oligosaccharide is cleaved to remove the glucose residues of each attached Glc.sub.3Man.sub.9GlcNAc.sub.2 oligosaccharide (FIG. 1A). The additional removal of a mannose group results in a Man.sub.8GlcNAc.sub.2 core structure (FIG. 1B). This core structure is further processed upon translocation of the glycoprotein to the Golgi. In yeast Golgi, this processing involves the activity of OCH1, an alpha-1,6 mannosyltransferase that acts on Man.sub.8GlcNAc.sub.2 core structures in a step necessary to initiate the further addition of mannosyl groups that can ultimately give rise to hypermannosylated glycan groups on the fully processed protein. (FIG. 1D) illustrates Man.sub.5GlcNAc.sub.2, a possible product upon cleavage of Man.sub.8GlcNAc.sub.2 at alpha-1,2 glycosidic bonds by an alpha-1,2 mannosidase. Unlike Man.sub.8GlcNAc.sub.2, OCH1 does not carry out efficient alpha-1,6 mannosyltransferase activity on Man.sub.5GlcNAc.sub.2 as a substrate. Triangle—glucose; square—N-acetylglucosamine; circle-Mannose.

[0089] Herein a “transformation” of a microorganism refers to the introduction of polynucleotides into a microorganism.

[0090] Herein a “transformant” refers to a microorganism that has been transformed.

[0091] Herein a “transgene” refers to a polynucleotide that can form a gene product if contained in a microorganism.

[0092] Herein an “expression cassette” is any polynucleotide that contains a subsequence that codes for a transgene and can confer expression of that subsequence when contained in a microorganism and is heterologous to that microorganism.

[0093] Herein a “promoter” refers to a polynucleotide subsequence of an expression cassette that is located upstream or 5′ to a transgene and is involved in initiating transcription from that transgene when the expression cassette is contained in a microorganism.

[0094] Herein a “glycoprotein” refers to a protein that carry carbohydrates covalently bound to their peptide backbone.

[0095] Herein a “glycoform” refers to any of several different forms of a glycoprotein where each is differentiated from the other by the different structures of peptide-bound polysaccharides.

[0096] In some embodiments the host microorganism carries one or more stably integrated heterologous transgenes that when expressed as proteins in the host are intended targets for alterations of their glycan groups by the heterologous alpha-1,2 mannosidase. Herein such transgenes are referred as the “target proteins”.

[0097] A. Synthesis of Vectors Containing Expression Cassettes:

[0098] First a vector carrying an expression cassette, containing an alpha-1,2 mannosidase to be transformed is made. In some embodiments multiple different alpha-1,2 mannosidases could be transformed, either on vectors carrying multiple expression cassettes, or on separate vectors. The expression cassettes described herein can be obtained using chemical synthesis, molecular cloning or recombinant methods, DNA or gene assembly methods, artificial gene synthesis, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence. For preparing polynucleotides using recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable cloning or expression vector, and the cloning or expression vector in turn can be introduced into a suitable host cell for replication and amplification. Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally may the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the expression vector. Methods for obtaining cloning and expression vectors are well-known (see, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, New York (2012)).

[0099] FIG. 2 provides examples of a vectors created by these means; FIG. 2 describes a vector containing (A) a promoter (FBA1 promoter in FIG. 2) operably linked to a transgene (T. reesei alpha-1,6 mannosidase 1—T.R. MDS1 in FIG. 2). The vector further comprises a C-terminus sequence encoding an HDEL ER retention signal fused in frame with the transgene (HDEL FIG. 2). The vector further comprises a Terminator Element (AOX1 terminator in FIG. 2). These elements are collectively referred to herein as an “Expression Cassette”, although in some embodiments a signal peptide can also be included in the design. In some embodiments the ER retention signal may or may not be present. To aide in the amplification of the vector prior to transformation into the host microorganism, those skilled in the art may rely on a replication origin (E) contained in the vector (ORI in FIG. 2). To aide in the selection of a microorganism stably transformed with the expression vector from those microorganisms that don't contain the expression vector, those skilled in the art may rely on a selection marker (F) contained in the vector downstream of a promoter element (Zeocin resistance gene in FIG. 2) The expression vector can also contain a restriction enzyme site (G) (SwaI in FIG. 2) that allows for linearization of the expression vector prior to transformation into the host microorganism to facilitate the expression vectors stable integration into the host genome. In FIG. 2, elements E,F may be removed from their genomic location post transformation by one skilled in the art due to the presence flanking LoxP sites that can catalyze excision of the intervening region by the CRE/lox recombination (https://en.wikipedia.org/wiki/Cre-Lox recombination). In general, the expression cassette is designed to mediate the transcription of the transgene when integrated into the genome of a cognate host microorganism. For the elements comprising the expression vectors in FIG. 2, this host microorganism is Pichia Pastoris although in other embodiments this host organism can be any microorganism where one skilled in the art can introduce the expression vector into its genome such that the elements in the expression vector are recognized by the cell to sufficiently induce the transcription and subsequent processing of transcript into the intended full-length protein. In some embodiments the transgene may be codon optimized for optimal expression in the host organism.

[0100] The genetic elements of the expression vector can be designed to be suitable for expression in the intended microorganism host by one trained in the art. In some embodiments an additional vector and or additional elements may be designed to aide (as deemed necessary by one skilled in the art) for the particular method of transformation (e.g. CAS9 and gRNA vectors for a CRISPR/CAS9 based method).

[0101] The Promoter Element (A) may include, but is not limited to, a constitutive promoter, inducible promoter, and hybrid promoter. Promoters include, but are not limited to, acu-5, adh1+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, α-amylase, alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbh1), ccg-1, cDNA1, cellular filament polypeptide (cfp), cpc-2, ctr4+, CUP1, dihydroxyacetone synthase (DAS), enolase (ENO, ENO1), formaldehyde dehydrogenase (FLD1), FMD, formate dehydrogenase (FMDH), G1, G6, GAA, GAL1, GAL2, GAL3, GAL4, GAL5, GAL6, GAL7, GAL8, GAL9, GAL10, GCW14, gdhA, gla-1, α-glucoamylase (glaA), glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUT1), HSP82, inv1+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, β-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmt1, NSP, pcbC, PETS, peroxin 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), pho1, PHO5, PH089, phosphatidylinositol synthase (PIS1), PYK1, pyruvate kinase (pki1), RPS7, sorbitol dehydrogenase (SDH), 3-phosphoserine aminotransferase (SER1), SSA4, SV40, TEF, translation elongation factor 1 alpha-(TEF1), THI11, homoserine kinase (THR1), tpi, TPS1, triose phosphate isomerase (TPI1), XRP2, YPT1, GCW14, GAP, a sequence or subsequence chosen from SEQ ID Nos: 31 to 47, and any combination thereof. In some embodiments, the nucleotides used may have a sequence that has 80% or more sequence identity with any of SEQ ID Nos 31 to 47. In some cases, the sequence identity may be greater than 90%, 95%, 98%.

[0102] A promoter used to express the mannosidases described herein may be heterologous to the host cell. A promoter used to express the mannosidases described herein may be native to the host cell. A promoter used to express the mannosidases described herein may be constitutive or inducible. A strong promoter may be used to drive the expression of the α-1,2-mannosidase. For instance, if a higher protein content is desired, the vector may comprise a strong promoter to increase the degree of deglycosylation of the recombinant protein. Alternatively, a weaker promoter may be used to drive the expression of the α-1,2-mannosidase. For instance, if a lower degree of deglycosylation is required, a weaker promoter may be used to drive the expression of the mannosidase.

[0103] A host cell may comprise a first promoter driving the expression of the recombinant nutritional protein and a second promoter driving the expression of the α-1,2-mannosidase. The first and second promoter may be selected from the list of promoters provided herein. In some cases, the expression of α-1,2-mannosidase and the recombinant nutritional protein may be derived from the same promoters. Alternatively, the first and the second promoter may be different.

[0104] The Signal peptide (B) A signal peptide, also known as a signal sequence, targeting signal, localization signal, localization sequence, signal peptide, transit peptide, leader sequence, or leader peptide, may support secretion of a protein or polynucleotide. Extracellular secretion of a recombinant or heterologously expressed protein from a host cell may facilitate protein purification. A signal peptide may be derived from a precursor (e.g., prepropeptide, preprotein) of a protein. Signal peptides may be derived from a precursor of a protein including, but not limited to, acid phosphatase (e.g., Pichia pastoris PHO1), albumin (e.g., chicken), alkaline extracellular protease (e.g., Yarrowia lipolytica XRP2), α-mating factor (α-MF, MATa) (e.g., Saccharomyces cerevisiae), amylase (e.g., α-amylase, Rhizopus oryzae, Schizosaccharomyces pombe putative amylase SPCC63.02c (Amyl)), β-casein (e.g., bovine), carbohydrate binding module family 21 (CBM21)-starch binding domain, carboxypeptidase Y (e.g., Schizosaccharomyces pombe Cpy1), cellobiohydrolase I (e.g., Trichoderma reesei CBH1), dipeptidyl protease (e.g., Schizosaccharomyces pombe putative dipeptidyl protease SPBC1711.12 (Dpp1)), glucoamylase (e.g., Aspergillus awamori), heat shock protein (e.g., bacterial Hsp70), hydrophobin (e.g., Trichoderma reesei HBFI, Trichoderma reesei HBFII), inulase, invertase (e.g., Saccharomyces cerevisiae SUC2), killer protein or killer toxin (e.g., 128 kDa pGKL killer protein, α-subunit of the K1 killer toxin (e.g., Kluyveromyces lactis), K1 toxin KILM1, K28 pre-pro-toxin, Pichia acaciae), leucine-rich artificial signal peptide CLY-L8, lysozyme (e.g., chicken CLY), phytohemagglutinin (PHA-E) (e.g., Phaseolus vulgaris), maltose binding protein (MBP) (e.g., Escherichia coli), P-factor (e.g., Schizosaccharomyces pombe P3), Pichia pastoris Dse, Pichia pastoris Exg, Pichia pastoris Pir1, Pichia pastoris Scw, and cell wall protein Pir4 (protein with internal repeats). Examples of signal peptides can also comprise a sequence or subsequence chosen from SEQ ID Nos 48 to 144, and any combination thereof. In some embodiments a signal peptide is not present. In some embodiments, the signal proteins or peptides may have a sequence that has 80% or more sequence identity with any of SEQ ID Nos 48 to 144. In some cases, the sequence identity may be greater than 90%, 95%, 98%.

ER Targeting/Retention Signal

[0105] This motif will signal the retention of the resultant protein to the ER. An ER retention signal may be derived from a precursor (e.g., prepropeptide, preprotein) of a protein. ER retention signals may be derived from a precursor of a protein including, but not limited to, polynucleotides that encode the amino acid sequence KDEL, HDEL, or transmembrane domains that may be encoded by subsequences contained in SEQ ID Nos 1 to 10 or 145 to 149. The ER retention signal is typically fused in frame on the C-terminus of the transgene ORF, although in some embodiments it may be fused in frame on the transgene N-terminus immediately downstream of the cleavage site of the signal peptide if it is present. In some embodiments an ER retention signal is not present. In some embodiments, the expressed protein, such as an alpha-1,2 mannosidase, will be retained in the ER or otherwise not require an ER retention signal to provide intracellular deglycosylation of a heterologous protein.

[0106] The Transgene (C) may include, but is not limited to, nucleic acids encoding polypeptides such as those polynucleotides chosen from the list comprised of SEQ ID Nos: 1 to 30 or 145 to 150. These sequences can be designed to be altered to encode the same protein, and be optimized for expression in the chosen host (i.e. codon optimized); for example, the nucleic acid sequence encoding an alpha-1,2 mannosidase and a codon optimized form SEQ ID Nos. 151-152.

[0107] The Terminator Element (D) in this example is the AOX1 terminator, but it may chosen to be any suitable sequences that serves to abort continuing elongation of the nascent transcript containing the mRNA corresponding to the transgene.

[0108] The Selectable Marker (F) may include, but is not limited to: an antibiotic resistance gene (e.g. zeocin, ampicillin, blasticidin, kanamycin, nurseothricin, chloroamphenicol, tetracycline, triclosan, ganciclovir, and any combination thereof), an auxotrophic marker (e.g. f ade1, arg4, his4, ura3, met2, and any combination thereof).

Transformation of Microorganism Host with Vectors

[0109] Next, expression vectors or polynucleotides (DNA or RNA) containing genetic information encoding expression cassettes derived from expression vectors are inserted into host cells and clonal populations of successful transformants may be isolated by any means known in the art.

[0110] Microorganisms that are suitable for transformation with a polynucleotide carrying an expression cassette that contains a subsequence that encodes for an alpha-1,2 mannosidase by someone trained in the art. These can include but are not limited to: Arxula spp., Arxula adeninivorans, Kluyveromyces spp., Kluyveromyces lactis, Pichia spp., Pichia angusta, Pichia pastoris, Saccharomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces spp., Schizosaccharomyces pombe, Yarrowia spp., Yarrowia hpolytica, Agaricus spp., Agaricus bisporus, Aspergillus spp., Aspergillus awamori, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Colletotrichum spp., Colletotrichum gloeosporiodes, Endothia spp., Endothia parasitica, Fusarium spp., Fusarium graminearum, Fusarium solani, Mucor spp., Mucor miehei, Mucor pusillus, Myceliophthora spp., Myceliophthora thermophila, Neurospora spp., Neurospora crassa, Penicillium spp., Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium (Talaromyces) emersonii, Penicillium funiculosum, Penicillium purpurogenum, Penicillium roqueforti, Pleurotus spp., Pleurotus ostreatus, Rhizomucor spp., Rhizomucor miehei, Rhizomucor pusillus, Rhizopus spp., Rhizopus arrhizus, Rhizopus oligosporus, Rhizopus oryzae, Trichoderma spp., Trichoderma altroviride, Trichoderma reesei, Trichoderma vireus, Aspergillus oryzae, Bacillus subtilis, Escherichia coli, Myceliophthora thermophila, Neurospora crassa, Pichia pastoris, Komagatella phaffii and Komagatella pastoris.

[0111] Cells may be transformed by introducing an exogenous polynucleotide, for example, by direct uptake, endocytosis, transfection, F-mating, PEG-mediated protoplast fusion, Agrobacterium tumefaciens-mediated transformation, biolistic transformation, chemical transformation, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated expression vector (such as a plasmid) or integrated into the host cell genome. The cell population can be selected for those cells that take up the exogeneous expression vectors (by virtue of resistance genes carried on the expression vectors) by plating onto agar plates containing some agent (e.g. the antibiotic Zeocin) that negatively selects cells that are not carrying a gene conferring resistance to that agent.

[0112] Alternatively, one can create an auxotrophic strain by knocking out a gene (e.g. URA3 gene in Pichia pastoris) required for synthesis of an essential metabolite (e.g. uracil), transform this strain using expression vectors that contain as a selection marker a gene that complements the knock out (i.e. the URA3 gene) and select for transformed cells by virtue of their ability to grow on a media that lacks this essential metabolite.

[0113] With either approach after incubating plates that have been spread with a population of cells containing putative transformants for time and temperature appropriate for growth of colonies that can be manually selected (as known to one trained in the art), individual colonies can be picked and verified for the integration of expression vectors into the host cell genome by standard molecular biological methods that are known to one trained in the art (i.e. colony PCR, genomic sequencing). Individual colonies from these plates can then be used to inoculate individual culture vessels containing appropriate growth medium for the cell line containing a selection agent chosen as appropriate for the selection marker(s) contained in the transformed expression vectors. After an appropriate amount of time (e.g. overnight at 30 degrees Celsius in a shaker flask; otherwise known to one trained in the art) The successful transformation of a cell line with recombinant vector can be determined in each culture vessel by the presence of protein coded by the transgene on the transformed expression cassettes (referred to henceforth as “recombinant protein”). This expression can be determined by standard molecular biology methods (e.g. Western blot, SDS-PAGE with known standard protein). Colonies from those plates that correspond to culture vessels that show the recombinant protein expression can then be used to inoculate vessels containing selection media appropriate for the transformed cell line to promote growth of the cell line and expression of the recombinant protein. Alternatively, colonies from those plates that correspond to culture vessels that showed recombinant protein expression can be stored for later use (e.g. at −80 degrees Celsius in a glycerol stock).

Determination of Efficacy of Transformed Strain

[0114] Resultant strains confirmed to be stably transformed with an integrated transgene encoding an alpha-1,2 mannosidase are tested for the effect of its expression on the glycosylation of either endogenous or heterologously expressed target proteins.

[0115] The expression and purification of proteins expressed in parental wild type strains or parental strains that contain a heterologous alpha-1,2 mannosidase are known to one trained in the art. For example, in a methylotrophic yeast strain (such as Pichia Pastoris) a target protein can be induced if it is operably linked to a methanol induced promoter (i.e. AOX1) for strong over expression. If this target protein also contains a signal peptide it can be recovered from the media, and be sufficiently purified for analysis using techniques known to one trained in the art. In general, one can compare the glycan groups present on a protein of interest (e.g. the target proteins) between protein samples purified from cells with and without (herein referred to as the “control proteins”) the alpha-1,2 mannosidases or as compared to the the same protein isolated from a native source. Such measures of sample preparation and comparison can be carried out using techniques included, but not limited to methods such as: capillary electrophoresis or SDS-PAGE for size comparison of protein of interest, immunostaining techniques (e.g. Western blotting) using glycan specific antibodies, and quantitative mass spectrometry methods to identify glycan groups within a sample (e.g. N-linked glycan profiling by MALDI-TOF/TOF MS). See, e.g., Ziv Roth, Galit Yehezkel, and Isam Khalaila International Journal of Carbohydrate Chemistry Volume 2012 (2012).

[0116] In some embodiments, a ratio for Man.sub.xGlcNAc.sub.2 and Man.sub.yGlcNAc.sub.2 values may be calculated for a recombinantly expressed egg white protein. In some cases, the x value may be less than or equal to 1, 2, 3, 4 or 5. In some cases, they value may be greater than or equal to 6, 7, 8, 9 or 10. In some cases, the ratio of Man.sub.xGlcNAc.sub.2:Man.sub.yGlcNAc.sub.2 may be greater than 1. In some embodiments, a recombinantly expressed egg white protein may have a degree of polymerization that is less than or equal to 9. In some cases, the degree of polymerization may be less than 9, 8, 7 or 6.

[0117] The following example outlines the preparation and analysis of samples for determining the glycan groups present on a target protein (namely the protein corresponding to SEQ ID NO: 12). In some embodiments, the target proteins or peptides may have a sequence that has 80% or more sequence identity with any of SEQ ID No. 12. In some cases, the sequence identity may be greater than 90%, 95%, or 98%.

[0118] In some embodiments, the recombinant egg white protein may have a nitrogen to carbon (N to C) ratio greater than 0.25. In some cases, the N to C ratio for the recombinantly expressed protein may be greater than about 0.25, about 0.3, about 0.35 or about 0.4.

[0119] N-Linked Glycan Profiling by MALDI-TOF/TOF MS

[0120] An aliquot of each sample corresponding to 300 μg can be used for analysis. The glycoprotein is reduced, alkylated, then digested with trypsin in Tris-HCl buffer overnight. After protease digestion, the sample is passed through a C18 sep pak cartridge, washed with a low w/w percentage acetic acid and the glycopeptides are eluted with a blend of isopropanol in low concentration acetic acid, before being dried by SpeedVac. The dried glycopeptides eluate are treated with PNGase F to release the N-linked glycans and the digest is passed through a C18 sep pak cartridge to recover the N-glycans.

[0121] Per-O-Methylation of N-Linked Glycans

[0122] The N-linked glycans is permethylated for structural characterization by mass spectrometry (Anumula and Taylor, 1992). Briefly, the dried eluate is dissolved with dimethyl sulfoxide and methylated with NaOH and methyl iodide. The reaction is quenched with water and per-O-methylated carbohydrates is extracted with methylene chloride and dried under N.sub.2.

[0123] Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF/TOF MS)

[0124] The permethylated glycans is dissolved with methanol and crystallized with α-dihyroxybenzoic acid (DHBA) matrix. Analysis of glycans present in the samples is performed by MALDI-TOF/TOF-MS using AB SCIEX TOF/TOF 5800 (Applied Biosystems).

[0125] FIGS. 3A and 3B illustrate a sample mass spectra results from the above procedure, intended to inform the practitioner of the relative amounts of each glycoform present in a control sample (FIG. 3A) relative to a sample obtained from a cell line expressing a heterologous alpha-1,2 mannosidase (FIG. 3B). The relative amounts for each identified glycoform are laid out in Tables 1 and 2 corresponding to the control sample and alpha-1,2 mannosidase sample respectively. The data presented in this figure represents a prophetic result in which the activity of the mannosidase is effecting an increase in the relative presence of Man.sub.5GlcNAc.sub.2 type structures relative to other glycan structures within the sample relative to the control sample. In sample 2, Man.sub.5GlcNAc.sub.2 comprises 77.1% of identified glycoforms (Table 1), while in sample 1, Man.sub.5GlcNAc.sub.2 is not represented among the identified glycoforms (Table 2). Square—N-acetylglucosamine (GlcNac); green circle Mannose (Man); white circle—Hexose (Hex).

TABLE-US-00001 TABLE 1 N-linked glycans from Sample 1 (rOVD expressed in Pichia) detected by MALDI TOF/TOF MS. Permethylated Text description of Cartoon representation mass (m/z).sup.1 structures of possible structures Percentage 1988.0 Man.sub.7GlcNAc.sub.2 [00001]  8.0 2192.1 Man.sub.8GlcNAc.sub.2 [00002]  8.6 2396.2 Man.sub.9 GlcNAc.sub.2 [00003] 14.2 2600.3 Man.sub.9 GlcNAc.sub.2 Hex [00004] 17.8 2804.4 Man.sub.9 GlcNAc.sub.2Hex.sub.2 [00005] 18.9 3008.5 Man.sub.9 GlcNAc.sub.2Hex.sub.3 [00006] 13.7 3212.6 Man.sub.9 GlcNAc.sub.2Hex.sub.4 [00007] 10.0 3416.7 Man.sub.9 GlcNAc.sub.2Hex.sub.5 [00008]  8.7 .sup.1All masses (mass + Na) are single-charged. .sup.2Calculated from the area units of detected N-linked glycans.

TABLE-US-00002 TABLE 2 N-linked glycans from Sample 2 (rOVD expressed in a modified Pichia strain) detected by MALDI TOF/TOF MS. Theoretical Permethylated Text description of Cartoon representation mass (m/z).sup.1 structures of possible structures Percentage  967.5 Man.sub.2GlcNAc.sub.2 [00009]  1.4 1171.6 Man.sub.3GlcNAc.sub.2 [00010]  1.7 1375.7 Man.sub.4GlcNAc.sub.2 [00011] 15.4 1579.8 Man.sub.5GlcNAc.sub.2 [00012] 77.1 1783.9 Man.sub.6GlcNAc.sub.2 [00013]  2.3 1988.0 Man.sub.7GlcNAc.sub.2 [00014]  1.1 2192.1 Man.sub.8GlcNAc.sub.2 [00015]  1.1

EXAMPLES

Example 1: Identification of alpha-1,2 mannosidases

[0126] Blast P was used to search for protein sequences with identity to known alpha-1,2 mannosidases that could confer modification of the glycan structures on proteins expressed heterologously in Pichia sp. (currently reclassified as Komagataella species). Exemplary fungal alpha-1,2 mannosidase protein sequences identified including SEQ ID Nos. 1-10. A further search was performed for sequences in Gallus gallus. Exemplary Gallus gallus alpha-1,2 mannosidase protein sequences include SEQ ID Nos. 145-150.

Example 2: Construction of Expression Vectors for Alpha-1,2 Mannosidase Expression in Pichia

[0127] A fungal alpha-1,2 mannosidase protein sequence, SEQ ID NO. 7 (referred to as TrMDS2), was selected for expression, along with a Gallus gallus alpha-1,2 mannosidase protein sequence, SEQ ID NO. 150 (referred to as GgMAN1A1). For GgMAN1A1, the cDNA (SEQ ID NO. 152) was codon optimized to increase expression in Pichia (SEQ ID NO. 153, referred to as GgMAN1A1C).

[0128] Each cDNA, TrMDS2 and GgMAN1A1C was cloned into a Pichia expression vector downstream of a methanol inducible promoter, the vectors containing the selectable marker for zeocin resistance, The alpha-1,2 mannosidase expression vectors were transformed by electroporation into a K. phaffii strain (Strain 1) previously confirmed to be secreting OVD. Expression cassettes for the 2 alpha-1,2 mannosidase enzymes were transformed both individually and together into the OVD-expressing strain. Transformed cells were selected on zeocin containing agar plates and individual colonies were grown up in a microtiter 96 well plate format to evaluate quality of secreted OVD.

Example 3: Expression of Alpha-1,2 Mannosidase in Pichia

[0129] Bradford protein assays were conducted in a high throughput format to confirm presence of secreted protein in the growth media. The supernatant from select wells were then screened by SDS-PAGE. Clones displaying desired protein patterns from SDS-PAGE were then scaled up in 40 mL shake flask format and/or up to 40 L bioreactor to confirm activity of transformed deglycosidase. External glycan analysis by LC/MS was conducted on one strain expressing TrMDS2 (Strain 2) using material generated in shake flask format. Inspection of SDS-PAGE results from TrMDS2-expressing Pichia indicated that this heterologous protein was not secreted under the conditions tested. This means that the native TrMDS2 protein sequence contains intracellular localization signals that were recognized by Pichia. TrMDS2 protein is large enough that it would run well above OVD and should be visible on the protein gel.

Example 4: Activity Analysis of Heterologous Expression of TrMDS2 in Pichia

[0130] Heterologous expression of TrMDS2 in Strain 2 did not significantly reduce OVD expression compared to its parent strain Strain 1 in shake flask experiments. In its initial shake flask run, SF17, Strain 2 made 95% secreted OVD compared to the average secretion level of a Strain 1 duplicate (FIG. 4A). However, this difference is within the error of shake flask experiments. In a subsequent run, SF22, a duplicate of Strain 2 made 109% secreted OVD compared to a duplicate of Strain 1 (FIG. 4B).

[0131] In all experiments, Strain 2 produced a visible band pattern downshift in the secreted OVD as seen by SDS-PAGE analysis (FIGS. 4A-B). This band shift indicated a decrease in the apparent molecular weight of OVD from Strain 1 to Strain 2, theorized to be a result of reduction in glycan presence on the protein.

[0132] The reduction of OVD glycosylation in the Strain 2 strain was confirmed by external LC/MS (Table 3). Almost all glycans found on Strain 1 produced OVD have a branch pattern of 9 mannose or more. In contrast, the majority of glycans found on Strain 2 produced OVD contain branches of 8 mannose or less. The known branching patterns of K. phaffii mannosylation are shown in FIG. 5.

TABLE-US-00003 TABLE 3 Summary of relative distribution of glycans found on OVD secreted by Strain 1 and Strain 2. Glycosylation Fragment Distribution Man16 Man15 Man14 Man13 Man12 Man11 Man10 Man9 Man8 Man7 Man6 Man5 STRAIN1 2 4 4 6 8 10 8 4 0 0 1 0 STRAIN2 1 0 1 0 3 2 3 3 7 3 11 12

Example 5: Heterologous Expression of GgMAN1A1 in Pichia

[0133] Heterologous expression of GgMAN1A1 in Strain 1 produce a range of deglycosylation effect, the strongest of which approach the band pattern of Strain 2, the weakest of which approximate Strain 1 band pattern with a very slight downshift.

[0134] SDS-PAGE analysis was conducted to compare the two extremes of GgMAN1A1 functionality with TrMDS2 as well as Strain 1 pattern (FIG. 6). In the analysis, Strain 3, a derivative strain of Strain 1 making more OVD but maintaining the same glycosylation pattern, was used as the standard OVD band pattern. While TrMDS2 expression varied between transformants, the weaker TrMDS2 clones still showed band patterning very close to that of Strain 2. A “weak” MDS2 clone was included in the comparison in FIG. 6 as well. There were minute differences in the band patterning of TrMDS2 vs GgMAN1A1.

Example 6: Localization of GgMAN1A1 in Pichia

[0135] The sample GgMAN1A1.a represents the strongest deglycosylation effect found during screening, and GgMAN1A1.b represents the weakest. There is a progressive upward band shift from MDS2 to GgMAN1A1.b on the left side of the gel, indicating a range of deglycosylation function. Each sample is then compared to Strain 3 individually on the right side of the gel to confirm deglycosylation. Inspection of SDS-PAGE results from GgMAN1A1-expressing Pichia indicated that this heterologous protein was not secreted under the conditions tested. GgMAN1A1 protein is large enough that it would run well above OVD and should be visible on the protein gel. This means that the native GgMAN1A1 protein sequence contains intracellular localization signals that were recognized by Pichia.

[0136] The major difference between the strong and weak TrMDS2 deglycosylation is seen in the band marked by an asterisk. This band appears to be a close doublet. In the strong TrMDS2 pattern, the doublet favors the bottom band, while the weak TrMDS2 pattern favors the top band. GgMAN1A1.a displays a band pattern close to that of MDS2, with the exception of the asterisk-marked band. This band in GgMAN1A1.a appears to be sized between the doublet. GgMAN1A1.b displays a further upward shift of all the bands. When compared immediately next to the standard OVD pattern on the right side of the gel, it is very slightly downshifted and displays the characteristic disappearance of the topmost band seen in TrMDS2 deglycosylated patterns.

[0137] TrMDS2 and GgMAN1A1 were coexpressed in Strain 1 and the glycosylation patterns examined by SDS-PAGE analysis. A range of deglycosylation patterns were seen, including that of TrMDS2 alone. (FIG. 7).

Example 7: Deglycosylation of HsORM1

[0138] Human serum glycoprotein, “Orosomucoid 1” (Homo sapiens ORM1; HsORM1; uniport P02763) possesses five predicted N-glycosylation consensus motifs at asparagine residues 33, 56, 72, 93 and 103. An HsORM1 coding sequence was placed downstream of a methanol-inducible promoter. An alpha-mating factor signal sequence was fused to the N-terminus of the HsORM1 coding sequence. The translated fusion provided the polypeptide sequence SEQ ID NO: 154 (bold indicating the HsORM1 sequences and the non-bolded indicating the signal sequence amino acids).

[0139] The expression construct was transformed into a Pichia pastoris (also referred to as K. phaffii) mutS strain, primary transformants were selected and then subjected to a 96 h time course using methanol as an inducer of HsORM1 transcription. Expression was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of culture supernatants. Pichia-expressed HsOrm1 migrated as six distinct polypeptide species (see FIG. 8, below); the lowest molecular weight species (21.5 kDa) is predicted to be the non-glycosylated form, and the other forms likely correspond to mono- through penta-glycosylated forms. To demonstrate that Pichia expressed HsORM1 possesses high mannose glycans, the HsOrm1-containing supernatant from Strain 4 was treated in vitro with 1000 units of Endoglucanase H (EH) for 1 h at 37° C. Following EH treatment, the sample was analyzed by SDS-PAGE and only the fully deglycosylated 21.5 kDa polypeptide species remained, further supporting the observation that this is the fully de-glycosylated form.

[0140] FIG. 8: Left panel—MW is a molecular weight protein reference ladder; the lanes to the right of MW are individual transformants expressing HsORM1. Right panel—lane 1 is the molecular weight protein reference ladder; lane 2 is an extract of a transformant expressing HsOrm1; lane 3 is extract of the same transformant treated with endoglycosidase H. Black arrow indicates exogenously added Endo H enzyme; grey arrow indicates in vitro deglycosylated HsOrm1 protein species at 21.5 kDa.

[0141] Following strain purification, Strain 4 (corresponding to well C11 supernatant; red arrow above) was made competent for DNA electroporation and subsequently transformed with the TrMDS2 cDNA expression construct under control of the methanol inducible promoter (SEQ ID NO: 38) and a methanol-inducible transcriptional terminator. HsORM1.sup.+/Pex11-TrMDS2 co-expressors were selected for by their HsORM1 band-shifting patterns following a 96 h time course experiment in methanol-containing induction media. FIGS. 9A and 9B show the banding pattern of HsORM1 on SDS-PAGE of the putative TrMDS2 transformants.

[0142] For a subset of the above tested transformants, the presence of TrMDS2 was verified by PCR using primers to amplify an internal 1066 bp PCR product in the open reading frame, as shown in FIG. 9C.

[0143] PCR produced a 1066 bp product is all of the tested transformants A2, A8, B3, C3, C7, D3, E4, F4, G8, whereas the PCR product was not found in an untransformed control.

[0144] Following the initial induction experiments, a subset of the HsORM1+/TrMDS2 co-expressors were compared for degree of HsORM1 deglycosylation (FIG. 10 below. From left to right, PCR-genotyped strains (positive for the TrMDS2 construct) displayed varying levels of HsOrm1 deglycosylation from very slight to significant deglycosylation, as observed by the increase in smaller HsORM1 polypeptide species on SDS-PAGE. The comparison of these strains indicated that the extent of deglycosylation of an expressed animal protein (such as HsOrm1) can be fine-tuned by selection of a variety of levels of deglycosylation patterns, such as created by differing levels of TrMDS2 expression.

Example 8: Deglycosylation of Ovalbumin (OVA)

[0145] Native G. gallus ovalbumin (OVA) is post-translationally modified by asparagine-linked (N-linked) glycosylation at amino acid residue 292 (SEQ ID NO: 26 in BOLD font) and it has also been noted in the literature that amino acid residue 311 is occasionally glycosylated (SEQ ID NO: 26 BOLD/underlined font).

[0146] An OVA expression construct was made containing the Pichia codon-biased ovalbumin cDNA under transcriptional control of an a methanol inducible promoter and a methanol-inducible terminator. This multicopy expression construct was subsequently transformed into a mutS Pichia strain Strain 5 to create Strain 6. Pichia strain Strain 6 was then subjected to antibiotic resistance marker (ARM) removal to create Strain 7, and this strain subsequently made competent for TrMDS2 transformation.

[0147] Following Pichia DNA transformation, expressed recombinant OVA (rOVA) appeared in culture supernatants of transformants as three distinct species following a 96 h timecourse in methanol-containing media; unglycosylated and mono- and diglycosylated that migrate together as a triplet on SDS-PAGE (see “Input” FIG. 11). To further characterize the OVA expressed by Pichia, supernatants were treated in vitro with commercially available endoglycosidases, EndoH (EH; New England Biolabs) and PNGase (PF; New England Biolabs) using both “native” (N) and “denaturing” (D) protocols for each, as described by the manufacturer (https://www.neb.com/protocols/2012/10/18/endo-hf-protocol; https://www.nebcom/protocols/2014/07/31/pngase-f-protocol). Treatment using either of the endoglycosidases leads to the band-shifted pattern of unglycosylated OVA. The black arrow indicates PNGase F added to the reaction and the grey arrow on the gel indicates the Endo H added to the reaction; the bands appearing above the grey and black arrows are the deglycosylated OVA protein.

[0148] An OVA-expressing Pichia strain (Strain 7; described above) was transformed with the Methanol-inducible-TrMDS2 construct (see Example 7). OVA.sup.+/TrMDS2.sup.+ transformants were subjected to 10% SDS-PAGE to visualize band-shifting patterns. Shown in FIG. 12, below, is a molecular weight (MW) ladder (lane 1, far left). Lanes labelled “C” contain rOVA produced by the parental OVA-expressing strain (no TrMDS2). Lanes A9, D10, F5, G5, G7, G10, H1 and H2 are from OVA strains transformed with the methanol inducible-TrMDS2 construct. These results suggest that TrMDS2 is capable of removing approximately 1.5-2.5 kDa in carbohydrate from each glycan chain on the Pichia-expressed rOVA.

[0149] Transformants were verified by PCR for the presence of TrMDS2 (see Example 7). Transformants A9, D10, F5, G5, G7, G10, H1 and H2 (all shown in the band-shifting gel above) were TrMDS2 positive transformants.

Example 9: Tr MDS1 Testing

[0150] Two different codon-biased TrMDS1 constructs were transformed into a strain expressing Gallus gallus OVD (GgOVD). For expression, the TrMDS1 was placed behind several inducible and constitutive promoters. Construct 1 was engineered for expression of a non-Pichia codon biased (NCO) TrMDS1 cDNA behind the constitutive promoter, construct 2 was engineered for expression of a Pichia codon-optimized (CO) TrMDS1 cDNA behind the constitutive GAP1 promoter, construct 3 was engineered for expression of a Pichia codon-optimized TrMDS1 cDNA behind a methanol-inducible promoter, construct 4 was engineered for expression of a Pichia codon-optimized TrMDS1 cDNA behind a methanol-inducible promoter, construct 5 was engineered for expression of non-Pichia codon-optimized TrMDS1 cDNA behind a methanol-inducible promoter and construct 6 was engineered for expression of a non-Pichia codon-optimized TrMDS1 cDNA behind a methanol-inducible promoter.

[0151] Following a timecourse under methanol induction, supernatants were analyzed for GgOVD band shifts. Despite efforts to express these many versions of MDS1, bandshift analysis indicated that the MDS1 was unable to deglycosylate GgOVD. This was in contrast to the new mannosidases exemplified above, MDS2 and the Gallus mannosidase.

[0152] Bandshift gels showing the lack of deglycosylation activity of MDS1 on GgOVD are shown in FIG. 13. Gel 1 (left to right): Molecular weight ladder, Construct 2 GAP-CO_TrMDS1 transformants 1-8, GgOVD strain alone (no mannosidase expression), Construct 1 constitutive-NCO_TrMDS1 transorformant 1, Construct 3 methanol-inducible-TrMDS1 transformants 1 and 2, GgOVD strain alone (no mannosidase expression), Construct 3 transorformant 3.

[0153] FIG. 14: Gel 2 (left to right): GgOVD strain alone (no mannosidase expression), Molecular weight ladder, Construct 4 methanol inducible-CO_TrMDS1 transformants 1-8, GgOVD strain alone (no mannosidase expression), Construct 5 methanol inducible-CO_TrMDS1 transformants 1-4.

[0154] In total, 240 separate transformants of MDS1 constructs were screened for the ability to deglycosylate GgOVD and none had activity.

Example 10: Comparison of OVD Glycosylation Patterns

[0155] Dry powders consisting of protein samples from Pichia fermentations and from a commercially available source of native chicken ovomucoid were analyzed for total crude protein using a standard combustion method. In this method, total crude protein is calculated from the nitrogen content of the feed material, based on sample type and presented as Percent Protein for the powder in Table 4. The protein factor applied to the nitrogen result is 6.25. The method has a detection limit of 0.1% protein (dry basis). MDS2 (Seq 7) was co-expressed in a Pichia cell along with chicken OVD and the resulting recombinant OVD (rOVD) was purified from the fermentation supernatant using standard protein chromatography methods. Non-protein contaminants were removed from the resulting protein solution using membrane filtration. The purified protein solution was dried to powder using lyophilization. The protein powder was then sent for total crude protein analysis. rOVD powder produced without any MDS2 function had 74% protein on average but that went up to 85% protein when MDS2 was co-expressed. The 85% MDS2-processed material was also a higher % protein relative to the native chicken OVD sample OVD, due to the function of MDS2 removing carbohydrate on the protein.

TABLE-US-00004 TABLE 4 Protein content of OVD samples Sample type Strain N (Total) % Protein rOVD with MDS2 Strain 2 13.7  85.625 rOVD no Strain 1 Not 74 deglycosylation available Native OVD repeat 1 — 12.35 77.1875 Native OVD repeat 2 — 12.44 77.75

TABLE-US-00005 TABLE 5 Sequences Protein SEQ ID NO Sequence MDS1 SEQ ID NO: 1 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN GLLFINTTIASIAAKEEGVSLEKREAEAATKRGSPNPTRAAAVKAAFQTSWNAYHHFAFP HDDLHPVSNSFDDERNGWGSSAIDGLDTAILMGDADIVNTILQYVPQINFTTTAVANQGS SVFETNIRYLGGLLSAYDLLRGPFSSLATNQTLVNSLLRQAQTLANGLKVAFTTPSGVPD PTVFFNPTVRRSGASSNNVAEIGSLVLEWTRLSDLTGNPQYAQLAQKGESYLLNRKGSPE AWPGLIGTFVSTSNGTFQDSSGSWSGLMDSFYEYLIKMYLYDPVAFAHYKDRWVLGAD STIGHLGSHPSTRKDLTFLSSYNGQSTSPNSGHLASFGGGNFILGGILLNEQKYIDFGIKLA SSYFGTYTQTASGIGPEGFAWVDSVTGAGGSPPSSQSGFYSSAGFWVTAPYYILRPETLES LYYAYRVTGDSKWQDLAWEALSAIEDACRAGSAYSSINDVTQANGGGASDDMESFWF AEALKYAYLIFAEESDVQVQATGGNKFVFNTEAHPFSIRSSSRRGGHLA* XP_417735.4 SEQ ID NO: 2 MVLPRKLPGMPGWPAALGLRLPQKFLFLLFLSGLLTLCFGALFLLPDSSRFKRLFLPRRA PREDICTED: TSSSSSSSSSSTRDTELPRSPPAAAEPRHASPAAPRRLREKLRARNAAPAAHTAPASRPQG mannosyl- PDGERPAEVGTGAPRESRAPFHFDYERFRQSLRHPVRGGRPDQDPDTRARKMKIKEMM oligosaccharide KFAWDNYKQYALGKNELRPLTKNGHIGNMFGGLRGATVVDALDTLYIMELEEEFQEAK 1,2-alpha- TWVEKSFDLNVNGEASLFEVNIRYIGGLLAAYYLTGEEVFKSKALELGEKLLPAFNTPTG mannosidase IC  IPRGVINLGSGMSWSWGWASAGSSILAEFGTLHLEFLHLSELSGNPVFAEKVLNIRKVLK [Gallus gallus] RVEKPQGLYPNFLSPVTGNWVQHHVSIGGLGDSFYEYLIKSWLMSDKKDSEAKKMYDD ALEAIEKHLVKKSAGGLTYIAEWRGGILDHKMGHLACFSGGMIALGAEHGGEERKQHY MDLAAEITNTCHESYARSDTKLGPEAFRFDAGTEAMATRLSERYYILRPEVVESYVYMW RLTHDVKYRQWGWEVVKALEKHCRVEAGFSGIRDVYTTVPTHDNMQQSFFLAETLKY LYLLFCEDDVLSLDDWVFNTEAHPLPVNHSNFKAKASVQ* no5ManI SEQ ID NO: 3 MRCSLFLRLHYESYFWTTLPTNYPPKQIRPLPTTSPLKFPKIQAASPSELPEALKTRLQRQT AVKDVFSKCWASYKRHAWKADELAPVSGGQKNPFGGWAATLVDSLDTLYLMDMKPE FDEAVAAAASIDFTKTDLDEVNVFETTIRYLGGFLSAYDLSADARLLSKAVEVGEMLYH AFDTPNRMPITRWAIHAAMAGKKQVAPAGLLVAEIGSLSMEFTRLSMLTRDPKWFDAV QRITEGMAAQQNATALPGLWPLVVSAQDEIYSVGDTFTLGAMADSVYEYLPKMSALTG GQLPVYREMYEAAMATALKHNLFRPMTPSNQDILVAGTVKADGGVKTTLEPQGQHLV CFLGGLLTLGGKLFGRQQDLDAARRLVDGCVWTYKALPRGIMPETFFMLPCPSSTCAW DEASWKRGVLARAAKDAADKASDDDDADAIISRDRLPKGFTSIPDRRYILRPEAIESVFV SYRATAEPSLMESAWDMFTAINATTSTRLANSAYWDVTRPMGEDPGMADSMESFWMG ETLKYFYLVFAAWDDVSLDEWVFNTEAHPFRRLLP* no4ManI SEQ ID NO: 4 MLNQLQGRVPRRYIALVAFAFFVAFLLWSGYDFVPRTATVGRFKYVPSSYDWSKAKVY YPVKDMKTLPQGTPVTFPRLQLRNQSEAQDDTTKARKQAVKDAFVKSWEAYKTYAWT KDQLQPLSLSGKETFSGWSAQLVDALDTLWIMDLKDDFFLAVKEVAVIDWSKTKDNKV INLFEVTIRYLGGLIAAYDLSQEPVLRAKAIELGDTLYATFDTPNRLPSHWLDYSKAKKG TQRADDSMSGAAGGTLCMEFTRLSQITGDPKYYDATERIKQFFYRFQNETTLPGMWFV MMNYREETMVESRYSMGGSADSLYEYLVKMPALLGGLDPQYPEMAIRALDTARDNLL FRPMTEKGDNILALGNALVDHGNVQRTTEMQHLTCFAGGMYAMAGKLFKRDDYVDLG SRISSGCVWAYDSFPSGIMPESADMAACAKLDGPCPYDEVKAPVDPDGRRPHGFIHVKS RHYLLRPEAIESVFYMWRITGDQVWRDTAWRMWENIVREAETEHAFAIVEDVTRTASK LTNNTYLLQTFWLAETLKYFYLIFDDESAIDLDKWVFNTEAHPFKRPAV* no3ManI SEQ ID NO: 5 MVMLVAIALAWLGCSLLRPVDAMRADYLAQLRQETVDMFYHGYSNYMEHAFPEDELR PISCTPLTRDRDNPGRISLNDALGNYSLTLIDSLSTLAILAGGPQNGPYTGPQALSDFQDG VAEFVRHYGDGRSGPSGAGIRARGFDLDSKVQVFETVIRGVGGLLSAHLFAIGELPITGY VPRPEGVAGDDPLELAPIPWPNGFRYDGQLLRLALDLSERLLPAFYTPTGIPYPRVNLRSG IPFYVNSPLHQNLGEAVEEQSGRPEITETCSAGAGSLVLEFTVLSRLTGDARFEQAAKRAF WEVWHRRSEIGLIGNGIDAERGLWIGPHAGIGAGMDSFFEYALKSHILLSGLGMPNASTS RRQSTTSWLDPNSLHPPLPPEMHTSDAFLQAWHQAHASVKRYLYTDRSHFPYYSNNHR ATGQPYAMWIDSLGAFYPGLLALAGEVEEAIEANLVYTALWTRYSALPERWSVREGNV EAGIGWWPGRPEFIESTYHIYRATRDPWYLHVGEMVLRDIRRRCYAECGWAGLQDVQT GEKQDRMESFFLGETAKYMYLLFDPDHPLNKLDAAYVFTTEGHPLIIPKSKRGSGSHNR QDRARKAKKSRDVAVYTYYDESFTNSCPAPRPPSEHHLIGSATAARPDLFSVSRFTDLYR TPNVHGPLEKVEMRDKKKGRVVRYRATSNHTIFPWTLPPAMLPENGTCAAPPERIISLIEF PANDITSGITSRFGNHLSWQTHLGPTVNILEGLRLQLEQVSDPATGEDKWRITHIGNTQLG RHETVFFHAEHVRHLKDEVFSCRRRRDAVEIELLVDKPSDTNNNNTLASSDDDVVVDAK AEEQDGMLADDDGDTLNAETLSSNSLFQSLLRAVSSVFEPVYTAIPESDPSAGTAKVYSF DAYTSTGPGAYPMPSLSDTPIPGNPFYNFRNPASNFPWSTVFLAGQACEGPLPASAPREHQ VTVMLRGGCSFSRKLDNIPSFSPHDRALQLVVVLDEPPPPPPPPPANDRRDVTRPLLDTEQ TTPKGMKRLHGIPMVLVRAARGDYELFGHAIGVGMRRKYRVESQGLVVENAVVL* no2ManI SEQ ID NO: 6 MRFPSSSVLALGLIGPALAYPKPGATKRGSPNPTRAAAVKAAFQTSWNAYHHFAFPHDD LHPVSNSFDDERNGWGSSAIDGLDTAILMGDADIVNTILQYVPQINFTTTAVANQGISVFE TNIRYLGGLLSAYDLLRGPFSSLATNQTLVNSLLRQAQTLANGLKVAFTTPSGVPDPTVF FNPTVRRSGASSNNVAEIGSLVLEWTRLSDLTGNPQYAQLAQKGESYLLNPKGSPEAWP GLIGTFVSTSNGTFQDSSGSWSGLMDSFYEYLIKMYLYDPVAFAHYKDRWVLAADSTIA HLASHPSTRKDLTFLSSYNGQSTSPNSGHLASFAGGNFILGGILLNEQKYIDFGIKLASSYF ATYNQTASGIGPEGFAWVDSVTGAGGSPPSSQSGFYSSAGFWVTAPYYILRPETLESLYY AYRVTGDSKWQDLAWEAFSAIEDACRAGSAYSSINDVTQANGGGASDDMESFWFAEAL KYAYLIFAEESDVQVQANGGNKFVFNTEAHPFSIRSSSRRGGHLA* no1ManI SEQ ID NO: 7 MARRRYRLFMICAAVILFLLYRVSQNTWDDSAHYATLRHPPASNPPAAGGESPLKPAAK PEHEHEHENGYAPESKPKPQSEPKPESKPAPEHAAGGQKSQGKPSYEDDEETGKNPPKSA VIPSDTRLPPDNKVHWRPVKEHFPVPSESVISLPTGKPLKVPRVQHEFGVESPEAKSRRVA RQERVGKEIERAWSGYKKFAWMHDELSPVSAKHRDPFCGWAATLVDSLDTLWIAGLKE QFDEAARAVEQIDFTTTPRNNIPVFETTIRYLGGLLGAFDVSGGHDGGYPMLLTKAVELA EILMGIFDTPNRMPILYYQWQPEYASQPHRAGSVGIAELGTLSMEFTRLAQLTSQYKYYD AVDRITDALIELQKQGTSIPGLFPENDASGCNHTATALRSSLSEAAQKQMDEDLSNKPE NYRPGKNSKADPQTVEKQPAKKQNEPVEKAKQVPTQQTAKRGKPPFGANGFTANWDC VPQGLVVGGYGFQQYHMGGGQDSAYEYFPKEYLLLGGLESKYQKLYVDAVEAINEWL LYRPMTDGDWDILFPAKVSTAGNPSQDLVATFEVTHLTCFIGGMYGLGGKIFGREKDLE TAKRLTDGCVWAYQSTVSGIMPEGSQVLACPTLEKCDFNETLWWEKLDPAKDWRDKQ YADDKDKATVGEALKETANSHDAAGGSKAVHKRAAVPLPKPGADDDVGSELPQSLKD KIGFKNGEQKKPTGSSVGIQRDPDAPVDSVLEAHRLPPQEPEEQQVILPDKPQTHEEFVK QRIAEMGFAPGVVHIQSRQYILRPEAIESVWYMYRITGDPIWMEKGWKMFEATIRATRTE INSAIDDVNSEEPGLKDEMESFWLAETLKYYYLLFSEPSVISLDEWVLNTEAHPFKRPG GSYIGHSI* patMannI SEQ ID NO: 8 MRFPSSSVLALGLIGPALAYPKPGATKRGSPNPTRAAAVKAAFQTSWNAYHHFAFPHDD LHPVSNSFDDERNGWGSSAIDGLDTAILMGDADIVNTILQYVPQINFTTTAVANQGISVFE TNIRYLGGLLSAYDLLRGPFSSLATNQTLVNSLLRQAQTLANGLKVAFTTPSGVPDPTVF FNPTVRRSGASSNNVAEIGSLVLEWTRLSDLTGNPQYAQLAQKGESYLLNPKGSPEAWP GLIGTFVSTSNGTFQDSSGSWSGLMDSFYEYLIKMYLYDPVAFAHYKDRWVLAADSTIA HLASHPSTRKDLTFLSSYNGQSTSPNSGHLASFAGGNFILGGILLNEQKYIDFGIKLASSYF ATYNQTASGIGPEGFAWVDSVTGAGGSPPSSQSGFYSSAGFWVTAPYYILRPETLESLYY AYRVTGDSKWQDLAWEAFSAIEDACRAGSAYSSINDVTQANGGGASDDMESFWFAEAL KYAYLIFAEESDVQVQANGGNKFVFNTEAHPFSIRSSSRRGGHLA* AAF34579.1 1,2-a- SEQ ID NO: 9 MRFPSSSVLALGLIGPALAYPKPGATKRGSPNPTRAAAVKAAFQTSWNAYHHFAFPHDD D-mannosidase LHPVSNSFDDERNGWGSSAIDGLDTAILMGDADIVNTILQYVPQINFTTTAVANQGSSVF [Trichoderma ETNIRYLGGLLSAYDLLRGPFSSLATNQTLVNSLLRQAQTLANGLKVAFTTPSGVPDPTV reesei] FFNPTVRRSGASSNNVAEIGSLVLEWTRLSDLTGNPQYAQLAQKGESYLLNPKGSPEAW PGLIGTFVSTSNGTFQDSSGSWSGLMDSFYEYLIKMYLYDPVAFAHYKDRWVLGADSTI GHLGSHPSTRKDLTFLSSYNGQSTSPNSGHLASFGGGNFILGGILLNEQKYIDFGIKLASSY FGTYTQTASGIGPEGFAWVDSVTGAGGSPPSSQSGFYSSAGFWVTAPYYILRPETLESLY YAYRVTGDSKWQDLAWEALSAIEDACRAGSAYSSINDVTQANGGGASDDMESFWFAE ALKYAYLIFAEESDVQVQATGGNKFVFNTEAHPFSIRSSSRRGGHLA* Hypacrea MDS1 SEQ ID NO: 10 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN GLLFINTTIASIAAKEEGVSLEKREAEAATKRGSPNPTRAAAVKAAFQTSWNAYHHFAFP HDDLHPVSNSFDDERNGWGSSAIDGLDTAILMGDADIVNTILQYVPQINFTTTAVANQGS SVFETNIRYLGGLLSAYDLLRGPFSSLATNQTLVNSLLRQAQTLANGLKVAFTTPSGVPD PTVFFNPTVRRSGASSNNVAEIGSLVLEWTRLSDLTGNPQYAQLAQKGESYLLNPKGSPE AWPGLIGTFVSTSNGTFQDSSGSWSGLMDSFYEYLIKMYLYDPVAFAHYKDRWVLGAD STIGHLGSHPSTRKDLTFLSSYNGQSTSPNSGHLASFGGGNFILGGILLNEQKYIDFGIKLA SSYFGTYTQTASGIGPEGFAWVDSVTGAGGSPPSSQSGFYSSAGFWVTAPYYILRPETLES LYYAYRVTGDSKWQDLAWEALSAIEDACRAGSAYSSINDVTQANGGGASDDMESFWF AEALKYAYLIFAEESDVQVQATGGNKFVFNTEAHPFSIRSSSRRGGHLA* α-ovomucin SEQ ID NO: 11 KEPVQIVQVSTVGRSECTTWGNFHFHTFDHVKFTFPGTCTYVFASHCNDSYQDFNIKIRR SDKNSHLIYFTVTTDGVILEVKETGITVNGNQIPLPFSLKSILIEDTCAYFQVTSKLGLTLK WNWADTLLLDLEETYKEKICGLCGNYDGNKKNDLILDGYKMHPRQFGNFHKVEDPSEK CPDVRPDDHTGRHPTEDDNRCSKYKKMCKKLLSRFGNCPKVVAFDDYVATCTEDMCN CVVNSSHSDLVSSCICSTLNQYSRDCVLSKGDPGEWRTKELCYQECPSNMEYMECGNSC ADTCADPERSKICKAPCTDGCFCPPGTILDDLGGKKCVPRDSCPCMFQGKVYSSGGTYST PCQNCTCKGGHWSCTSLPCSGSCSIDGGFHITTFDNKKFNFHGNCHYVLAKNTDDTFVVI GEIIQCGTSKT*MTCLKNVLVTLGRTTIKICSCGSIYMNNFIVKLPVSKDGITIFRPSTFFIKI LSSTGVQIRVQMKPVMQLSITVDHSYQNRTSGLCGNFNNIQTDDFRTATGAVEDSAAAF GNSWKTRASCFDVEDSFEDPCSNSVDKEKFAQHVVCALLSNISSTFAACHSVVDPSVYIKR CMYDTCNAEKSEVALCSVLSTYSRDCAAAGMTLKGWRQGICDPSEECPETMVYNYSVK YCNQSCRSLDEPDPLCKVQIAPMEGCGCPEGTYLNDEEECVTPDDCPCYYKGKIVQPGN SFQEDKLLCKCIQGRLDCIGETVLVKDCPAPMYYFNCSSAGPGAIGSECQKSCKTQDMH CYVTECVSGCMCPDGLVLDGSGGCIPKDQCPCVHGGHFYKPGETIRVDCNTCTCNKRQ WNCTDSPCKGTCTVYGNGHYMSFDGEKFDFLGDCDYILAQDFCPNNMDAGTFRIVIQN NACGKSLSICSLKITLIFESSEIRLLEGRIQEIATDPGAEKNYKVDLRGGYIVIETTQGMSFM WDQKTTVVVHVTPSFQGKVCGLCGDFDGRSRNDFTTRGQSVEMSIQEFGNSWKITSTCS NINMTDLCADQPFKSALGQKHCSIIKSSVFEACHSKVNPIPYYESCVSDFCGCDSVGDCEC FCTSVAAYARSCSTAGVCINWRTPAICPVFCDYYNPPDKHEWFYKPCGAPCLKTCRNPQ GKCGNILYSLEGCYPECSPDKPYFDEERRECVSLPDCTSCNPEEKLCTEDSKDCLCCYNG KTYPLNETIYSQTEGTKCGNAFCGPNGMIIETFIPCSTLSVPAQEQLMQPVTSAPLLSTEAT PCFCTDNGQLIQMGENVSLPMNISGHCAYSICNASCQIELIWAECKVVQTEALETCEPNSE ACPPTAAPNATSLVPATALAPMSDCLGLIPPRKFNESWDFGNCQIATCLGEENNIKLSSIT CPPQQLKLCVNGFPFMKHHDETGCCEVFECQCICSGWGNEHYVTFDGTYYHFKENCTY VLVELIQPSSEKFWIHIDNYYCGAADGAICSMSLLIFHSNSLVILTQAKEHGKGTNLVLFN DKKVVPDISKNGIRITSSGLYIIVEIPELEVYVSYSRLAFYIKLPFGKYYNNTMGLCGTCTN QKSDDARKRNGEVTDSFKEMALDWKAPVSTNRYCNPGISEPVKIENYQHCEPSELCKII WNLTECHRVVPPQPYYEACVASRCSQQHPSTECQSMQTYAALCGLHGICVDWRGQTNG QCEATCARDQVYKPCGEAKRNTCFSREVIVDTLLSRNNTPVFVEGCYCPDGNILLNEHD GICVSVCGCTAQDGSVKKPREAWEHDCQYCTCDEETLNISCFPRPCAKSPPINCTKEGFV RKIKPRLDDPCCTETVCECDIKTCIINKTACDLGFQPVVAISEDGCCPIFSCIPKGVCVSEG VEFKPGAVVPKSSCEDCVCTDEQDAVTGTNRIQCVPVKCQTTCQQGFRYVEKEGQCCSQ CQQVACVANFPFGSVTIEVGKSYKAPYDNCTQYTCTESGGQFSLTSTVKVCLPFEESNCV PGTVDVTSDGCCKTCIDLPHKCKRSMKEQYIVHKHCKSAAPVPVPFCEGTCSTYSVYSFE NNEMEHKCICCHEKKSHVEKVELVCSEHKTLKFSYVHVDECGCVETKCPMRRT* Ovomucoid SEQ ID NO: 12 AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDG (canonical) ECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGAS VDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNG TLTLSHFGKC* Ovomucoid SEQ ID NO: 13 AEVDCSRFPNATDMEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSVEFGTNISKEHD GECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQG ASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVES NGTLTLSHFGKC* Ovomucoid SEQ ID NO: 14 AEVDCSRFPNATDMEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSVEFGTNISKEHD G162MF167A GECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQG ASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYMNKCNACNAVVE SNGTLTLSHFGKC* Ovoglobulin G2 SEQ ID NO: 15 TRAPDCGGILTPLGLSYLAEVSKPHAEVVLRQDLMAQRASDLFLGSMEPSRNRITSVKVA DLWLSVIPEAGLRLGIEVELRIAPLHAVPMPVRISIRADLHVDMGPDGNLQLLTSACRPTV QAQSTREAESKSSRSILDKVVDVDKLCLDVSKLLLFPNEQLMSLTALFPVTPNCQLQYLP LAAPVFSKQGIALSLQTTFQVAGAVVPVPVSPVPFSMPELASTSTSHLILALSEHFYTSLYF TLERAGAFNMTIPSMLTTATLAQKITQVGSLYHEDLPITLSAALRSSPRVVLEEGRAALKL FLTVHIGAGSPDFQSFLSVSADVTAGLQLSVSDTRMMISTAVIEDAELSLAASNVGLVRA ALLEELFLAPVCQQVPAWMDDVLREGVHLPHLSHFTYTDVNVVVHKDYVLVPCKLKLR STMA* Ovoglobulin G3 SEQ ID NO: 16 MDSISVTNAKFCFDVFNEMKVHHVNENILYCPLSILTALAMVYLGARGNTESQMKKVL HFDSITGAGSTTDSQCGSSEYVHNLFKELLSEITRPNATYSLEIADKLYVDKTFSVLPEYLS CARKFYTGGVEEVNFKTAAEEARQLINSWVEKETNGQIKDLLVSSSIDFGTTMVFINTIYF KGIWKIAFNTEDTREMPFSMTKEESKPVQMMCMNNSFNVATLPAEKMKILELPYASGDL SMLVLLPDEVSGLERIEKTINFDKLREWTSTNAMAKKSMKVYLPRMKIEEKYNLTSILM ALGMTDLFSRSANLTGISSVDNLMISDAVHGVFMEVNEEGTEATGSTGAIGNIKHSLELE EFRADHPFLFFIRYNPTNAILFFGRYWSP* β-ovomucin SEQ ID NO: 17 CSTWGGGHFSTFDKYQYDFTGTCNYIFATVCDESSPDFNIQFRRGLDKKIARIIIELGPSVII VEKDSISVRSVGVIKLPYASNGIQIAPYGRSVRLVAKLMEMELVVMWNNEDYLMVLTE KKYMGKTCGMCGNYDGYELNDFVSEGKLLDTYKFAALQKMDDPSEICLSEEISIPAIPH KKYAVICSQLLNLVSPTCSVPKDGFVTRCQLDMQDCSEPGQKNCTCSTLSEYSRQCAMS HQVVFNWRTENFCSVGKCSANQIYEECGSPCIKTCSNPEYSCSSHCTYGCFCPEGTVLDD ISKNRTCVHLEQCPCTLNGETYAPGDTMKAACRTCKCTMGQWNCKELPCPGRCSLEGG SFVTTFDSRSYRFHGVCTYILMKSSSLPHNGTLMAIYEKSGYSHSETSLSAIIYLSTKDKIVI SQNELLTDDDELKRLPYKSGDITIFKQSSMFIQMHTEFGLELVVQTSPVFQAYVKVSAQF QGRTLGLCGNYNGDTTDDFMTSMDITEGTASLFVDSWRAGNCLPAMERETDPCALSQL NKISAETHCSILTKKGTVFETCHAVVNPTPFYKRCVYQACNYEETFPYICSALGSYARTCS SMGLILENWRNSMDNCTITCTGNQTFSYNTQACERTCLSLSNPTLECHPTDIPIEGCNCPK GMYLNHKNECVRKSHCPCYLEDRKYILPDQSTMTGGITCYCVNGRLSCTGKLQNPAESC KAPKKYISCSDSLENKYGATCAPTCQMLATGIECIPTKCESGCVCADGLYENLDGRCVPP EECPCEYGGLSYGKGEQIQTECEICTCRKGKWKCVQKSRCSSTCNLYGEGHITTFDGQRF VFDGNCEYILAMDGCNVNRPLSSFKIVTENVICGKSGVTCSRSISIYLGNLTIILRDETYSIS GKNLQVKYNVKKNALHLMFDIIIPGKYNMTLIWNKHMNFFIKISRETQETICGLCGNYNG NMKDDFETRSKYVASNELEFVNSWKENPLCGDVYFVVDPCSKNPYRKAWAEKTCSIINS QVFSACHNKVNRMPYYEACVRDSCGCDIGGDCECMCDAIAVYAMACLDKGICIDWRTP EFCPVYCEYYNSHRKTGSGGAYSYGSSVNCTWHYRPCNCPNQYYKYVNIEGCYNCSHD EYFDYEKEKCMPCAMQPTSVTLPTATQPTSPSTSSASTVLTETTNPPV* Lysozyme SEQ ID NO: 18 KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQI NSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCK GTDVQAWIRGCRL* Lysozyme SEQ ID NO: 19 KVFGRCELAAAMKRHGLDNYRGYSLGNWVCVAKFESNFNTQATNRNTDGSTDYGILQI NSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMSAWVAWRNRCK GTDVQAWIRGCRL* Lysozyme C SEQ ID NO: 20 KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIF (Human) QINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRN RCQNRDVRQYVQGCGV* Lysozyme C (Bos SEQ ID NO: 21 KVFERCELARTLKKLGLDGYKGVSLANWLCLTKWESSYNTKATNYNPSSESTDYGIFQI taurus) NSKWWCNDGKTPNAVDGCHVSCRELMENDIAKAVACAKHIVSEQGITAWVAWKSHCR DHDVSSYVEGCTL* Ovoinhibitor SEQ ID NO: 22 IEVNCSLYASGIGKDGTSWVACPRNLKPVCGTDGSTYSNECGICLYNREHGANVEKEYD GECRPKHVMIDCSPYLQVVRDGNTMVACPRILKPVCGSDSFTYDNECGICAYNAEHHTN ISKLHDGECKLEIGSVDCSKYPSTVSKDGRTLVACPRILSPVCGTDGFTYDNECGICAHNA EQRTHVSKKHDGKCRQEIPEIDCDQYPTRKTTGGKLLVRCPRILLPVCGTDGFTYDNECG ICAHNAQHGTEVKKSHDGRCKERSTPLDCTQYLSNTQNGEAITACPFILQEVCGTDGVTY SNDCSLCAHNIELGTSVAKKHDGRCREEVPELDCSKYKTSTLKDGRQVVACTMIYDPVC ATNGVTYASECTLCAHNLEQRTNLGKRKNGRCEEDITKEHCREFQKVSPICTMEYVPHC GSDGVTYSNRCFFCNAYVQSNRTLNLVSMAAC* Cystatin SEQ ID NO: 23 MAGARGCVVLLAAALMLVGAVLGSEDRSRLLGAPVPVDENDEGLQRALQFAMAEYNR ASNDKYSSRVVRVISAKRQLVSGIKYILQVEIGRTTCPKSSGDLQSCEFHDEPEMAKYTTC TFVVYSIPWLNQIKLLESKCQ* Ovalbumin related SEQ ID NO: 24 MFFYNTDFRMGSISAANAEFCFDVFNELKVQHTNENILYSPLSIIVALAMVYMGARGNTE protein X YQMEKALHFDSIAGLGGSTQTKVQKPKCGKSVNIHLLLFKELLSDITASKANYSLRIANRL YAEKSRPILPIYLKCVKKLYRAGLETVNFKTASDQARQLINSWVEKQTEGQIKDLLVSSS TDLDTTLVLVNAIYFKGMWKTAFNAEDTREMPFHVTKEESKPVQMMCMNNSFNVATL PAEKMKILELPFASGDLSMLVLLPDEVSGLERIEKTINFEKLTEWTNPNTMEKRRVKVYL PQMKIEEKYNLTSVLMALGMTDLFIPSANLTGISSAESLKISQAVHGAFMELSEDGIEMA GSTGVIEDIKHSPELEQFRADHPFLFLIKHNPTNTIVYFGRYWSP* Ovalbumin related SEQ ID NO: 25 MDSISVTNAKFCFDVFNEMKVHHVNENILYCPLSILTALAMVYLGARGNTESQMKKVL protein Y HFDSITGAGSTTDSQCGSSEYVHNLFKELLSEITRPNATYSLEIADKLYVDKTFSVLPEYLS CARKFYTGGVEEVNFKTAAEEARQLINSWVEKETNGQIKDLLVSSSIDFGTTMVFINTIYF KGIWKIAFNTEDTREMPFSMTKEESKPVQMMCMNNSFNVATLPAEKMKILELPYASGDL SMLVLLPDEVSGLERIEKTINFDKLREWTSTNAMAKKSMKVYLPRMKIEEKYNLTSILM ALGMTDLFSRSANLTGISSVDNLMISDAVHGVFMEVNEEGTEATGSTGAIGNIKHSLELE EFRADHPFLFFIRYNPTNAILFFGRYWSP* Ovalbumin SEQ ID NO: 26 MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVR FDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQC VKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVF KGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGT MSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVL MAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEE FRADHPFLFCIKHIATNAVLFFGRCVSP* Porcine Lipase SEQ ID NO: 27 SEVCFPRLGCFSDDAPWAGIVQRPLKILPWSPKDVDTRFLLYTNQNQNNYQELVADPSTI TNSNFRMDRKTRFIIHGFIDKGEEDWLSNICKNLFKVESVNCICVDWKGGSRTGYTQASQ NIRIVGAEVAYFVEVLKSSLGYSPSNVHVIGHSLGSHAAGEAGRRTNGTIERITGLDPAEP CFQGTPELVRLDPSDAKFVDVIHTDAAPIIPNLGFGMSQTVGHLDFFPNGGKQMPGCQK NILSQIVDIDGIWEGTRDFVACNHLRSYKYYADSILNPDGFAGFPCDSYNVFTANKCFPCP SEGCPQMGHYADRFPGKTNGVSQVFYLNTGDASNFARWRYKVSVTLSGKKVTGHILVS LFGNEGNSRQYEIYKGTLQPDNTHSDEFDSDVEVGDLQKVKFIWYNNNVINPTLPRVGA SKITVERNDGKVYDFCSQETVREEVLLTLNPC* Kid Lipase SEQ ID NO: 28 GLVAADRITGGKDFRDIESKFALRTPEDTAEDTCHLIPGVTESVANCHFNHSSKTFVVIHG WTVTGMYESWVPKLVAALYKREPDSNVIVVDWLSRAQQHYPVSAGYTKLVGQDVAKF MNWMADEFNYPLGNVHLLGYSLGAHAAGIAGSLTSKKVNRITGLDPAGPNFEYAEAPS RLSPDDADFVDVLHTFTRGSPGRSIGIQKPVGHVDIYPNGGTFQPGCNIGEALRVIAERGL GDVDQLVKCSHERSVHLFIDSLLNEENPSKAYRCNSKEAFEKGLCLSCRKNRCNNMGYE INKVRAKRSSKMYLKTRSQMPYKVFHYQVKRIFSGTESNTYTNQAFEISLYGTVAESENI PFTLPEVSTNKTYSFLLYTEVDIGELLMLKLKWISDSYFSWSNWWSSPGFDIGKIRVKAG ETQKKVIFCSREKMSYLQKGKSPVIFVKCHDKSLNRKSG* Porcine SEQ ID NO: 29 APKKGVRWCVISTAEYSKCRQWQSKIRRTNPMFCIRRASPTDCIRAIAAKRADAVTLDG Lactoferrin GLVFEADQYKLRPVAAEIYGTEENPQTYYYAVAVVKKGFNFQLNQLQGRKSCHTGLGR SAGWNIPIGLLRRFLDWAGPPEPLQKAVAKFFSQSCVPCADGNAYPNLCQLCIGKGKDK CACSSQEPYFGYSGAFNCLHKGIGDVAFVKESTVFENLPQKADRDKYELLCPDNTRKPV EAFRECHLARVPSHAVVARSVNGKENSIWELLYQSQKKFGKSNPQEFQLFGSPGQQKDL LFRDATIGFLKIPSKIDSKLYLGLPYLTAIQGLRETAAEVEARQAKVVWCAVGPEELRKC RQWSSQSSQNLNCSLASTTEDCIVQVLKGEADAMSLDGGFIYTAGKCGLVPVLAENQKS RQSSSSDCVHRPTQGYFAVAVVRKANGGITWNSVRGTKSCHTAVDRTAGWNIPMGLLV NQTGSCKFDEFFSQSCAPGSQPGSNLCALCVGNDQGVDKCVPNSNERYYGYTGAFRCLA ENAGDVAFVKDVTVLDNTNGQNTEEWARELRSDDFELLCLDGTRKPVTEAQNCHLAV APSHAVVSRKEKAAQVEQVLLTEQAQFGRYGKDCPDKFCLFRSETKNLLFNDNTEVLA QLQGKTTYEKYLGSEYVTAIANLKQCSVSPLLEACAFMMR* Bovine SEQ ID NO: 30 APRKNVRWCTISQPEWFKCRRWQWRMKKLGAPSITCVRRAFALECIRAIAEKKADAVT Lactoferrin LDGGMVFEAGRDPYKLRPVAAEIYGTKESPQTHYYAVAVVKKGSNFQLDQLQGRKSCH TGLGRSAGWIIPMGILRPYLSWTESLEPLQGAVAKFFSASCVPCIDRQAYPNLCQLCKGE GENQCACSSREPYFGYSGAFKCLQDGAGDVAFVKETTVFENLPEKADRDQYELLCLNNS RAPVDAFKECHLAQVPSHAVVARSVDGKEDLIWKLLSKAQEKFGKNKSRSFQLFGSPPG QRDLLFKDSALGFLRIPSKVDSALYLGSRYLTTLKNLRETAEEVKARYTRVVWCAVGPE EQKKCQQWSQQSGQNVTCATASTTDDCIVLVLKGEADALNLDGGYIYTAGKCGLVPVL AENRKSSKHSSLDCVLRPTEGYLAVAVVKKANEGLTWNSLKDKKSCHTAVDRTAGWNI PMGLIVNQTGSCAFDEFFSQSCAPGADPKSRLCALCAGDDQGLDKCVPNSKEKYYGYTG AFRCLAEDVGDVAFVKNDTVWENTNGESTADWAKNLNREDFRLLCLDGTRKPVTEAQ SCHLAVAPNHAVVSRSDRAAHVKQVLLHQQALFGKNGKNCPDKFCLFKSETKNLLFND NTECLAKLGGRPTYEEYLGTEYVTAIANLKKCSTSPLLEACAFLTR* AOX1 SEQ ID NO: 31 GATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCAC AGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCA GACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATC GAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTAT TAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTT CATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAG ATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAA CTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAA GATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTT CCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATA ATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGC CGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATT GTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAA ATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCT GTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTC CAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAA ACAACTAATTATTGGATCCCGA DAS1 SEQ ID NO: 32 AAATCTGAACACGATGAAACCTCCCCGTAGATTCCACCGCCCCGTTACTTTTTTGGGC AATCCCGTTGATAAGATCCATTTTAGAGTTGTTTCTGAAAGGATTACAGGCGTTGAA GGGTCAGAGAGATGCCAGAGAACAGACCAATTGGTAGTTTGCTAAAGTGGACGTCT GGCAGGTGCTCTATCGTGTTCTTTATTTAGGGCGTTACACTTAGTAGGATTACGTAAC AATTTGGCTTAACCTTCTAAGTTAGAAAGAAACCAAGAGGGGTCCTCTTTAACGTTC AGCAGTATCTAAAACACAAAACCTGCCCTCATAATACATCATTCTATCTGTCAAGCT GTGCTACCCCACAGAAATACCCCCAAGAGTTAAAGTGAAAAGAAAAGCTAAATCTG TTAGACTTCACCCCATAACAAACTTGATAGTTCCTGTAGCCAATGAAAGTTAACCCC ATTCAATGTTCCGAGATCTAGTATGCTTGCTCCTATAAGGAACGAAGGGTTCCAGCTT CCTTACCCCATCAATGGAAATCTCCTATTTACCCCCCACTGGAAAGATCCGTCCGAAC GAACGGATAATAGAAAAAAGAAATTCGGACAAAATAGAACACTTATTTAGCCAATG AAATCCATTTCCAGCATCTCCTTCAACTGCCGTTCCATCCCCTTTGTTGAGCTACACC ATCGTCAGCCAGTACCGAATAGGAAACTTAACCGATATCTTGGAGAATTCTAATGCG CGAATGAGTTTAGCCTAGATATCCTTAGTGAAGGGTTGTTCCGATACTTCTCCACATT CAGTCATTTCAGATGGGCAGCATTGTTATCATGAAGAAACGGAAACGGGCAGTAAG GGTTAACCGCCAAATTATATAAAGACAACATGTCCCCAGTTTAAAGTTTTTCTTTCCT ATTCTTGTATCCTGAGTGACCGTTGTGTTTAAAATAACAAGTTCGTTTTAACTTAAGA CCAAAACCAGTTACAACAAATTATTCCCCAACTAAACACTAAAGTTCACTCTTATCA AACTATCAAACATCAAAG DAS2 SEQ ID NO: 33 CCTGTTGATAAGACGCATTCTAGAGTTGTTTCATGAAAGGGTTACGGGTGTTGATTG GTTTGAGATATGCCAGAGGACAGATCAATCTGTGGTTTGCTAAACTGGAAGTCTGGT AAGGACTCTAGCAAGTCCGTTACTCAAAAAGTCATACCAAGTAAGATTACGTAACAC CTGGGCATGACTTTCTAAGTTAGCAAGTCACCAAGAGGGTCCTATTTAACGTTTGGC GGTATCTGAAACACAAGACTTGCCTATCCCATAGTACATCATATTACCTGTCAAGCT ATGCTACCCCACAGAAATACCCCAAAAGTTGAAGTGAAAAAATGAAAATTACTGGT AACTTCACCCCATAACAAACTTAATAATTTCTGTAGCCAATGAAAGTAAACCCCATT CAATGTTCCGAGATTTAGTATACTTGCCCCTATAAGAAACGAAGGATTTCAGCTTCCT TACCCCATGAACAGAAATCTTCCATTTACCCCCCACTGGAGAGATCCGCCCAAACGA ACAGATAATAGAAAAAAGAAATTCGGACAAATAGAACACTTTCTCAGCCAATTAAA GTCATTCCATGCACTCCCTTTAGCTGCCGTTCCATCCCTTTGTTGAGCAACACCATCG TTAGCCAGTACGAAAGAGGAAACTTAACCGATACCTTGGAGAAATCTAAGGCGCGA ATGAGTTTAGCCTAGATATCCTTAGTGAAGGGTTGTTCCGATACTTCTCCACATTCAG TCATAGATGGGCAGCTTTGTTATCATGAAGAGACGGAAACGGGCATTAAGGGTTAAC CGCCAAATTATATAAAGACAACATGTCCCCAGTTTAAAGTTTTTCTTTCCTATTCTTG TATCCTGAGTGACCGTTGTGTTTAATATAACAAGTTCGTTTTAACTTAAGACCAAAAC CAGTTACAACAAATTATAACCCCTCTAAACACTAAAGTTCACTCTTATCAAACTATCA AACATCAAAAGAATTCGCG FLD1 SEQ ID NO: 34 AAATCAGCCATTAATCTCACCTCAGTTTTMAATCAGTAGAATTITCAATGAAACAA ACGGTTGGTATATTATTTGATAGGGTAGCCAAATTTCCAAAAATGAACTTTTCATCAG GTAATATCTTGAATACCGTAATGTAGTGACTATTGGAAGAAACTGCTATCAAATTAT ATTTCGGATAGAAATCCAAACCCCAGACTGATCTCTTGAGTCTCAACTCTAAGTCAG CCGCGACTCTAATTATCTGTGGATTAGGAGTTAGTGTGGACAAAGCATCAGTATAGT ATAACTTTACGGTTCCATTATCAGACGCTATTGCAAGAACTTCCTTTCCATTGATCTC TCCAATTCGACAGTAATTGATATCATAAGGTAGGTCTGGAAACACACTGGCGCTTGT ATCCCATTCTGCAGGAATTTCTGGAACGGTGGTAATGGTAGTTATCCAACGGAGTTG GGGTAGTTGGTATATCTGGATATGCCGCCTATAGGATAAAAACAGGAGAGAGTGAA CCTTGCTTACGGCTACTAGATTGTTCTTGTACTCGGAATTGTCGTTATCGGAAACTAG ACTAATCTCATCTGTGTGTTGCAGTACTATTGAGTCGTTGTAGTATCTACCAGGAGGG CATTCCATGAACTAGTGAGACAAATGAGTTGGATTTTCTCAATAGACATATGCAAGA ATGCTACACAACGGATGTCGCACTCTTTTTCTTAGTTGATAATATCATCCAATCAGAA GACACGGGCTAGAAGGACTTGCTCCCGAAGGATAATCCACTGCTACTATCTCCCTTV CTCACATATAGTCTTGCAGGGCTCATGCCCCTTTCTCCTTCGAACTGCCCGATGAGGA AGTCTTTAGCCTATCAAGGAATTCGGGACCATCATCAATTTTTAGAGCCTTACCTGAT CGCAATCAGGATTIVACTACTCATATAAATACATCACTCAAACTCCAACTTTGCTTGT TCATACAATTCTTGATATTCACAGGATC PEX8 SEQ ID NO: 35 AAATTAACCAGTGTTTTCTTATCTATTTGTCTTTTTACACTAAAGTGAAGTACGAATC CATGCGATTGATTCCTCCTCAGATATCAGCTGAATTCTTGCTTATGTAATACTTGCGC GAACTACATGTGAACTTAGGATTCGATAAGGCTGGGGGGTCAACCAACCCCACTTCA AAGAGCCGACCCGTATAAATAGCCTCTGCGTCCTCAGATCAACAAGACGAAGCAATT TTTTTTTACCTATCTTCAGGTGCCTGTTAG SHB17 SEQ ID NO: 36 AAATTCTTTTTACGTGGTGCGCATACTGGACAGAGGCAGAGTCTCAATTTCTTCTTTT GAGACAGGCTACTACAGCCTGTGATTCCTCTTGGTACTMGATTTGCTTTTATCTGGC TCCGTTGGGAACTGTGCCTGGGTTTTGAAGTATCTTGTGGATGTGTTTCTAACACTTT TTCAATCTTCTTGGAGTGAGAATGCAGGACTTTGAACATCGTCTAGCTCGTTGGTAGG TGAACCGTTTTACCTTGCATGTGGTTAGGAGTTTTCTGGAGTAACCAAGACCGTCTTA TCATCGCCGTAAAATCGCTCTTACTGTCGCTAATAATCCCGCTGGAAGAGAAGTTCG AACAGAAGTAGCACGCAAAGCTCTTGTCAAATGAGAATTGTTAATCGTTTGACAGGT CACACTCGTGGGCTATGTACGATCAACTTGCCGGCTGTTGCTGGAGAGATGACACCA GTTGTGGCATGGCCAATTGGTATTCAGCCGTACCACTGTATGGAAAATGAGATTATC TTGTTCTTGATCTAGTTTCTTGCCATTTTAGAGTTGCCACATTCGTAGGTTTCAGTACC AATAATGGTAACTTCCAAACTTCCAACGCAGATACCAGAGATCTGCCGATCCTTCCC CAACAATAGGAGCTTACTACGCCATACATATAGCCTATCTATTTTCACTTTCGCGTGG GTGCTTCTATATAAACGGTTCCCCATCTTCCGTITCATACTACTTGAATTTTAAGCACT AAAGAATT FGH1 SEQ ID NO: 37 GTGAATTTGTCACGGAATTGACCAAGAGGTCAGACGATCCTGTATCCCATTGAGCCG TTATGCTTTGTGGGGGAAACCCTATTTCTATCGTACTAAGAAAACCAATGGTGAACT CATATTCGGTATCAATGGCGACGATTCCAGCATAGCCTGTAGACAGTAACAACACTA GGGCAACAGCAACTAACATATCTTCATTGATGAAACGTTGTGATCGGTGTGACTTTT ATAGTAAAAGCTACAACTGTTTGAAATACCAAGATATCATTGTGAATGGCTCAAAAG GGTAATACATCTGAAAAACCTGAAGTGTGGAAAATTCCGATGGAGCCAACTCATGAT AACGCAGAAGTCCCATTTTGCCATCTTCTCTTGGTATGAAACGGTAGAAAATGATCC GAGTATGCCAATTGATACTCTTGATTCATGCCCTATAGTTTGCGTAGGGTTTAATTGA TCTCCTGGTCTATCGATCTGGGACGCAATGTAGACCCCATTAGTGGAAACACTGAAA GGGATCCAACACTCTAGGCGGACCCGCTCACAGTCATTTCAGGACAATCACCACAGG AATCAACTACTTCTCCCAGTCTTCCTTGCGTGAAGCTTCAAGCCTACAACATAACACT TCTTACTTAATCTTTGATTCTCGAATTGTTTACCCAATCTTGACAACTTAGCCTAAGC AATACTCTGGGGTTATATATAGCAATTGCTCTTCCTCGCTGTAGCGTTCATTCCATCT TTCTAGAATTCGT Methanol SEQ ID NO: 38 CTTCCCCATTTCACTGACAGTTTGTAGAAATAGGGCAACAATTGATGCAAATCGATTT inducible TCAACGCATTGGTTTTGATAGCATTGATGATCTTGGAGCTGTAAAAGTCCGGCTGGA promoter TAAGCTCAATGAAATAGGTTGGTTGATCTGGATCTTCTTTTGGGTCATTTTGTTCGCT CTGTATTTCACAAATTGCCAGAATCTCTGCCAACCACAGTGGTAGGTCCAACTTGGT GTTCTGAATCACAGGCTTCCCCGGGTTGTTCTCTAAATAACCGAGGCCCGGCACAGA AATCGTAAACCGACACGGTATCTTTTGTCCGTCCGCCAGTATCTCATCAAGGTCGTAG TAGCCCATGATGAGTATCAAAGGGGATTTGGTTATGCGATGCAACGAGAGATTGTTT ATCCCAGATGCTGATGTAAAAACCTTAACCAGCGTGACAGTAGAAATAAGACACGTT AAAATTACCCGCGCTTCCCTAACAATTGGCTCTGCCTTTCGGCAAGTTTCTAACTGCC CTCCCCTCTCACATGCACCACGAACTTACCGTTCGCTCCTAGCAGAACCACCCCAAA GTTTAATCAGGACCGCATTTTAGCCTATTGCTGTAGAACCCCACAACATAACCTGGTC CAGAGCCAGCCCTTTATATATGGTAAATCCCGTTTGAACTTCGAAGTGGAATCGGAA TTTTTACATCAAAGAAACTGATACTGAAACTTTTGGCTTCGACTTGGACTTTCTCTTA ATCGAATTCGT PMP20 SEQ ID NO: 39 ACACAGTTATTATTCATTTAAATGTCAAAACAGTAGTGATAAAAGGCTATGAAGGAG GTTGTCTAGGGGCTCGCGGAGGAAAGTGATTCAAACAGACCTGCCAAAAAGAGAAA AAAGAGGGAATCCCTGTTCTTTCCAATGGAAATGACGTAACTTTAACTTGAAAAATA CCCCAACCAGAAGGGTTCAAACTCAACAAGGATTGCGTAATTCCTACAAGTAGCTTA GAGCTGGGGGAGAGACAACTGAAGGCAGCTTAACGATAACGCGGGGGGATTGGTGC ACGACTCGAAAGGAGGTATCTTAGTCTTGTAACCTCTTTTTTCCAGAGGCTATTCAAG ATTCATAGGCGATATCGATGTGGAGAAGGGTGAACAATATAAAAGGCTGGAGAGAT GTCAATGAAGCAGCTGGATAGATTTCAAATTTTCTAGATTTCAGAGTAATCGCACAA AACGAAGGAATCCCACCAAGACAAAAAAAAAAATTCTAAGG AATTCCGAAACG DAK2 SEQ ID NO: 40 AAATAAGCATGTTTGTTTCAGATCAAAGATTAGCGTTTCAAAGTTGTGGAAAAGTGA CCATGCAACAATATGCAACACATTCGGATTATCTGATAAGTTTCAAAGCTACTAAGT AAGCCCGTTTCAAGTCTCCAGACCGACATCTGCCATCCAGTGATTTTCTTAGTCCTGA AAAATACGATGTGTAAACATAAACCACAAAGATCGGCCTCCGAGGTTGAACCCTTAC GAAAGAGACATCTGGTAGCGCCAATGCCAAAAAAAAATCACACCAGAAGGACAATT CCCTTCCCCCCCAGCCCATTAAAGCTTACCATTTCCTATTCCAATACGTTCCATAGAG GGCATCGCTCGGCTCATTTTCGCGTGGGTCATACTAGAGCGGCTAGCTAGTCGGCTG TTTGAGCTCTCTAATCGAGGGGTAAGGATGTCTAATATGTCATAATGGCTCACTATAT AAAGAACCCGCTTGCTCAACCTTCGACTCCTTTCCCGATCCTTTGCTTGTTGCTTCTTC TTTTATAACAGGAAACAAAGGAATTTATACACTTTAAGAATT GCW14 SEQ ID NO: 41 CAGGTGAACCCACCTAACTATTTTTAACTGGCATCCAGTGAGCTCGCTGGGTGAAAG CCAACCATCTTTTGTTTCGGGGAACCGTGCTCGCCCCGTAAAGTTAATTTTTTTTTCCC GCGCAGCTTTAATCTTTCGGCAGAGAAGGCGTTTTCATCGTAGCGTGGGAACAGAAT AATCAGTTCATGTGCTATACAGGCACATGGCAGCAGTCACTATTTTGCTTTTTAACCT TAAAGTCGTTCATCAATCATTAACTGACCAATCAGATTTTTTGCATTTGCCACTTATC TAAAAATACTTTTGTATCTCGCAGATACGTTCAGTGGTTTCCAGGACAACACCCAAA AAAAGGTATCAATGCCACTAGGCAGTCGGTTTTATTTTTGGTCACCCACGCAAAGAA GCACCCACCTCTTTTAGGTTTTAAGTTGTGGGAACAGTAACACCGCCTAGAGCTTCA GGAAAAACCAGTACCTGTGACCGCAATTCACCATGATGCAGAATGTTAATTTAAACG AGTGCCAAATCAAGATTTCAACAGACAAATCAATCGATCCATAGTTACCCATTCCAG CCTTTTCGTCGTCGAGCCTGCTTCATTCCTGCCTCAGGTGCATAACTTTGCATGAAAA GTCCAGATTAGGGCAGATTTTGAGTTTAAAATAGGAAATATAAACAAATATACCGCG AAAAAGGTTTGTTTATAGCTTTTCGCCTGGTGCCGTACGGTATAAATACATACTCTCC TCCCCCCCCTGGTTCTCTTTTTCTTTTGTTACTTACATTTTACCGTTCCGT FDH1 SEQ ID NO: 42 AAATAAATGGCAGAAGGATCAGCCTGGACGAAGCAACCAGTTCCAACTGCTAAGTA AAGAAGATGCTAGACGAAGGAGACTTCAGAGGTGAAAAGTTTGCAAGAAGAGAGCT GCGGGAAATAAATTTTCAATTTAAGGACTTGAGTGCGTCCATATTCGTGTACGTGTCC AACTGTTTTCCATTACCTAAGAAAAACATAAAGATTAAAAAGATAAACCCAATCGGG AAACTTTAGCGTGCCGTTTCGGATTCCGAAAAACTTTTGGAGCGCCAGATGACTATG GAAAGAGGAGTGTACCAAAATGGCAAGTCGGGGGCTACTCACCGGATAGCCAATAC ATTCTCTAGGAACCAGGGATGAATCCAGGTTTTTGTTGTCAGGTAGGTCAAGCATT CACTTCTTAGGAATATCTCGTTGAAAGCTACTTGAAATCCCATTGGGTGCGGAACCA GCTTCTAATTAAATAGTTCGATGATGTTCTCTAAGTGGGACTCTACGGCTCAAACTTC TACACAGCATCATCTTAGTAGTCCCTTCCCAAAACACCATTCTAGGTTTCGGAACGTA ACGAAACAATGTTCCTCTCTTCACATTGGGCCGTTACTCTAGCCTTCCGAAGAACCAA TAAAAGGGACCGGCTGAAACGGGTGTGGAAACTCCTGTCCAGTTTATGGCAAAGGCT ACAGAAATCCCAATCTTGTCGGGATGTTGCTCCTCCCAAACGCCATATTGTACTGCA GTTGGTGCGCATTTTAGGGAAAATTTACCCCAGATGTCCTGATTTTCGAGGGCTACCC CCAACTCCCTGTGCTTATACTTAGTCTAATTCTATTCAGTGTGCTGACCTACACGTAA TGATGTCGTAACCCAGTTAAATGGCCGAAAAACTATTTAAGTAAGTTTATTTCTCCTC CAGATGAGACTCTCCTTCTTTTCTCCGCTAGTTATCAAACTATAAACCTATTTTACCTC AAATACCTCCAACATCACCCACTTAAACAGAATT FBA1 SEQ ID NO: 43 TGCTTAAGTAATTGAAAACAGTGTTGTGATTATATAAGCATGGTATTTGAATAGAAC TACTGGGGTTAACTTATCTAGTAGGATGGAAGTTGAGGGAGATCAAGATGCTTAAAG AAAAGGATTGGCCAATATGAAAGCCATAATTAGCAATACTTATTTAATCAGATAATT GTGGGGCATTGTGACTTGACTTTTACCAGGACTTCAAACCTCAACCATTTAAACAGTT ATAGAAGACGTACCGTCACTTTTGCTTTTAATGTGATCTAAATGTGATCACATGAACT CAAACTAAAATGATATCTTTTACTGGACAAAAATGTTATCCTGCAAACAGAAAGCTT TCTTCTATTCTAAGAAGAACATTTACATTGGTGGGAAACCTGAAAACAGAAAATAAA TACTCCCCAGTGACCCTATGAGCAGGATTTTTGCATCCCTATTGTAGGCCTTTCAAAC TCACACCTAATATTTCCCGCCACTCACACTATCAATGATCACTTCCCAGTTCTCTTCTT CCCCTATTCGTACCATGCAACCCTTACACGCCTTTTCCATTTCGGTTCGGATGCGACT TCCAGTCTGTGGGGTACGTAGCCTATTCTCTTAGCCGGTATTTAAACATACAAATTCA CCCAAATTCTACCTTGATAAGGTAATTGATTAATTTCATAAATGAATTCGCG GAP SEQ ID No: 44 TTTTTGTAGAAATGTCTTGGTGTCCTCGTCCAATCAGGTAGCCATCTCTGAAATATCT GGCTCCGTTGCAACTCCGAACGACCTGCTGGCAACGTAAAATTCTCCGGGGTAAAAC TTAAATGTGGAGTAATGGAACCAGAAACGTCTCTTCCCTTCTCTCTCCTTCCACCGCC CGTTACCGTCCCTAGGAAATTTTACTCTGCTGGAGAGCTTCTIVTACGGCCCCCTTGC AGCAATGCTCTTCCCAGCATTACGTTGCGGGTAAAACGGAGGTCGTGTACCCGACCT AGCAGCCCAGGGATGGAAAAGTCCCGGCCGTCGCTGGCAATAATAGCGGGCGGACG CATGTCATGAGATTATTGGAAACCACCAGAATCGAATATAAAAGGCGAACACCTTTC CCAATTTMGTTTCTCCTGACCCAAAGACTTTAAATTTAATTFATTTGTCCCTATTTCA ATCAATTGAACAACTAT PGK SEQ ID No: 45 AAATAGCAGTTTGCGGTTTCTTGATTTCATGGGGGGAACAAACAATAGTGTTGCCTT AATTCTAATTGGCATTGTTGCTTGGAATCGAAATTGGGGGATAACGTCATATCTGAA AAGTAAACAACTTCGGGAAATCAGGCTGTTTGAATGGCTTGGAAGCGAGATAGAAA GGGGATAGCGAGATAGAGGGGGCGGAGTAGACGAAGGGTGTTAAACTGCTGAAATC TCTCAATCTGGAAGAAACGGAATAAATTAACTCCTTGCGATAATAAAATCCGAGTCC GTTATGACCCCACACCGTGTTGACCACGGCATACCCCATGGAATCTGGTACAAAGCG TCAGTCTTGAAGACACCATCACGTGTAGGAGACTGATTGTCTGACCGTCCAGCAAAA AGGGCATTATAAATCTTGCTGTTAAAGGGGTGAGGGGAGATGCAGGTTGTTCTTTTA TTCGCCTTGAACTTTITAATTTTCCCGGGGTTGCGGAGCGTGAACAGTTAGCCCGATC TGATAGCTTGCAAGATTCAACAGTTTATCCACTACAGGTCAGAGAGATCGCCGCAGA AGAAATGCTCGTCTCGTGTTCCAGCACACATACTGGTGAAGTCGTTATTTTGCCGAA GGGGGGGTAATAAGGTTATGCACCCCCTCTCCACACCCCAGAATCATTTTTTAGCTG GGTTCAAGGCATTAGACTTTGCACATTTTTCCCTTAAACACCCTTGAAACGCGGATAA ACAGTTGCATGTGCATCCTAAAACTAGGTGAGATGCGTACTCCGTGCTCCGATAATA ACAGTGGTGTTGGGGTTGCTGCTAGCTCACGCACTCCGTTCTTTTTTTTCAACCAGCA AAATTCGATGGGGAGAAACTTGGGGTACTTTGCCGACTCCTCCACCATGCTGGTATA TAAATAATACTCGCCCACTTTTCGTTTGCTGCTTTTATATTTCATAGACTGAAAAAGA CTCTTCTTCTACTTTTTCATAATATATCTCAGATATCACTACTATAG AOX2_PRO SEQ ID NO: 46 cgcATTTAAATtgacttccttacaaaggggcttctgtttttgaggttccagttttctc ataaactccaaccctgtagctctctctaatgcttctaatggtacttcaaaatctgtga gtttgacagaatttggtattggctcgtttggaaggacgaaagctgccagcgcaacatc accagggtttcgtctattcttcgggtcctcggctacgaccaatttaaagaaatgcgtc ggcactgcaactgatggcggacttccaatgagttcatatgttaccttccatttaccat cattaccatcctgcttaggcaaaaaaagaggacctgtaacaatgcgaactgatcgaaa atattgagttagagtacgagtaaagtactccaaatgagcccaataatctctgttaaaa ccatctccaacttggggtgacatgttggtcaaaaaaaagtttcatccattgcgttttg agagaacttagcgtttgccgctggtgcttgatgccctctatcataaccagatcgaaaa tagtcctttaatcttgccctaaatatgcttggaatttgctcatcttccttaaaaaaac aattctttctatcagcattgtgactggctaaagaatctggggtcaaatgttcaacgac ataatatggattccgggtttgacggttgtatactgagacaaattctgctctggtttgt aaatcatggatgggaccaggaaaaccatacttgaaaaaatcagaaggtctcactattg gagtctctagcgaaacagatgttgttggaggagataatgagctaggacttatggtagt tggatttgcaactatagtgtcctttgccttactccaaaacattgatctggcaaaagct gagtatatagggaaagttactggtggaattgactaacctgcttagtttctggagcgcg ctaaaacttcaattctttttccccgcgacaaaactttcaagtgtttgaaaccaaagct agcaccttcgaatagtcaaattagcGAATTCgcg TEFg_PRO SEQ ID NO: 47 GCGatttaaattcgcgaaagaacagcctaataaactccgaagcatgatggcctctatc cggaaaacgttaagagatgtggcaacaggagggcacatagaatttttaaagacgctga agaatgctatcatagtccgtaaaaatgtgatagtactttgtttagtgcgtacgccact tattcggggccaatagctaaacccaggtttgctggcagcaaattcaactgtagattga atctctctaacaataatggtgttcaatcccctggctggtcacggggaggactatcttg cgtgatccgcttggaaaatgttgtgtatccctttctcaattgcggaaagcatctgcta cttcccataggcaccagttacccaattgatatttccaaaaaagattaccatatgttca tctagaagtataaatacaagtggacattcaatgaatatttcattcaattagtcattga cactttcatcaacttactacgtcttattcaacaatGAATTCgcg SEQ ID NO: 48 MQVKSIVNLLLACSLAVA SEQ ID NO: 49 MQFNWMKTVASILSALTLAQA SEQ ID NO: 50 MYRNLIIATALTCGAYSAYVPSEPWSTLTPDASLESALKDYSQTFGIAIKSLDADKIKR SEQ ID NO: 51 MNLYLITLLFASLCSAITLPKR SEQ ID NO: 52 MFEKSKFVVSFLLLLQLFCVLGVHG SEQ ID NO: 53 MQFNSVVISQLLLTLASVSMG SEQ ID NO: 54 MKSQLIFMALASLVASAPLEHQQQHHKHEKR SEQ ID NO: 55 MKFAISTLLIILQAAAVFA SEQ ID NO: 56 MKLLNFLLSFVTLFGLLSGSVFA SEQ ID NO: 57 MIFNLKTLAAVAISISQVSA SEQ ID NO: 58 MKISALTACAVTLAGLAIAAPAPKPEDCTTTVQKRHQHKR SEQ ID NO: 59 MSYLKISALLSVLSVALA SEQ ID NO: 60 MLSTILNIFILLLFIQASLQ SEQ ID NO: 61 MKLSTNLILAIAAASAVVSAAPVAPAEEAANHLHKR SEQ ID NO: 62 MFKSLCMLIGSCLLSSVLA SEQ ID NO: 63 MKLAALSTIALTILPVALA SEQ ID NO: 64 MSFSSNVPQLFLLLVLLTNIVSG SEQ ID NO: 65 MQLQYLAVLCALLLNVQSKNVVDFSRFGDAKISPDDTDLESRERKR SEQ ID NO: 66 MKIHSLLLWNLFFIPSILG SEQ ID NO: 67 MSTLTLLAVLLSLQNSALA SEQ ID NO: 68 MINLNSFLILTVTLLSPALALPKNVLEEQQAKDDLAKR SEQ ID NO: 69 MFSLAVGALLLTQAFG SEQ ID NO: 70 MKILSALLLLFTLAFA SEQ ID NO: 71 MKVSTTKFLAVFLLVRLVCA SEQ ID NO: 72 MQFGKVLFAISALAVTALG SEQ ID NO: 73 MWSLFISGLLIFYPLVLG SEQ ID NO: 74 MRNHLNDLVVLFLLLTVAAQA SEQ ID NO: 75 MFLKSLLSFASILTLCKA SEQ ID NO: 76 MFVFEPVLLAVLVASTCVTA SEQ ID NO: 77 MVSLRSIFTSSILAAGLTRAHG SEQ ID NO: 78 MFSPILSLEIILALATLQSVFA SEQ ID NO: 79 MIINHLVLTALSIALA SEQ ID NO: 80 MLALVRISTLLLLALTASA SEQ ID NO: 81 MRPVLSLLLLLASSVLA SEQ ID NO: 82 MVLIQNFLPLFAYTLFFNQRAALA SEQ ID NO: 83 MKFPVPLLFLLQLFFIIATQG SEQ ID NO: 84 MVSLTRLLITGIATALQVNA SEQ ID NO: 85 MIFDGTTMSIAIGLLSTLGIGAEA SEQ ID NO: 86 MVLVGLLTRLVPLVLLAGTVLLLVFVVLSGG SEQ ID NO: 87 MLSILSALTLLGLSCA SEQ ID NO: 88 MRLLHISLLSIISVLTKANA SEQ ID NO: 89 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNS TNNGLLFINTTIASIAAKEEGVSLDKREAEA SEQ ID NO: 90 MFKSVVYSILAASLANA SEQ ID NO: 91 MLLQAFLFLLAGFAAKISA SEQ ID NO: 92 MASSNLLSLALFLVLLTHANS SEQ ID NO: 93 MNIFYIFLFLLSFVQGLEHTHRRGSLVKR SEQ ID NO: 94 MLIIVLLFLATLANSLDCSGDVFFGYTRGDKTDVHKSQALTAVKNIKR SEQ ID NO: 95 MESVSSLFNIFSTIMVNYKSLVLALLSVSNLKYARGMPTSERQQGLEER SEQ ID NO: 96 MFAFYFLTACISLKGVFG SEQ ID NO: 97 MRFSTTLATAATALFFTASQVSA SEQ ID NO: 98 MKFAYSLLLPLAGVSASVINYKR SEQ ID NO: 99 MKFFAIAALFAAAAVAQPLEDR SEQ ID NO: 100 MQFFAVALFATSALA SEQ ID NO: 101 MKWVTFISLLFLFSSAYSRGVFRR SEQ ID NO: 102 MRSLLILVLCFLPLAALG SEQ ID NO: 103 MKVLILACLVALALA SEQ ID NO: 104 MFNLKTILISTLASIAVA SEQ ID NO: 105 MYRKLAVISAFLATARAQSA WT SEQ ID NO: 106 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNS TNNGLLFINTTIASIAAKEEGVQLDKR App3 SEQ ID NO: 107 MRFPPIFTAALFAASSALAAPANTTTEDETAQIPAEAVIGYLDSEGDSDVAVLPFSNS TNNGLSFINTTIASIAAKEEGVQLDKR App8 SEQ ID NO: 108 MRFPSIFTAVLFAASSALAAPANTTTEDETAQIPAEAVISYSDLEGDFDAAALPLSNS TNNGLSSTNTTIASIAAKEEGVQLDKR App9 SEQ ID NO: 109 MRPPSIFTAVLFAASSALAAPANTTTEDETTQIPAEAVATYLDLEGDVDVAVLPFSSS TNNGLSFINTTIASIAAKEEGVQLDKR App10 SEQ ID NO: 110 MRFPSIFFAALFAASSALAAPANTTTEGETAQTPAEAVIGYRDLEGDFDVAVLPFPNS TNNGLLFTNTTTASIAAKEEGVQLDKR appS1 SEQ ID NO: 111 MRFPSIFTAVLLAAPSALAAPANATTEDEAAQIPAEAVIGYLDLEGDFDAAVLPFSNS TNNGLLSINTTIASIAAKEEGVQLDKR appS4 SEQ ID NO: 112 MRFPSIFTAVVFAASSALAAPANTTAEDETAQIPAEAVIGYLGLEGDSDVAALPLSDS TNNGSLSTNTTIASIAAKEEGVQLDKR appS6 SEQ ID NO: 113 MRLPSIFTAAVFAASSALAAPANTTTEDETAQIPAEAAIGYLDLEGDSDVAVLPLSNS TNNGLLFINTTIASIAAKEEGVQLDKR appS8 SEQ ID NO: 114 MRFPSIFTAVLFAASSALAAPANTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNS TNDGLSFINTTTASIAAKEEGVQLDKR a-Factor SEQ ID NO: 115 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPA PpScw11p SEQ ID NO: 116 MLSTILNIFILLLFIQASLQ APIPVVTKYVTEGIANV PpDse4p SEQ ID NO: 117 MSFSSNVPQLFLLLVLLTNIVSGAVISVWSTSKVTK PpExg1p SEQ ID NO: 118 MNLYLITLLFASLCSAITLPKRDIIWDYSSEKIMG a-EGFP SEQ ID NO: 119 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPA S-EGFP SEQ ID NO: 120 MLSTILNIFILLLFIQASLQEFDYKDDDDKMVSKG D-EGFP SEQ ID NO: 121 MSFSSNVPQLFLLLVLLTNIVSGEFDYKDDDDKMV E-EGFP SEQ ID NO: 122 MNLYLITLLFASLCSAEFDYKDDDDKMVSKGEELF a-CALB SEQ ID NO: 123 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPA S-CALB SEQ ID NO: 124 MLSTILNIFILLLFIQASLQEFLPSGSDPAFSQPK D-CALB SEQ ID NO: 125 MSFSSNVPQLFLLLVLLTNIVSGEFLPSGSDPAFS E-CALB SEQ ID NO: 126 MNLYLITLLFASLCSAEFLPSGSDPAFSQPKSVLD Amylase (AA) SEQ ID NO: 127 MVAWWSLFLYGLQVAAPALAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTY TNDCLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCG TDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLC GSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC Alpha K (AK) SEQ ID NO: 128 MRFPSIFTAVLFAASSALAAPVNTTTEDELEGDFDVAVLPFSASIAAKEEGVSLEKRAE VDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGE CKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASVD KRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLT LSHFGKC Alpha T (AT) SEQ ID NO: 129 MRFPSIFTAVLFAASSALAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTND CLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDG VTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSD NKTYGNKCNFCNAVVESNGTLTLSHFGKC Lysozyme (LZ) SEQ ID NO: 130 MLGKNDPMCLVLVLLGLTALLGICQGAEVDCSRFPNATDKEGKDVLVCNKDLRPICGT DGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAF NPVCGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAE DRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC Killer Protein SEQ ID NO: 131 MTKPTQVLVRSVSILFFITLLHLVVAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDG (KP) VTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNP VCGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAED RPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC Invertase (IV) SEQ ID NO: 132 MLLQAFLFLLAGFAAKISAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTND CLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDG VTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSD NKTYGNKCNFCNAVVESNGTLTLSHFGKC Serum Albumin SEQ ID NO: 133 MKWVTFISLLFLFSSAYSAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDC (SA) LLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGV TYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDN KTYGNKCNFCNAVVESNGTLTLSHFGKC Glucoamyl (GA) SEQ ID NO: 134 MSFRSLLALSGLVCSGLAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDC LLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGV TYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDN KTYGNKCNFCNAVVESNGTLTLSHFGKC Inulase (IN) - IC SEQ ID NO: 135 MKLAYSLLLPLAGVSAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLL CAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTY DNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKT YGNKCNFCNAVVESNGTLTLSHFGKC Alpha KS (AKS) SEQ ID NO: 136 MRFPSIFTAVLFAASSALAAPVNTTTEDELEGDFDVAVLPFSASIAAKEEGVSLEKREA EAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKE HDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQG ASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESN GTLTLSHFGKC Ovomucoid signal SEQ ID NO: 137 MAMAGVFVLFSFVLCGFLPDAAFG peptide Lysozyme signal SEQ ID NO: 138 MRSLLILVLCFLPLAALG peptide Ovalbumin Signal SEQ ID NO: 139 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNST Peptide NNGLLFINTTIASIAAKEEGVSLDKREAEA Ovotransferrin SEQ ID NO: 140 MKLILCTVLSLGIAAVCFA Signal Peptide Bovine SEQ ID NO: 141 MKLFVPALLSLGALGLCLA Lactoferrin Signal Peptide Porcine SEQ ID NO: 142 MKLFIPALLFLGTLGLCLA Lactoferrin Signal Peptide Kid Lipase Signal SEQ ID NO: 143 MESKALLLLALSVWLQSLTVSHG Peptide Porcine Lipase SEQ ID NO: 144 MLLIWTLSLLLGAVLG Signal Peptide XP_015135086.1 SEQ ID NO: 145 MYAAAAAAVAASPPRRDFISVTLSPEEAVGAGGYNNSKAWRRRSCWRKWKQLSRLQR PREDICTED: SIILFLFAFLTVC endoplasmic GVISYTSVREPWKSLTSKSSDEHGTEPDAPGLRLANPAVLPAPQKADANAGDYPELSPQK reticulum PKLPHGRRNP mannosyl- SNFQIKPPWGDVRLQTRHDTRKAVEEPAQADKQEKTEKSVISWRGAVIEPDQSSEPPSSR oligosaccharide VKEPEKPSSV 1,2-alpha-  EGESQKEPVPINERQMAVIEAFRHAWKGYKDFAWGHDELKPLSKSYSEWFGLGLTLIDA mannosidase LDTMWILGLRE isoform X2 EFEEARKWVANDLAFDKNVDVNLFESTIRILGGLLSTYHLSGDSLFLEKAKDIGNRLMPA [Gallus gallus] FKTPSKIPYS DVNIGRGTAHPPRWTSDSTVAEVTSIQLEFRELSRLTGDEKYQKAVDEVMKHVHTLSGK NDGLVPMFINT NSGQFTHLGVYTLGARADSYYEYLLKQWIQGGKTENELLEDYMKAIEGVKKHLLQRSQ PKKLTFVGELAH GHFSAKMDHLVCFLPGTLALGAHNGLTADHMKLAEALIETCYQMYAQVETGLSPEIVH FNLHAQKGHKDV EIKPADRHNLLRPETVESLFYMYRFTGDKKYQDWGWEILQNFNKYTRVPTGGYTSINNV QNPSNPEPRDK MESEPLGETLKYMFLLFSDDIDLINLDKYVFNTEAHPLPIWVPA XP_015135085.1 SEQ ID NO: 146 MYAAAAAAVAASPPRRDFISVTLSPEEAVGAGGYNNSKAWRRRSCWRKWKQLSRLQR PREDICTED: SIILFLFAFLTVC endoplasmic GVISYTSVREPWKSLTSKSSDEHGTEPDAPGLRLANPAVLPAPQKADANAGDYPELSPQK reticulum KPKLPHGRRN mannosyl- PSNFQIKPPWGDVRLQTRHDTRKAVEEPAQADKQEKTEKSVISWRGAVIEPDQSSEPPSS oligosaccharide RVKEPEKPSS 1,2-alpha- VEGESQKEPVPINERQMAVIEAFRHAWKGYKDFAWGHDELKPLSKSYSEWFGLGLTLID mannosidase ALDTMWILGLR isoform X1  EEFEEARKWVANDLAFDKNVDVNLFESTIRILGGLLSTYHLSGDSLFLEKAKDIGNRLMP [Gallus gallus] AFKTPSKIPY SDVNIGRGTAHPPRWTSDSTVAEVTSIQLEFRELSRLTGDEKYQKAVDEVMKHVHTLSG KNDGLVPMFIN TNSGQFTHLGVYTLGARADSYYEYLLKQWIQGGKTENELLEDYMKAIEGVKKHLLQRS QPKKLTFVGELA HGHFSAKMDHLVCFLPGTLALGAHNGLTADHMKLAEALIETCYQMYAQVETGLSPEIV HFNLHAQKGHKD VEIKPADRHNLLRPETVESLFYMYRFTGDKKYQDWGWEILQNFNKYTRVPTGGYTSINN VQNPSNPEPRD KMESFFLGETLKYMFLLFSDDIDLINLDKYVFNTEAHPLPIWVPA XP_416490.2 SEQ ID NO: 147 MSAPALLPLAGRRLPALNLGASSFPHHRATLRLSEKFILLLILSAFITLCFGAFFFLPDS PREDICTED: SKHKRFDLGL mannosyl- EDVLIPHVDTSKGGKHLGSFLIHGQGHDEHRHREEEERLRNKIRADHEKALEEAKEKLK oligosaccharide KSRDEIQAEIQ 1,2-alpha- TEKNKVVQELKKKDSKPLPPVPLPNLVGINSGEPADPDIREKRNKIKEMMKHAWDNYRQ mannosidase IB YGWGHNELKPI [Gallus gallus] ARKGHSTNIFGNSQMGATIVDALDTLYIMGLRDEFREGQEWIDKNLDFSVNSEVSVFEV NIRFIGGLLAA YYLSGQEVFKIKAVQLAGKLLPAFNTPTGIPWAMVNLKSGVGRNWGWASAGSS1LAEF GTLHMEFVHLSY LTGDPVYYNKVMHIRKLLQKMDRPNGLYPNYLNPRTGRWGQHHTSVGGLGDSFYEYL LKAWLMSDKTDTE ARKMYDDALEAIEKHLIRKSNGGLTFIGEWKNGHLERKMGHLTCFAGGMFALGADGSR DDKAGHYLQLGA EIAHTCHESYDRTTLKLGPEAFKFDGGVEAVAVRQNEKYYILRPEVIETYWYMWRFTHD PKYRQWGWEAT QAIDKYCRVSGGFSGVKDVYSSSPTYDDVQQSFFLAETLKYLYLLFSNDDLLPLDNWVF NTEAHPLPVLH LANTTLSGNPAYR XP_422293.5 SEQ ID NO: 148 MSGAAGCRGGGGERGPRWRRPWKLLALGLLSASSVLAAAPGAGAMSKEEKRRLGNQV PREDICTED: ER LEMFDHAYSNYMD degradation- IIAYPADELMPLTCRGRVRGQEPSRGDVDDALGKFSLTLIDTLDTLVVLNKTKEFEEAVK enhancing alpha- KVIKDVNLDND mannosidase- IVVSVFETNIRVLGGLLGGHSVAIMLKDKGEYMQWYNGELLHMAKELGYKLLPAFNTT like protein 3 SGLPYPRVNLKF isoform X2 GVRHPEARTGTETDTCTACAGTLILEFAALSRFTGTSIFEEYARKALDFIWEKRQRSSNLV [Gallus gallus] GVTINIFITG DWVRKDSGVGAGIDSYYEYLLKAYVLLGDDSFLERFNTHYDAIMKYISQPPLLLDVHIH KPMLNARTWMD SLLAFFPGLQVLKGDIRPAIETHEMLYQVIKKHNFLPEAFTTDFRVHWAQHPLRPEFAEST YFLYKATGD PYYLEVGKTLIENLNKYARVPCGFAAMKDVRTGSHEDRMDSFFLAEMFKYLYLLFADK EDMIFDIEDYIF TTEAHLLPLWLSTTNQTISKKNTTTEYTELDDSNFDWTCPNTQILFPNDPMFAQSIREPLK NVVDKSCPR SISRAEESLGTGPKPPLRARDFMASNPEHLEILKKMGVSLIHLKDGRVQLVQHAVQAASS LDAEDGLRFM QEMIELSSQQQKEQQLPPRAVQIVSHPFFGRVVLTAGPAQFGMDLSKHKSGTRGFVATIK PYNGCSEITN PEAVKEKIALMQRGQCMFAEKARNIQKAGAIGGIVIDDNEGSSSDTAPLFQMAGDGKNT DDITIPMLFLF NKEGNIILDAIREYEAVEVLLSDKAKDRDLEMENMDQKLSENDSHKQNSEEASSASQDV GAVSEEPEEGE SSDVSDLDSLPPAQADTDSVSTSDQDSSIPGPGEAGAPEPACTQGDEQPQEQQTETESDSK VNWDNKVQP MESILADWNEDIEAFEMMEKDEL O46432.1 SEQ ID NO: 149 MGADARPLGVRAGGGGRGAARPGTSSRALPPPLPPLSFLLLLLAAPGARAAGYETCPMV Lysosomal HPDMLNVHLVA alpha- HTHDDVGWLKTVDQYFYGIFINDVQHAGVQYILDSVISSLLVEPTRRFIYVEIAFFSRWW mannosidase HQQTNATQEVV RDLVRQGRLEFANGGWVMNDEAATHYGAIIDQMTLGLRFLEDTFGKDGRPRVAWHIDP FGHSREQASLFA QMGFDGLFFGRLDYQDKRVREENLGLEQVWRASASLKPPAADLFTSVLPNIYNPPEKLC WDTLCADKPFV EDRRSPEYNAEELVNYFLQLATAQGQHFRTNHTIMTMGSDFQYENANMWFRNLDRLIQ LVNAQQQANGSR VNVLYSTPACYLWELNKANLTWSVKQDDFFPYADGPHQFWSGYFSSRPALKRYERLSY NFLQVCNQLEAL AGPAANVGPYGSGDSAPLNQAMAVLQHHDAVSGTSKQHVADDYARQLAAGWDPCEV LLSNALARLSGSKE DFTYCRNLNVSVCPLSQTAKNFQVTIYNPLGRKIDWMVRLPVSKHGFVVRDPNGTVVPS DVVILPSSDGQ ELLFPASVPALGFSIYSVSQVPGQRPHAHKPQPRSQRPWSRVLAIQNEHIRARFDPDTGLL VEMENLDQN LLLPVRQAFYWYNASVGNNLSTQVSGAYIFRPNQEKPLMVSHWAQTRLVKTPLVQEVH QNFSAWCSQVVR LYRGQRHLELEWTVGPIPVGDGWGKEIISRFDTVLETKGLFYTDSNGREILERRRDYRPT WKLNQTETVA GNYYPVNSRIYIRDGNMQLTVLTDRSQGGSSLRDGSMELMVHRRLLKDDGRGVGEALL EDGLGRWVRGRH LVLLDKVRTAATGHRLQAEKEVLTPQVVLAPGGGAPYHLKVAPRKQFSGLRRELPPSVH LLTLARWDQKT LLLRLEHQFAVGEDSGNLSSPVTLDLTDLFSAFTITYLQETTLVANQLRASASRLKWTPN TGPTPLPSPS RLDPATITLQPMEIRTFLASVQWEEHG XP_419762.5 SEQ ID NO: 150 MPAASLLPLFGSAAGPGALGGPAGGGAGGGGRKAAGPGAFRLTEKFVLLLVFSAFITLC PREDICTED: FGAIFFLPDSS mannosyl- KLLSGVFFHSAALQPPPPPPGFQPRAPPQPGAGPAMPEEAGGAGSLERIRADHERALREA oligosaccharide KETLQKLPEE 1,2-alpha- IRRDIRQDKEKLLQDARGRKEAAAAGLPQRPFRQPVGAVGREPADLAVRQRRDKIKEM mannosidase IA MKYAWDNYKRYA [Gallus gallus] WGLNELKPISKQGHSSNLFGNIQGATIVDALDTLFIMEMKEEFKEAKEWVEKNLDFNVN AEISVFEVNIR FVGGLLSAYYLSGEEIFRKKAVELGEKLLPAFNTPTGIPWALLNIKSGIGRNWPWASGGS SILAEFGTLH LEFVHLSHLSGNPVFAEKVMNIRKVLSRLDKPEGLYPNYLNPSSGQWGQHHVSIGGLGD SFYEYLLKAWL MSDKTDEEGKKMYYDAVQAIETHLIRKSSGGLTYIAEWKGGLLEHKMGHLTCFAGGMF ALGADGAPSDKT GHHIELGAEIARTCHESYDRTSMKLGPEAFRFDGGVEAIATRQNEKYYILRPEVIETYMY MWRLTHDPKY RQWAWEAVEALEKHCRVDGGYSGIRDVYSNHESHDDVQQSFFLSETLKYLYLLFSDDD LLPFEHWVFNTE AHPFPILRKEDGSKEEKEK NoManIB SEQ ID NO: 153 MARRRYRLFMICAAVILFLLYRVSQNTWDDSAHYATLRHPPASNPPAAGGESPLKPAAK PEHEHEHENGYAPESKPKPQSEPKPESKPAPEHAAGGQKSQGKPSYEDDEETGKNPPKSA VIPSDTRLPPDNKVHWRPVKEHFPVPSESVISLPTGKPLKVPRVQHEFGVESPEAKSRRVA RQERVGKEIERAWSGYKKFAWMHDELSPVSAKHRDPFCGWAATLVDSLDTLWIAGLKE QFDEAARAVEQIDFTTTPRNNIPVFETTIRYLGGLLGAFDVSGGHDGGYPMLLTKAVELA EILMGIFDTPNRMPILYYQWQPEYASQPHRAGSVGIAELGTLSMEFTRLAQLTSQYKYYD AVDRITDALIELQKQGTSIPGLFPENLDASGCNHTATALRSSLSEAAQKQMDEDLSNKPE NYRPGKNSKADPQTVEKQPAKKQNEPVEKAKQVPTQQTAKRGKPPFGANGFTANWDC VPQGLVVGGYGFQQYHMGGGQDSAYEYFPKEYLLLGGLESKYQKLYVDAVEAINEWL LYRPMTDGDWDILFPAKVSTAGNPSQDLVATFEVTHLTCFIGGMYGLGGKIFGREKDLE TAKRLTDGCVWAYQSTVSGIMPEGSQVLACPTLEKCDFNETLWWEKLDPAKDWRDKQ VADDKDKATVGEALKETANSHDAAGGSKAVHKRAAVPLPKPGADDDVGSELPQSLKD KIGFKNGEQKKPTGSSVGIQRDPDAPVDSVLEAHRLPPQEPEEQQVILPDKPQTHEEFVK QRIAEMGFAPGVVHIQSRQYILRPEAIESVWYMYRITGDPIWMEKGWKMFEATIRATRTE IANSAIDDVNSEEPGLKDEMESFWLAETLKYYYLLFSEPSVISLDEWVLNTEAHPFKRPG GSVIGHSI cDNA sequence of SEQ ID NO: 152 ATG CCA GCT GCT TCT TTG TTG CCA TTG TTT GGT TCT GCT GCT GGT CCA GGT G Gallus gallus CT TTG GGT GGT CCA GCT GGT GGT GGT GCT GGT GGT GGT GGT AGA AAGGCT G protein sequence CT GGT CCA GGT GCT TTT AGA TTG ACT GAA AAG TTT GTT TTG TTG TTG GTT TT chosen for T TCT GCT TTT ATT ACT TTG TGT TTT GGT GCT ATT TTT TTT TTGCCA GAT TCT expression TCT AAG TTG TTG TCT GGT GTT TTT TTT CAT TCT GCT GCT TTG CAA CCA CCA CCA CCA CCA CCA GGT TTT CAA CCA AGA GCT CCA CCA CAACCA GGT GCT GGT CCA GCT ATG CCA GAA GAA GCT GGT GGT GCT GGT TCT TTG GAA AGA ATT AGA GCT GAT CAT GAA AGA GCT TTG AGA GAA GCT AAG GAAACT TTG CAA AAG TTG CCA GAA GAA ATT AGA AGA GAT ATT AGA CAA GAT AAG GAA AAG TTG TTG CAA GA T GCT AGA GGT AGA AAG GAA GCT GCT GCT GCTGGT TTG CCA CAA AGA CCA TT T AGA CAA CCA GTT GGT GCT GTT GGT AGA GAA CCA GCT GAT TTG GCT GTT AG A CAA AGA AGA GAT AAG ATT AAG GAA ATGATG AAG TAG GCT TGG GAT AAC T AC AAG AGA TAC GCT TGG GGT TTG AAC GAA TTG AAG CCA ATT TCT AAG CAA GGT CAT TCT TCT AAC TTG TTT GGT AACATT CAA GGT GCT ACT ATT GTT GAT G CT TTG GAT ACT TTG TTT ATT ATG GAA ATG AAG GAA GAA TTT AAG GAA GCT A AG GAA TGG GTT GAA AAG AAC TTGGAT TTT AAC GTT AAC GCT GAA ATT TCT G TT TTT GAA GTT AAC ATT AGA TTT GTT GGT GGT TTG TTG TCT GCT TAC TAC TTG TCT GGT GAA GAA ATT TTTAGA AAG AAG GCT GTT GAA TTG GGT GAA AAG TTG TTG CCA GCT TTT AAC ACT CCA ACT GGT ATT CCA TGG GCT TTG TTG AAC ATT A AG TCT GGT ATT GGTAGA AAC TGG CCA TGG GCT TCT GGT GGT TCT TCT ATT TT G GCT GAA TTT GGT ACT TTG CAT TTG GAA TTT GTT CAT TTG TCT CAT TTG TCT GGT AAC CCAGTT TTT GCT GAA AAG GTT ATG AAC ATT AGA AAG GTT TTG TCT A GA TTG GAT AAG CCA GAA GGT TTG TAC CCA AAC TAC TTG AAC CCA TCT TCT G GT CAATGG GGT CAA CAT CAT GTT TCT ATT GGT GGT TTG GGT GAT TCT TTT TA C GAA TAC TTG TTG AAG GCT TGG TTG ATG TCT GAT AAG ACT GAT GAA GAA GG TAAG AAG ATG TAC TAC GAT GCT GTT CAA GCT ATT GAA ACT CAT TTG ATT AG A AAG TCT TCT GGT GGT TTG ACT TAC ATT GCT GAA TGG AAG GGT GGT TTGTTG GAA CAT AAG ATG GGT CAT TTG ACT TGT TTT GCT GGT GGT ATG TTT GCT TTG GGT GCT GAT GGT GCT CCA TCT GAT AAG ACT GGT CAT CAT ATT GAATTG GGT G CT GAA ATT GCT AGA ACT TGT CAT GAA TCT TAC GAT AGA ACT TCT ATG AAG T TG GGT CCA GAA GCT TTT AGA TTT GAT GGT GGT GTT GAA GCTATT GCT ACT AG A CAA AAC GAA AAG TAC TAC ATT TTG AGA CCA GAA GTT ATT GAA ACT TAC AT G TAC ATG TGG AGA TTG ACT CAT GAT CCA AAG TAC AGACAA TGG GCT TGG GA A GCT GTT GAA GCT TTG GAA AAG CAT TGT AGA GTT GAT GGT GGT TAC TCT GG T ATT AGA GAT GTT TAC TCT AAC CAT GAA TCT CATGAT GAT GTT CAA CAA TCT TTT TTT TTG TCT GAA ACT TTG AAG TAC TTG TAC TTG TTG TTT TCT GAT GAT GA T TTG TTG CCA TTT GAA CAT TGG GTT TTTAAC ACT GAA GCT CAT CCA TTT CCA ATT TTG AGA AAG GAA GAT GGT TCT AAG GAA GAA AAG GAA AAG Codon optimized SEQ ID NO: 153 ATG CCA GCA GCA TCC TTA CTT CCA TTA TTT GGC TCC GCA GCT GCA CCT GGC Gallus gallus GCT TTA GGT GGT CCT GCT GGC GGC GGA GCC GGA GGC GGC GGC CGT AAAGCC cDNA GCA GGT CCT GGT GCA TTC AGG CTG ACC GAG AAA TTC GTC CTG CTA CTT GTC TTT TCA GCT TTT ATA ACG CTG TGT TTC GGC GCA ATT TTT TTT CTTCCT GAT TC C TCC AAA CTT CTT TCA GGT GTC TTT TTC CAT AGT GCA GCA CTT CAA CCT CCT CCC CCC CCT CCA GGT TTC CAA CCC AGA GCT CCT CCA CAACCA GGA GCT GGA CCT GCC ATG CCC GAA GAG GCA GGA GGT GCC GGT AGT CTA GAA AGA ATA AG G GCA GAC CAC GAA AGA GCA CTT CGT GAG GCT AAA GAAACC CTA CAG AAA C TT CCC GAG GAG ATC CGT AGG GAC ATA AGG CAA GAT AAA GAA AAA CTT TTA CAA GAC GCA CGT GGT CGT AAA GAA GCC GCC GCC GCAGGA CTA CCC CAA AGA CCA TTT CGT CAG CCT GTT GGC GCT GTC GGA AGG GAA CCC GCT GAT CTT GCA GTA AGA CAG AGA AGA GAC AAA ATC AAG GAG ATGATG AAG TAT GCC TGG GA C AAT TAT AAG CGT TAT GCC TGG GGA CTA AAT GAG CTA AAA CCT ATT TCT AA A CAG GGA CAC ACT TCT AAT TTA TTT GGA AACATC CAA GGT GCC ACC ATA GT T GAT GCA CTT GAT ACT CTG TTC ATA ATG GAG ATG AAA GAA GAG TTC AAA GA G GCA AAA GAA TGG GTA GAG AAA AAC CTTGAT TTC AAC GTA AAC GCA GAA A TC AGT GTC TTC GAA GTA AAT ATA AGA TTC GTT GGA GGC CTA CTT TCC GCT TA T TAT TTA TCA GGA GAG GAA ATA TTTCGT AAG AAG GCC GTG GAA TTA GGT GA A AAA CTT TTG CCA GCT TTT AAC ACC CCA ACA GGA ATT CCT TGG GCT TTG TTG AAT ATC AAG AGT GGA ATC GGTAGA AAC TGG CCT TGG GCT TCT GGT GGA AGT TCA ATA TTG GCC GAA TTT GGA ACT CTT CAT TTA GAA TTC GTC CAT TTA TCC C AT CTA AGT GGT AAC CCAGTT TTC GCC GAG AAA GTA ATG AAT ATT CGT AAA G TT TTG TCT CGT CTT GAT AAG CCT GAG GGC CTG TAC CCT AAC TAG CTT AAT CC C TCT TCA GGC CAATGG GGC CAG CAC CAC GTG TCC ATC GGC GGT CTT GGA GA T AGT TTT TAT GAG TAT CTG CTG AAG GCT TGG TTA ATG TCC GAC AAG ACT GA C GAA GAG GGCAAA AAG ATG TAT TAT GAT GCC GTC CAA GCT ATC GAG ACT CA C TTA ATT AGG AAG TCT AGT GGT GGT CTG ACC TAT ATA GCC GAA TGG AAG GG C GGC CTTCTT GAA CAC AAA ATG GGT CAC TTA ACC TGC TTT GCA GGA GGT AT G TTT GCT TTA GGC GCA GAC GGC GCC CCC TCA GAT AAA ACG GGA CAT CAT AT T GAGTTA GGA GCC GAG ATT GCC AGG ACA TGC CAC GAA TCA TAT GAT AGG AC G AGT ATG AAG TTA GGT CCT GAG GCA TTC AGA TTT GAT GGC GGC GTT GAG GC AATC GCT ACC AGA CAA AAT GAG AAA TAC TAC ATT TTA AGA CCA GAA GTC AT T GAG ACC TAC ATG TAC ATG TGG CGT CTA ACT CAT GAC CCC AAA TAT CGTCA G TGG GCA TGG GAG GCC GTT GAA GCC CTA GAA AAA CAT TGC AGA GTT GAC G GC GGT TAT AGT GGC ATA CGT GAT GTC TAT TCA AAC CAT GAG TCC CACGAC G AC GTA CAA CAG TCT TTT TTT CTT TCA GAG ACA CTT AAG TAC CTA TAC CTA CT A TTC AGT GAC GAC GAT CTT CTA CCT TTC GAA CAT TGG GTT TTCAAC ACC GAA GCT CAT CCC TTC CCC ATC TTA CGT AAG GAG GAC GGT TCC AAA GAG GAA AAA GAG AAA Homo sapiens SEQ ID NO: 154 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN ORM1; HsORM1; GLLFINTTIASIAAKEEGVSLDKREAEAQIPLCANLVPVPITNATLDQITGKWFYIASAF uniport P02763 RNEEYNKSVQEIQATFFYFIPNKTEDTIFLREYQTRQDQCIYNTTYLNVQRENGTIS RYVGGQEHFAHLLILRDTKTYMLAFDVNDEKNWGLSVYADKPETTKEQLGEFYEA LDCLRIPKSDVVYTDWKKDKCEPLEKQHEKERKQEEGES*

[0156] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.